Immunization with outer membrane protein A from Salmonella enterica serovar Enteritidis induces humoral immune response but no protection against homologous challenge in chickens.

Vaccination of poultry is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well. We evaluated the efficacy of outer membrane protein A (OmpA) as a novel vaccine candidate against Salmonella in poultry. Native OmpA purified from Salmonella enterica serovar Enteritidis was mixed with adjuvant and administered intramuscularly to 41-d-old chicks. The vaccinated birds showed no decrease in cecal excretion and tissue colonization compared with the unvaccinated birds after oral challenge with 10(9) cfu of the homologous strain at 28 d postimmunization. However, this vaccination induced an increased level of serum anti-OmpA IgG. Similar results were obtained in the replication experiments using a recombinant OmpA with single and double doses. For the development of more effective component vaccines for avian salmonellosis, the vaccine efficacy of outer membrane proteins other than OmpA and route of immunization other than parenteral administration should be evaluated with regard to protection and immune responses, including mucosal IgA.

Morphology of the cockerel's comb after androgen administration.

1. Androgen receptor (AR) expression and morphological changes in blood capillaries were investigated in the comb of cockerels, both untreated controls and after the administration of testosterone (T) or the androgen antagonist flutamide (F) for 7 weeks. 2. Twenty-six male Single Comb White Leghorn roosters were divided into T-treated, F-treated and untreated groups. Tissue sections were examined histologically, immunohistochemically, and comb blood vessel castings were analysed by scanning electron microscopy. 3. Histologically, the capillaries of the peripheral dermis layer in the T-treated group were dilated compared with controls. Many red blood cells were seen in the lumen. Although the capillary diameter in the F-treated group did not show a significant difference as compared with control, blood capillaries with small diameters were often observed, and there were few red blood cells in the capillaries. Some capillary castings were extended markedly in the T treated group, and small blood vessels were observed arborising from the extended blood capillaries. In contrast, all capillaries were slender in the F-treated group, and the casting surface was rough. 4. Immunoreactivity for AR was found in capillary endothelial cells in the peripheral dermis layer of the comb. The intensity of staining in these cells was increased in the T-treated group but was reduced in the F-treated group. 5. It is concluded that immunoreactivity for AR was found in capillary endothelial cells in the peripheral dermis layer of the rooster's comb. The intensity of staining in these cells was increased in the T-treated group but reduced in the F-treated group. Thus, the capillary endothelial cells in the peripheral dermis layer of the comb are androgen targets.

Expression of the T cell receptor Vbeta repertoire in a human T cell resistant to asbestos-induced apoptosis and peripheral blood T cells from patients with silica and asbestos-related diseases.

To explore the effects of asbestos and silica on the human immune system, an experimental model of low-dose and long-term exposure was established using a human HTLV-1-immortalized polyclonal T cell line, MT-2 (MT-2Org). MT-2 cells were continuously exposed to asbestos at a concentration (10 microg/ml) which does not induce complete cell death during short-term exposure. After acquiring resistance to CB-induced apoptosis (designated MT-2Rst), an immunological comparison was made between the MT-2Org and MT-2Rst lines in terms of T cell receptor-Vbeta (TcR-Vbeta) expression. MT-2Rst cells showed excess expression of various TcR-Vbeta, although TcR-Vbeta-overpresenting cells were characterized as undergoing apoptosis due to first contact with CB. Patients with asbestos-related diseases (ARD), such as asbestosis and malignant mesothelioma, were compared with silicosis (SIL) patients as a disease control and with healthy donors (HD). SIL and ARD not only differed in their causative materials, silica and asbestos as mineral silicates, but also in terms of complications; autoimmune disorders in SIL and tumors in ARD. ARD patients showed a restricted overpresentation of TcR-Vbeta without clonal expansion, whereas SIL patients revealed significant overpresentation of TcR-Vbeta 7.2. These experimental and clinical analyses indicate the superantigenic and dysregulation of autoimmunity-inducing effects of asbestos and silica, respectively.

Characterization of the functional subunit combination of nicotinic acetylcholine receptors in bovine adrenal chromaffin cells.

The combination of nicotinic acetylcholine receptors (nAChRs) subunits connecting with the secretion of catecholamines in bovine adrenal chromaffin cells was pharmacologically investigated using selective agonists and antagonists for their nAChRs. The EC(50) values (microM) for the agonists that stimulate the catecholamine secretion and the rank order were as follows: nicotine (3.3)> or =1,1-dimethyl-4-phenylpiperazinium (3.5) > (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (14) > cytisine (23) > or =acetylcholine (25). However, because both the rank order and the EC(50) values differed considerably from those in the various subunits' combinations expressed in Xenopus oocytes or mammalian cells (e.g. alpha2beta2, alpha3beta4, alpha4beta4, etc.), we could not compare them. On the other hand, the IC(50) values (microM) for the antagonists that inhibit the secretion and the rank order were mecamylamine (0.08) > alpha-conotoxin-MII (alpha-CTX-MII) (0.71) > dihydro-beta-erythroidine (DHbetaE) (48) > alpha-bungarotoxin (alpha-BTX) (no effect). Mecamylamine is a highly selective antagonist for alpha3beta4 nAChRs, and alpha-CTX-MII and alpha-BTX are specific antagonists for alpha3beta2 and alpha7 nAChRs, respectively. DHbetaE is a selective antagonist for the alpha4beta2. It has already been shown that the mRNAs for alpha3, alpha5, alpha7 and beta4 subunits are expressed in the chromaffin cells. Therefore, the subunit combination of nAChRs associated with the catecholamine secretion from bovine adrenal chromaffin cells is suggested to be at least alpha3beta4 or alpha3beta4alpha5. Further, the results indicate that the utilization of the nicotinic agonists as selective ligands for the subunit combination of nAChRs may be not suitable for the characterization of nAChRs.

Characterization of ginseng saponin ginsenoside-Rg(3) inhibition of catecholamine secretion in bovine adrenal chromaffin cells.

Since ginsenoside-Rg(3), one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg(3) inhibition were investigated. Although ginsenoside-Rg(3) inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K(+) or veratridine, an activator of the voltage-sensitive Ca(2+) or Na(+) channels, only slightly. The ACh-induced Na(+) and Ca(2+) influxes into the cells were also reduced by ginsenoside-Rg(3). The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca(2+). The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 microM ginsenoside-Rg(3) and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 microM of this compound. Above 30 microM ginsenoside-Rg(3) increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg(3) at 30 microM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 microM. These results strongly suggest that ginsenoside-Rg(3) blocks the nicotinic ACh receptor-operated cation channels, inhibits Na(+) influx through the channels, and consequently reduces both Ca(2+) influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.

Detection of dichromate (VI)-induced DNA strand breaks and formation of paramagnetic chromium in multiple mouse organs.

DNA single-strand breaks (and/or alkali-labile sites) induced by Cr(VI) were evaluated with the alkaline single cell gel electrophoresis (SCG) (Comet) assay in five organs (liver, kidney, spleen, lung, and brain) of male mice dosed with K(2)Cr(2)O(7) (20 mg Cr/kg) by a single ip injection in vivo, and the formation of paramagnetic Cr(V) in these organs was investigated by electron spin resonance (ESR) spectrometry. Furthermore, the in vivo effects of deferoxamine (DFO), an iron chelator, and dimethylthiourea (DMTU), a hydroxyl radical scavenger, on the formation of Cr(V) and DNA strand breaks induced by the metal in the liver and kidney were examined. SCG assay detected DNA strand breaks were detected in the liver and kidney at 15 min and showed that they were being repaired at 3 h after Cr(VI) injection. The ESR spectra of paramagnetic Cr(V) were also observed in the liver and kidney for 15 min to 24 h after Cr(VI) injection. In contrast, there were no significant levels of DNA strand breaks and Cr(V) in the spleen, lung, or brain. The pretreatment of mice with DFO reduced the formation of Cr(VI)-induced DNA strand breaks and Cr(V) complexes as well as the total contents of Cr in the liver and kidney at 15 min after the metal injection. In the case of the pretreatment with DMTU, DNA strand breaks induced by Cr(VI) were suppressed in the liver and kidney at 15 min, without any influence on the levels of Cr(V) complexes and total Cr contents in the organs. The in vitro study showed that DFO decreased the levels of Cr(V)-GSH complexes and Cr(V)-mediated hydroxyl radicals, while DMTU reduced only the levels of Cr(V)-mediated hydroxyl radicals without affecting the formation of Cr(V)-GSH complexes. These results demonstrated that the SCG assay may be useful for detecting DNA strand breaks and/or alkali-labile sites caused by Cr(VI) in vivo. The results also indicated that the in vivo formation of hydroxyl radicals during the reduction of Cr(VI) may play an important role in the induction of the DNA strand breaks caused by this metal and implied that the levels of Cr(V) inside the cells may not always be related to the induction of DNA strand breaks.

Effects of extract and ingredients isolated from Magnolia obovata thunberg on catecholamine secretion from bovine adrenal chromaffin cells.

The crude extract of magnolia bark, an herbal drug, inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh) in a concentration-dependent manner (200-900 microg/mL). The extract also diminished the secretion induced by high K(+), which is a stimulus directly depolarizing the plasma membranes, but its inhibition was weaker than that of ACh-evoked secretion. beta-Eudesmol, honokiol, magnolol, and bornyl acetate, but not alpha- and beta-pinenes, all of which are ingredients of magnolia bark, greatly reduced ACh-evoked secretion. beta-Eudesmol and magnolol also inhibited high K(+)-induced secretion to an extent similar to that of ACh-evoked secretion. However, honokiol and bornyl acetate inhibited the secretion induced by high K(+) much less than the secretion evoked by ACh. ACh-induced Na(+) influx and ACh- or high K(+)-induced Ca(2+) influx into the cells were diminished by beta-eudesmol or honokiol. These results indicate that magnolia bark contains some effective components inhibiting the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by ACh due to the antagonism of Na(+) and Ca(2+) influxes into the cells. However, inhibition by the extract of magnolia bark seems to be attributable to honokiol and bornyl acetate. Furthermore, the results indicate that the inhibitory effect of magnolia bark may be associated with its pharmacological effect on activities of the nervous system.

Activation of rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin.

The small GTPase Rho, which regulates a variety of cell functions, also serves as a specific substrate for bacterial toxins. Here we demonstrate that Bordetella dermonecrotizing toxin (DNT) catalyzes cross-linking of Rho with ubiquitous polyamines such as putrescine, spermidine and spermine. Mass spectrometric analyses revealed that the cross-link occurred at Gln63, which had been reported to be deamidated by DNT in the absence of polyamines. Rac1 and Cdc42, other members of the Rho family GTPases, were also polyaminated by DNT. The polyamination, like the deamidation, markedly reduced the GTPase activity of Rho without affecting its GTP-binding activity, indicating that polyaminated Rho behaves as a constitutively active analog. Moreover, polyamine-linked Rho, even in the GDP-bound form, associated more effectively with its effector ROCK than deamidated Rho in the GTP-bound form and, when microinjected into cells, induced the anomalous formation of stress fibers indistinguishable from those seen in DNT-treated cells. The results imply that the polyamine-linked Rho, transducing signals to downstream ROCK in a novel GTP-independent manner, plays an important role in DNT cell toxicity.

Identification of functional domains of Bordetella dermonecrotizing toxin.

Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants. Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; consequently, DNT contains an additional 13 amino acids at its N-terminal end. All of the N-terminally truncated mutants were found to modify Rho. The shortest fragment of DNT possessing the Rho modification activity consists of amino acids from Ile1176 to the C-terminal end. This fragment overlaps the region homologous to Escherichia coli cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT. The introduction of a mutation at Cys1305 located in the highly conserved region between CNFs and DNT eliminated the activity, indicating that this domain is the catalytic center of DNT. The N-terminal fragment (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynucleation in MC3T3-E1 cells when simultaneously added with the holotoxin, suggesting competitive inhibition in the receptor-binding or internalizing step. Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facilitate analysis of the overall action of the toxin on the mammalian target cells.

Effects of interferons on cortisol production in bovine adrenal fasciculata cells stimulated by adrenocorticotropin.

The effects of interferons (IFNs) IFN-alpha, IFN-beta and IFN-gamma on the production of cortisol in bovine adrenal fasciculata cells have been investigated. Pretreatment of the fasciculata cells with recombinant interferon-alpha-2b from man (over 300 units mL(-1)), but not with fibroblast IFN-beta or recombinant IFN-gamma from man, reduced the production of cortisol in cells stimulated with adrenocorticotropin (ACTH) (1 nM). IFN-alpha-2b inhibited ACTH-induced cortisol production in a concentration- (300-15000 units mL(-1)) and time- (2-24h) dependent manner. The inhibitory effect of IFN-alpha-2b on the production was abolished when the cells were simultaneously treated with anti-IFN-alpha antibody, and it was reversible. IFN-alpha-2b also inhibited dibutyryl cyclic AMP-induced production of cortisol but not pregnenolone-induced production. The effect of IFN-alpha-2b was not influenced by increases in external ACTH and Ca2+ concentrations and IFN-alpha-2b did not affect the ACTH-induced increase in cyclic AMP level in the cells. These results strongly suggest that IFN-alpha-2b reduces ACTH-induced production of cortisol in bovine adrenal fasciculata cells by affecting the early process of cortisol synthesis. The results also indicate that IFNs might not directly affect steroidogenesis in the adrenal cortex in-vivo, because of the requirement of high concentrations of IFN-alpha-2b for inhibition, and because of the ineffectiveness of IFN-beta and IFN-gamma.

Effects of ginseng saponins on responses induced by various receptor stimuli.

We investigated the effects of four ginseng saponins, ginsenoside-Rb1, -Rg2, -Rg3 and -Ro, on the responses induced by receptor stimulation of various stimuli. Ginsenoside-Rg2 (1-100 microM) reduced the secretions of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine and gamma-aminobutyric acid but not by angiotensin II, bradykinin, histamine and neurotensin. In guinea-pig, the ginsenoside also diminished the nicotine-induced secretion of catecholamines from the adrenal chromaffin cells, but it did not affect the muscarine- and the histamine-induced ileum contractions. On the other hand, ginsenoside-Rg3 (1-100 microM) reduced not only the acetylcholine-, the gamma-aminobutyric acid- and the neurotensin-induced secretions but also, at a higher concentration (100 microM), the angiotensin II-, the bradykinin- and the histamine-induced secretions from the bovine chromaffin cells. Furthermore, the saponin (3-100 microM) significantly inhibited the muscarine- and the histamine-induced ileum contractions of the guinea-pig. Ginsenoside-Rb1 and -Ro had no marked effect on their responses. These results strongly suggest that ginsenoside-Rg2 is a potent selective blocker of nicotinic acetylcholine and gamma-aminobutyric acid receptors (ionotropic receptors) and ginsenoside-Rg3 is not only a blocker of ionotropic receptors but also an antagonist of muscarinic or histamine receptors.

Asymmetrical membrane fluidity of bovine adrenal chromaffin cells and granules and effect of trichosporin-B-VIa.

We examined membrane fluidity of bovine adrenal chromaffin cells and chromaffin granules using cationic trimethylammonium derivative of diphenylhexatriene (TMA-DPH) as a fluorescence probe. After adding TMA-DPH to the suspension of chromaffin cells and that of granules, it first bound to the outer layer of the plasma membrane of the cells and that of the granule membrane, then gradually penetrated the inner layer of each membrane and distributed to both leaflets of the respective membranes. Accompanying increases in the ratio of incorporated probe on the cytoplasmic side of the chromaffin cell membrane, its fluorescence anisotropy gradually decreased. However, in chromaffin granules, the fluorescence anisotropy gradually increased with increases in the ratio of incorporated probe. These findings suggest that the inner layer of the plasma membrane and outer layer of the granular membrane are more fluid than the corresponding side of each membrane, which is suitable for the fusion between both membranes. We also examined the effect of trichosporin-B-VIa, a fungal ion channel forming alpha-aminoisobutyric acid-containing peptide, on the fluidity of chromaffin cells using TMA-DPH. The peptide decreased the fluorescence anisotropy and increased the fluorescence intensity in the concentration range that induced Ca2+ dependent catecholamine secretion, suggesting that a change in lipid dynamics of the lipid bilayer of the plasma membrane was induced by this peptide.

Properties of ginseng saponin inhibition of catecholamine secretion in bovine adrenal chromaffin cells.

To investigate the relationship between the inhibitory effects of ginseng saponins (ginsenosides) on acetylcholine-evoked secretion of catecholamines and the structures of ginsenosides, we examined the effects of ginsenoside-Rg3 and -Rh2, which are panaxadiol saponins, 20(R)- and 20(S)-ginsenoside-Rg2, which are epimers involving the hydroxyl group at C-20 of sapogenin, and other plant saponins on the acetylcholine-evoked secretion of catecholamines from cultured bovine adrenal chromaffin cells. The ginsenoside-Rg3 (1-100 microM) and -Rh2 (10-100 microM) greatly reduced the acetylcholine-evoked secretion in a concentration-dependent manner comparable to that of ginsenoside-Rg2, a panaxatriol saponin, which was the most potent inhibitor in our previous study. 20(R)- and 20(S)-ginsenoside-Rg2 (1-100 microM) similarly reduced the acetylcholine-evoked secretion. In contrast, saikosaponin-a, glycyrrhizin and the cardiac glycosides (100 nM-100 microM), digitoxin and digoxin, had no significant inhibitory effect on catecholamine secretion. Saikosaponin-c (10-100 microM), however, had an inhibitory effect, which was less than that of ginsenoside-Rg2 and -Rg3. These results strongly suggest that the inhibitory effects of ginsenosides on the acetylcholine-evoked secretion of catecholamines from bovine adrenal chromaffin cells are a unique property of ginseng. Further, the relationship between the inhibitory effects and the structures of ginsenosides is discussed.

Down-regulation of the noradrenaline transporter by interferon-alpha in cultured bovine adrenal medullary cells.

The aim of this study was to investigate the effect of long-term treatment with interferon (IFN)-alpha on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN-alpha caused a decrease in uptake of [3H]noradrenaline by the cells in time (4-48 h)- and concentration (300-1,000 U/ml)-dependent manners. IFN-beta also inhibited [3H]noradrenaline uptake to a lesser extent than did IFN-alpha, whereas IFN-gamma had little effect. An anti-IFN-alpha antibody reduced the effect of IFN-alpha on [3H]noradrenaline uptake. Saturation analysis of [3H]noradrenaline uptake showed that the inhibitory effect of IFN-alpha was due to a reduction in the maximal uptake velocity (Vmax) values without altering apparent Michaelis constant (Km) values. Incubation of cells with IFN-alpha caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN-alpha on [3H]noradrenaline uptake was diminished in protein kinase C-down-regulated cells. Incubation of cells with IFN-alpha for 48 h significantly reduced the specific binding of [3H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [3H]desipramine binding revealed that IFN-alpha decreased the maximal binding (Bmax) values without any change in the dissociation constant (K(D)) values. These findings suggest that IFN-alpha suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.

Bordetella bronchiseptica dermonecrotizing toxin induces reorganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho.

Bordetella dermonecrotizing toxin causes assembly of actin stress fibers and focal adhesions in some cultured cells and induces mobility shifts of the small GTP-binding protein Rho on electrophoresis. We attempted to clarify the molecular basis of the toxin action on Rho. Analysis of the amino acid sequence of toxin-treated RhoA revealed the deamidation of Gln-63 to Glu. The substitution of Glu for Gln-63 of RhoA by site-directed mutagenesis caused a mobility shift on electrophoresis, which was indistinguishable from that of the toxin-treated RhoA. Neither mutant RhoA-bearing Glu-63 nor toxin-treated RhoA significantly differed from untreated wild type RhoA in guanosine 5'-[gamma-thio]triphosphate binding activity but both showed a 10-fold reduction in GTP hydrolysis activity relative to untreated RhoA. C3H10T1/2 cells transfected with cDNA of the mutant RhoA bearing Glu-63 showed extensive formation of actin stress fibers similar to the toxin-treated cells. These results indicate that the toxin catalyzes deamidation of Gln-63 of Rho and renders it constitutively active, leading to formation of actin stress fibers.

Interferon-alpha reduces catecholamine secretion from bovine adrenal chromaffin cells stimulated by acetylcholine.

A long-term pretreatment (72 h) of bovine adrenal chromaffin cells with recombinant human interferon (IFN) -alpha-2b (1500 units/ml) produced a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine (ACh) (25 micromol/l) but not that with human fibloblast IFN-beta (3000 units/ml) or recombinant human IFN-gamma (3000 units/ml). IFN-alpha-2b inhibited the ACh-induced secretion in a concentration- (30-1500 units/ml) and time-dependent manner (18-72 h). The content of catecholamines in the cells treated with IFN-alpha-2b for 72 h did not change. The inhibitory effect of IFN-alpha-2b on the secretion was abolished when the cells were simultaneously treated with anti-IFN-alpha antibody, and it was overcome by the increase in the external ACh concentration. IFN-alpha-2b also inhibited ACh-induced Ca2+ influx into the cells in a concentration-dependent manner similar to that of the IFN-alpha-2b inhibiting ACh-induced secretion. On the other hand, IFN-alpha-2b failed to reduce the secretion from the cells induced by high K+. These results strongly suggest that IFN-alpha-2b reduces the ACh-induced secretion of catecholamines from bovine adrenal chromaffin cells due to modulating the gene expression of the nicotinic ACh receptor-operated cation channels rather than due to directly affecting the channels. The results further indicate that the IFN-alpha-2b inhibition may be associated with the psychiatric side effects of IFN-alpha (depression, neurasthenica and somnolence, etc.), and that immune systems may regulate the function of (autonomic) nervous systems or adrenal medulla via IFN-alpha in vivo.

[Effects of ginseng saponins on receptor stimulation-responses].

We investigated the effects of root of Panax ginseng C. A. Meyer on the secretion of catecholamines (CAs) from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh). In two major parts, nonsaponin and crude saponin fractions from the root, the crude saponin but not the non-saponin greatly reduced the ACh-evoked secretion. Furthermore, various purified ginseng saponins (ginsenosides) had a tendency to reduce the secretion. Most effective saponin was ginsenoside (G) RG2. GRg2 also inhibited the ACh-evoked Na+ and Ca2+ influxes into the cells. The GRg2 inhibition of the secretion was overcome by increasing the external Na+ but not Ca2+ concentrations. However, GRg2 did not affect the secretion from the cells induced by high K+, which is regarded as directly depolarizing the cell membranes and causing Ca2+ influx through voltage-sensitive Ca2+ channels. Therefore, the root of Panax ginseng contains ingredients, that is saponins, which inhibit the secretion of catecholamines from the cells stimulated by ACh. The inhibition is probably due to the antagonism of nicotinic acetylcholine receptor-operated cation channels. GRg2 had no effects on other receptor stimulation-responses but GRg3 inhibited them. These may be why the root of Panax gingseng has a variety of pharmacological effects.

Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum.

Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.

Isolation of a novel lectin from the globiferous pedicellariae of the sea urchin Toxopneustes pileolus.

Sea urchin lectin-I (SUL-I), a 32 kDa lectin was purified from the large globiferous pedicellariae of the sea urchin, Toxopneustes pileolus by using gel permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. SDS-PAGE showed that SUL-I is a monomeric protein with a molecular mass of 32 kDa. Amino acid analysis indicates SUL-I to contain 294 residues. SUL-I was shown to have chemotactic properties for guinea-pig neutrophils at concentrations of 0.625 microgram/ml. These data suggest that a 32 kDa lectin from T. pileolus may be related to defensive role.

Dimethylsulfoxide potentiates the suppressive effects of lidocaine on synaptic transmission in bullfrog sympathetic ganglion.

The effects of dimethylsulfoxide used as a solvent on the suppressive effects of lidocaine on synaptic transmission were examined in the bullfrog sympathetic ganglion. Compound action potentials (CAP) in response to orthodromic stimulation of the presynaptic nerve trunk were recorded extracellularly from the bullfrog sympathetic ganglion. Application of lidocaine dissolved in normal Ringer's solution significantly decreased the amplitude of CAP. This suppressive effect of lidocaine was markedly potentiated when lidocaine was dissolved in 0.1% DMSO solution. A similar potentiating effect of DMSO was observed on the suppressive action of lidocaine on acetylcholine (ACh)-induced depolarizing response recorded intracellularly from the sympathetic ganglion cell. On the other hand, 0.1% DMSO solution alone had no effect on synaptic transmission, resting membrane potential, resting membrane resistance or ACh receptor activity. The present findings suggest that DMSO as solvent should be used carefully because of its potentiating effect on drug actions. Since the effect of lidocaine is due to the suppression of nicotinic ACh receptor at the postsynaptic membrane, DMSO may allosterically act on the nicotinic ACh receptor causing an increase in efficacy of lidocaine.