The release of [3H]‐noradrenaline ([3H]‐NA) in response to nicotinic acetylcholine receptor (nAChR) agonists was compared with agonist‐induced currents in cultured rat superior cervical ganglion (SCG) neurones. [3H]‐NA release in response to high concentrations of nicotinic agonists was reduced, but not fully inhibited, by the presence of either tetrodotoxin (TTX) or Cd2+ to block voltage‐gated Na+ or Ca2+ channels, respectively. We used the component of transmitter release that remained in the presence of these substances (named TTX‐ or Cd2+‐insensitive release) to pharmacologically characterize nAChRs in proximity to the sites of vesicular exocytosis (prejunctional receptors). Prejunctional nAChRs were activated by nicotinic agonists with a rank order of potency of dimethylphenylpiperazinium iodide (DMPP) > nicotine > cytisine > ACh, and with EC50 values ranging from 22 μM (DMPP) to 110 μM (ACh). [3H]‐NA release in response to low concentrations of nAChR agonists was fully inhibited by the presence of either TTX or Cd2+ (named TTX‐ or Cd2+‐sensitive release). TTX‐sensitive release was triggered by nicotinic agonists with a rank order of potency of DMPP > cytisine ≈ nicotine ≈ ACh, which due to its similarity to TTX‐insensitive release indicates that it might also be triggered by prejunctional‐type nAChRs. The EC50 values for TTX (Cd2+)‐sensitive release were less than 10 μM for all four agonists. By contrast to transmitter release, somatic nAChRs as seen by patch clamp recordings were most potently activated by cytisine, with a rank order of potency of cytisine > nicotine ≈ DMPP > ACh. EC50 values for the induction of currents exceeded 20 μM for all four agonists. The nicotinic antagonist mecamylamine potently inhibited all transmitter release in response to nicotine. α‐Bungarotoxin (α‐BuTX) was, on the other hand, without significant effect on nicotine‐induced TTX‐insensitive release. The competitive antagonist dihydro‐β‐erythroidine (DHβE) caused rightward shifts of the dose‐response curves for both TTX‐sensitive and TTX‐insensitive transmitter release as well as for currents in response to nicotine, with pA2 values ranging from 4.03 to 4.58. Due to clear differences in the pharmacology of agonists we propose that nAChRs of distinct subunit composition are differentially targeted to somatic or axonal domains.
Trichoderma reesei was studied for its ability to produce fl-mannanase activity on a variety of carbon sources. The highest fl-mannanase activity was produced on cellulose, whereas fl-mannan-containing carbon sources (such as konjac powder or locust bean gum) gave lower enzyme titres. The enzyme responsible for the major fl-mannanolytic activity from T. reesei was purified to physical homogeneity by preparative chromatofocusing and anion exchange fast protein liquid chromatography. This fl-mannanase is a glycoprotein, with a molecular mass of 46 (+ 2) kDa and an isoelectric point of 5.2. It has an optimal pH at 5.0 and broad pH stability (2.5-7.0). It is stable for 60 min at 55 ° C, and has an optimal temperature for activity at 75 ° C. During incubation with locust bean gum, the enzyme releases mainly tri-and disaccharides.
Reuptake of extracellular noradrenaline (NA) into superior cervical ganglion (SCG) neurones is mediated by means of the noradrenaline transporter (NAT, uptake 1). We now demonstrate by single‐cell RT‐PCR that mRNA of the organic cation transporter 3 (OCT3, uptake 2) occurs in rat SCG neurones as well. Furthermore, our RT‐PCR analyses reveal the presence of mRNA for novel organic cation transporters 1 and 2 (OCTN1 and OCTN2), but not for OCT1 or OCT2 in the ganglion. Making use of the NAT as a powerful, neurone‐specific transporter system, we loaded[3H]‐N‐methyl‐4‐phenylpyridinium ([3H]‐MPP+) into cultured rat SCG neurones. The ensuing radioactive outflow from these cultures was enhanced by desipramine and reserpine, but reduced (in the presence of desipramine) by the OCT3 inhibitors cyanine 863, oestradiol and corticosterone. In contrast, cyanine 863 enhanced the radioactive outflow from cultures preloaded with [3H]‐NA. Two observations suggest that a depletion of storage vesicles by cyanine 863 accounts for the latter phenomenon: first, the primary radioactive product isolated from supernatants of cultures loaded with [3H]‐NA was the metabolite [3H]‐DHPG; and second, inhibition of MAO significantly reduced the radioactive outflow in response to cyanine 863. The outflow of [3H]‐MPP+ was significantly enhanced by MPP+, guanidine, choline and amantadine as potential substrates for OCT‐related transmembrane transporters. However, desipramine at a low concentration essentially blocked the radioactive outflow induced by all of these substances with the exception of MPP+, indicating the NAT and not an OCT as their primary site of action. The MPP+‐induced release of [3H]‐MPP+ was fully prevented by a combined application of desipramine and cyanine 863. No trans‐stimulation of [3H]‐MPP+ outflow was observed by the OCTN1 and OCTN2 substrate carnitine at 100 μM. Our observations indicate an OCT‐mediated transmembrane transport of [3H]‐MPP+. Amongst the three OCTs expressed in the SCG, OCT3 best fits the profile of substrates and antagonists that cause trans‐stimulation and trans‐inhibition, respectively, of [3H]‐MPP+ release.
Trichoderma reesei RUT C-30 formed an extracellular a-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain a-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced at-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of ac-galactosidase formation by galactose. a-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 ± 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several a-galactosides (p-nitrophenyl-t-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei 1-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50°C resulted in recovery of 70%o of activity), and its highest level of activity occurred at 60°C. Its action on galactomannan was increased by the presence of 13-mannanase. Hemicelluloses are the second most abundant polysaccharides in nature, and they seem to be linked to lignin in wood (23). The major constituents of hemicellulose are the hetero-1,4-0-D-xylans and the hetero-1,4-,B-D-mannans (galactoglucomannans and glucomannans). While heteroxylans are found mainly in grasses, cereals, and hardwoods, P-mannans are more abundant in softwoods (8, 23). The biotechnological interest in the hydrolysis of hemicelluloses for the pulp and paper industry and the feedstock industry has recently revived interest in the enzymology of hemicellulose degradation. In the case of galactomannans, this enzymatic hydrolysis requires the concerted action of the following hydrolytic enzymes: endo-1
The addition of Ca2+-antagonizers (La2+), Ca2+-ionophores (A23187) and Ca2+-complexing agents (EGTA) inhibited the formation of xylanase activity in resting mycelia of Trichoderma reesei. The inhibition by the ionophore was reversed by the addition of Ca2+ ions. A similar inhibitory effect was obtained by the addition of the calmodulin inhibitors, trifluoroperazine, chlorpromazine and quinacrine, hence suggesting that the observed effect of Ca2+ on xylanase formation occurred via calmodulin. The inhibition of xylanase formation by trifluoroperazine was accompanied by an inhibition of formation of the xyn2 transcript, and of the hph (hygromycin B-phosphotransferase-encoding) gene when fused downstream of the 5'-regulatory signals of the T. reesei xyn2 gene, indicating that calmodulin is required for xyn2 induction. At trifluoroperazine concentrations, which inhibited extracellular xylanase formation only slightly (about 30%), the cell-free extracts exhibited slightly increased xylanase activities. Subcellular fractionation showed that in these mycelia, the XYN II protein was distributed over a range of light vesicular fractions. This accumulated XYN II protein had the same Mr as the secreted, extracellular enzyme, indicating that it had already passed Golgi-located preprotein processing. Trifluoroperazine also specifically interfered with the endogenous, Ca2+-dependent phosphorylation of a 20-kDa protein, which was predominantly observed in cell-free extracts from mycelia growing on xylan. From these data, we conclude that calmodulin is required for xylanase II formation by T. reesei both at a transcriptional level as well as at a post-Golgi step of the secretory pathway. We also suggest that at least one of these two steps may be mediated via Ca2+-calmodulin-dependent phosphorylation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.