Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).     

Gangliosides prevent reduction in exploratory activity and delayed neuronal death in gerbils subjected to transitory cerebral ischemia

INTRODUCTION: The ganglioside GM1 has been shown to be effective in the treatment of experimental cerebral ischemia. Gangliosides from bovine brain have not been used in the treatment of ischemic cerebral accidents. There is evidence suggesting that they may also be effective. RESULTS: Ten minutes of bilateral occlusion of the carotid arteries of Mongolian gerbils leads a week later to reduced spontaneous exploratory activity, assessed by counting the number of times they stood up in an open field over a period of three minutes, and retarded neuronal death in the pyramidal stratum of the CA1 sector of the hippocampus, evaluated on the density of normal neurons in this region of both hemispheres. Treatment with 30 mg/kg of intra-peritoneal bovine cerebral gangliosides during the first six days following occlusion of the carotid arteries, leads to conservation of both exploratory activity and density of pyramidal neurons observed in the control animals. CONCLUSIONS: Bovine cerebral gangliosides have a short term cytoprotector effect on neurons sensitive to the ischemia-reperfusion phenomenon. This effect may be due to more than one mechanism, in which other gangliosides (together with GM1) may be present due to transient permeability of the blood-brain barrier.     

Protective effect of a prostaglandin I2 analog, TEI-7165, on ischemic neuronal damage in gerbils

In this study we have analyzed short- and long-term changes in extracellular signal-regulated kinase (ERK) 1 and 2 activity during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of human promyelocytic leukemia cells. Immunoprecipitation of HL-60 cellular extracts with an ERK antibody followed by in vitro myelin basic protein phosphorylation demonstrated a rapid reduction in total ERK activity by 70%. Mitogen-activated protein kinase substrate peptide phosphorylation also demonstrated that this reduction was sustained during differentiation. Immunoblot analysis revealed that ERK1 and ERK2 are the predominant ERK isoforms present in HL-60 cells and that over a 96-h period ERK1 protein was gradually reduced by 60% while ERK2 protein showed only a small, insignificant reduction. Therefore, the large, rapid decrease in total ERK activity could not be attributed to the gradual reductions in ERK1 or ERK2 amounts. Immunoblot analysis with two different phosphotyrosine antibodies revealed a rapid decrease in ERK1 phosphotyrosine and a concurrent transient increase in ERK2 phosphotyrosine. These contrasting changes in phosphorylated ERKs were paralleled by respective shifts in mobility during SDS-PAGE analysis. Together these results indicate that the rapid reduction in total ERK activity is due to rapid tyrosine and possible threonine dephosphorylation of ERK1 but not of ERK2. These results also indicate that ERK1 and ERK2 are regulated by distinct mechanisms during TPA-induced HL-60 differentiation, suggesting that their biological roles are nonredundant.     

Protective effect of clonazepam on ischemic brain damage induced by 10-minute bilateral carotid occlusion in Mongolian gerbils

Phenotypic and functional aspects of melanoma-hyaluronate interactions were investigated by studying the expression of CD44, cell migration, and transmembrane penetration of human melanoma cell lines on hyaluronate-coated substrates. Expression of CD44 was tested by flow cytometry on seven human melanoma cell lines. Strong reactivity with anti-CD44 monoclonal antibody was observed in four of seven of the cell lines. Migration studies of CD44(+) cell lines on hyaluronic acid- and chondroitin-6-sulfate-coated substrates, using time-lapse video-microscopy, showed a dramatic dose-dependent increase in migration rate on hyaluronate but not on chondroitin-6-sulfate. Moreover, CD44(-) cell lines showed no modification in migration rate on either substrate. Addition of soluble hyaluronate produced a dose-dependent inhibition of acceleration of CD44(+)cells on hyaluronate-coated substrates, whereas addition of chondroitin-6-sulfate had no effect. Migration inhibition experiments with soluble CD44 (CD44 receptor globulin) also showed specific blocking of the migration of CD44(+) cells on hyaluronate. Haptotactic invasion was increased in CD44(+) cell lines through hyaluronate-coated polycarbonate membranes, whereas no change was detected on chondroitin-6-sulfate-coated membranes. CD44(-) cell lines showed no response to either type of coating. In the melanoma cell lines tested, the expression of CD44 correlated with in vitro migration and invasiveness on hyaluronate substrates. Taken together, our data are consistent with the suggestion that CD44 may play a role in stimulating in vivo aggressiveness of tumors through hyaluronate-rich stroma.     

Antidepressant effects of pramipexole, a novel dopamine receptor agonist

These studies compared the effects of the 5-HT1B/1D receptor agonists sumatriptan, CP-122 288 ((R)-N-methyl-[3-(1-methyl-2-pyrrolidinylmethyl)-1H-indol-5-yl] methanesulphonamide succinate) and CP-93 129 (3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one dihydrochloride) on neurogenic dural extra-vasation and vasodilation in anaesthetized rats. Dural extravasation, evoked by high intensity (1.2 mA) stimulation of the trigeminal ganglion, was measured using the radioactive plasma marker 125I-labelled bovine serum albumin. Dural vasodilation produced by lower intensity (50-300 microA) stimulation of trigeminal fibres, was measured through a closed cranial window using intravital microscopy. All compounds inhibited dural extravasation (rank order of potency: CP-122 288 > > sumatriptan > CP-93 129) and dural vasodilation (rank order of potency: CP-93 129 > > sumatriptan = CP-122 288). Comparison of the potency of these compounds with their potencies in an in vitro functional model, agonist-induced [35S]GTP gamma S binding, suggests that blockade of dural extravasation was consistent with an action at rat 5-HT1D receptors, but activity at another, unknown, "extravasation receptor" could also be involved. In contrast, inhibition of dural vasodilation was consistent with an action at rat 5-HT1B receptors. We suggest that in our preparations, production of dural vasodilation involves activation of trigeminal A delta-fibres whereas production of dural extravasation involves activation of trigeminal C-fibres. The differential effects of compounds on dural extravasation and vasodilation may therefore be due to the different receptor subtypes involved and to the selective localization of these subtypes on different populations of trigeminal sensory fibre.     

Delayed recovery of auditory cortical evoked potentials is correlated with cortical neuronal death after transient cerebral ischemia in awake gerbils

1. Spermatozoa in semen samples from 8 individual male domestic fowls were shown to have a differential and characteristic ability to hydrolyse holes in the inner perivitelline layer from laid eggs in an in vitro assay. 2. The number of holes produced by samples of spermatozoa per unit area of inner perivitelline layer in vitro was linearly correlated with sperm ATP content (r = 0.85) and motility (r = 0.76). 3. The number of holes formed in the inner perivitelline layer in vitro was also linearly correlated with the numbers of holes formed in the inner perivitelline layer of eggs fertilised in vivo, in inseminated hens (r = 0.90); and was correlated logarithmically with the proportion of fertile eggs laid by these hens.     

Cerebral ischemia in gerbils: effects of acute and chronic treatment with adenosine A2A receptor agonist and antagonist

Despite significant progress in understanding of the potential of adenosine A1 receptor-based therapies in treatment of cerebral ischemia and stroke, very little is known about the effect of selective stimulation of adenosine A2A receptors on the outcome of a cerebrovascular arrest. In view of a major role played by adenosine A2 receptors in the regulation of cerebral blood flow, we have investigated the effect of both acute and chronic administration of the selective adenosine receptor agonist 2-[(2-aminoethylamino)-carbonylethylphenylethylamino]-5'-N- ethylcarboxoamidoadenosine (APEC) and antagonist 8-(3-chlorostyryl)caffeine (CSC) on the outcome of 10 min ischemia in gerbils. Acute treatment with APEC improved recovery of postischemic blood flow and survival without affecting neuronal preservation in the hippocampus. Acute treatment with CSC had no effect on the cerebral blood flow but resulted in a very significant protection of hippocampal neurons. Significant improvement of survival was present during the initial 10 days postischemia. Due to subsequent deaths of animals treated acutely with CSC, the end-point mortality (14 days postischemia) in this group did not differ statistically from that seen in the controls. It is, however, possible that the late mortality in the acute CSC group was caused by the systemic effects of brain ischemia that are not subject to the treatment with this drug. Chronic treatment with APEC resulted in a statistically significant improvement in all studied measures. Although chronic treatment with CSC improved postischemic blood flow, its effect on neuronal preservation was minimal and statistically insignificant. Mortality remained unaffected. The results indicate that the acute treatment with adenosine A2A receptor antagonists may have a limited value in treatment of global ischemia. However, since administered CSC has no effect on the reestablishment of postischemic blood flow, treatment of stroke with adenosine A2A receptor antagonists may not be advisable. Additional studies are necessary to elucidate whether chronically administered drugs acting at adenosine A2 receptors may be useful in treatment of stroke and other neurodegenerative disorders.     

Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia

Delayed neuronal death after transient cerebral ischemia may be mediated, in part, by the induction of apoptosis-regulatory gene products. Caspase-3 is a newly characterized mammalian cysteine protease that promotes cell death during brain development, in neuronal cultures, and in other cell types under many different conditions. To determine whether caspase-3 serves to regulate neuronal death after cerebral ischemia, we have (1) cloned a cDNA encoding the rat brain caspase-3; (2) examined caspase-3 mRNA and protein expression in the brain using in situ hybridization, Northern and Western blot analyses, and double-labeled immunohistochemistry; (3) determined caspase-3-like activity in brain cell extracts; and (4) studied the effect of caspase-3 inhibition on cell survival and DNA fragmentation in the hippocampus in a rat model of transient global ischemia. At 8-72 hr after ischemia, caspase-3 mRNA and protein were induced in the hippocampus and caudate-putamen (CPu), accompanied by increased caspase-3-like protease activity. In the hippocampus, caspase-3 mRNA and protein were predominantly increased in degenerating CA1 pyramidal neurons. Proteolytic activation of the caspase-3 precursor was detected in hippocampus and CPu but not in cortex at 4-72 hr after ischemia. Double-label experiments detected DNA fragmentation in the majority of CA1 neurons and selective CPu neurons that overexpressed caspase-3. Furthermore, ventricular infusion of Z-DEVD-FMK, a caspase-3 inhibitor, decreased caspase-3 activity in the hippocampus and significantly reduced cell death and DNA fragmentation in the CA1 sector up to 7 d after ischemia. These data strongly suggest that caspase-3 activity contributes to delayed neuronal death after transient ischemia.     

Protective effect of the insulin-like growth factor I receptor on apoptosis induced by okadaic acid

Okadaic acid (OKA), a potent inhibitor of serine phosphatases at concentrations as low as 20-25 nM, induces apoptosis of R- mouse embryo fibroblasts, which are 3T3-like cells devoid of type 1 insulin-like growth factor receptors (IGF-IRs). From R- cells, we have generated (by stable transfection) cell lines with IGF-IR numbers ranging from 0 (R- cells) to >10(6) receptors per cell. The wild-type IGF-IR protects R- cells from OKA-induced apoptosis, its protective effect being exquisitely dependent on the number of receptors. A small increment in wild-type receptor number (from 15 x 10(3) to 22 x 10(3) receptors/cell) is sufficient to change R(-)-derived cells from sensitive to resistant to apoptosis. We have also studied the effect of various mutations of the IGF-IR on its ability to protect R(-)-derived cells from OKA-induced apoptosis. Our data indicate a correlation between protection from apoptosis and the ability of the receptor to respond to insulin-like growth factor I with mitogenesis.     

Distinctions in agonist and antagonist specificity conferred by anionic residues of the nicotinic acetylcholine receptor

Two anionic residues in the nicotinic acetylcholine receptor, Asp-152 in the alpha-subunit and Asp-174 in the gamma-subunit or the corresponding Asp-180 in the delta-subunit, are presumed to reside near the two agonist binding sites at the alphagamma and alphadelta subunit interfaces of the receptor and have been implicated in electrostatic attraction of cationic ligands. Through site-directed mutagenesis and analysis of state changes in the receptor elicited by agonists, we have distinguished the roles of anionic residues in conferring ligand specificity and ligand-induced state changes. alphaAsp-152 affects agonist and antagonist affinity similarly, whereas gammaAsp-174 and deltaAsp-180 primarily affect agonist affinity. Combining charge neutralization on the alpha subunit with that on the gamma and delta subunits shows an additivity in free energy changes for carbamylcholine and d-tubocurarine, suggesting independent contributions of these residues to stabilizing the bound ligands. Since both aromatic and anionic residues stabilize cationic ligands, we substituted tyrosines (Y) for the aspartyl residues. While the substitution, alphaD152Y, reduced the affinities for agonists and antagonists, the gammaD174Y/deltaD180Y mutations reduced the affinity for agonist binding, but surprisingly enhanced the affinity for d-tubocurarine. To ascertain whether selective changes in agonist binding stem from the capacity of agonists to form the desensitized state of the receptor, carbamylcholine binding was measured in the presence of an allosteric inhibitor, proadifen. Mutant nAChRs carrying alphaD152Q or gammaD174N/deltaD180N show similar reductions in dissociation constants for the desensitized compared with activable receptor state and a similar proadifen concentration dependence. Hence, these mutations influence ligand recognition rather than the capacity of the receptor to desensitize. By contrast, the alphaD200Q mutation diminishes the ratio of dissociation constants for two states and requires higher proadifen concentrations to induce desensitization. Thus, the contributions of alphaAsp-152, gamma/deltaAsp-174/180, and alphaAsp-200 in stabilizing ligand binding can be distinguished by the interactions between agonists and allosteric inhibitors.     

Beneficial effects of S-adenosyl-L-methionine on blood-brain barrier breakdown and neuronal survival after transient cerebral ischemia in gerbils

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.     

Hemodynamic characterization of YM435, a novel dopamine DA1 receptor agonist, in anesthetized dogs

In previous studies on experimental renal failure, hypertrophy of cardiomyocytes, diminished capillarization, and increased intercapillary distances had been observed, abnormalities that will expose the heart to reduced ischemia tolerance. It has not been established, however, whether such structural alterations are unique for the heart (eg, as a consequence of left ventricular hypertrophy) or are demonstrable in other tissues as well. Clarification of this point is important to test hypotheses on some potential mechanisms for cardiac undercapillarization. To address this issue further, we compared capillary length density (by stereologic techniques) in perfusion-fixed skeletal muscle (m. psoas) and hearts of subtotally nephrectomized (SNX) rats with moderate renal failure to those in sham-operated pair-fed controls. The duration of renal failure was 8 weeks. SNX rats had significantly higher mean systolic blood pressure (128 mm Hg v 109 mm Hg), serum creatinine, and urea levels. Despite pair feeding, the mean body weight was significantly lower in the SNX rats (409 g v 471 g), but the left ventricular weight to body weight ratio tended to be higher than in the sham-operated controls (2.39 mg/g v 2.13 mg/g). In the heart, myocyte mean cross-sectional area (675 +/- 112 microm2 v 545 +/- 111 microm2) and volume density of nonvascular interstitial tissue (3.47 +/- 1.04 v 1.33 +/- 0.22) were significantly higher in the SNX rats than in the controls. In parallel, myocardial capillary length density was significantly reduced after subtotal nephrectomy (3,036 +/- 535 mm/mm3 v 3,916 +/- 615 mm/mm3). In contrast, in skeletal muscle, myocyte cross-sectional area (3,109 +/- 783 microm2 v 3,042 +/- 639 microm2), capillary length density (718 +/- 248 mm/mm3 v 717 +/- 184 mm/mm3), and three-dimensional capillary fiber ratio (2.10 +/- 0.26 v 2.13 +/- 0.4) were similar in SNX and control rats. These data document a selective defect of capillarization in the heart of animals with moderate renal failure, pointing to tissue-specific abnormalities of cardiac capillarogenesis.     

The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two-electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (< or = 200 nM). 2. Inhibition of the ACh-induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)-tubocurarine was studied as a function of temperature (over the range 4-25 degrees C). 3. The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)-tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)-tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage-dependence. 4. The van't Hoff plots (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non-linear for (+)-tubocurarine. From these plots, values for the changes in the standard Gibbs free energy delta G degrees water-->AChR, enthalpy delta H degree water-->AChR, entropy delta S degree water-->AChR and heat capacity delta Cp degree water-->AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding. 5. A comparison between the temperature-dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature-dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.     

An immunosuppressant, FK506, protects against neuronal dysfunction and death but has no effect on electrographic and behavioral activities induced by systemic kainate

Kainate is a potent agonist of an excitatory amino acid receptor subtype in the central nervous system, and causes neuronal death in several regions of the brain. Neurons are preferentially killed in the hippocampus, especially in the CA1 region, by systemic administration of kainate. It is speculated that functional alterations occur in the neurons preceding death. We examined the effect of FK506 on kainate-induced neuronal death and functional alterations in the rat hippocampal CA1 region. FK506 had no effect on electrographic and behavioral seizure activities induced by kainate; however, it prevented neuronal death measured seven days after administration. Although neither death nor morphological alterations of neurons were observed in the CA1 region 24 h after administration, the neurons exhibited decreased excitatory postsynaptic potentials and enhanced long-term potentiation. This functional alteration was not detected in the rats administered FK506 prior to kainate. Taken together, these observations indicate that functional alteration precedes neuronal death in rats systemically administered kainate and that FK506 prevents both. It is suggested that FK506 exerts its neuroprotective effect not by attenuating electrographic and behavioral seizure activities, but by protecting neurons from kainate-induced functional disorders.     

YM796, a novel muscarinic agonist, improves the impairment of learning behavior in a rat model of chronic focal cerebral ischemia

Four-layered microgyria is associated with many developmental disorders, including mental retardation, epilepsy, and developmental dyslexia. Freezing lesions to the newborn rodent neocortex result in the formation of four-layered microgyria. Previous research had suggested this type of injury acts as an hypoxic/ischemic event to the developing cortical plate. The current study examines the effectiveness of the non-competitive N-methyl-D-aspartate receptor antagonist dizocilpine (MK-801) in protecting against freezing injury to the newborn rat cortical plate. Three groups of rats received freezing injury to the cortical plate on the first day of life (postnatal day 1). Two groups were treated with MK-801 (1 or 2 mg/kg) 0.5 h before the lesion and 6 and 14 h after, while one group received saline injections. A fourth group received MK-801 injections, but did not have a freezing lesion. The volume of neocortical abnormality was determined for all three groups in rats killed after postnatal day 7. Treatment with the higher dose of MK-801 (3 x 2 mg/kg) dramatically reduced the effects of freezing injury but also resulted in over 50% mortality in both lesioned and unlesioned groups. Animals in the lesioned group, however, had a decreased volume of abnormal cortex, and there were fewer animals with microsulci than in the untreated group. This is the first demonstration of a significant anatomical neuroprotective effect in newborns leading to a reduction of cortical malformation.     

Protective effect of l-cis-diltiazem on hypercontracture of rat myocytes induced by veratridine

The protective effect of l-cis-diltiazem, the stereoisomer of d-cis-diltiazem, was studied against the veratridine-induced hypercontracture of rat myocytes. Veratridine increased both [Na+]i and [Ca2+]i, but did not cause hypercontracture in the absence of extracellular Ca2+. Both l-cis-diltiazem (0.1-10 microM) and d-cis-diltiazem (10-30 microM) inhibited the hypercontracture and the increase in [Ca2+]i in a concentration-dependent manner. However, l-cis-diltiazem did not exert a negative inotropic effect in K+ (20 mM)-depolarized rat papillary muscles even at a dose of 10 microM. As seen in the case of tetrodotoxin, l-cis-diltiazem and d-cis-diltiazem also suppressed the increase in [Na+]i. The results show that l-cis-diltiazem prevents the veratridine-induced hypercontracture of myocytes by suppression of the [Ca2+]i increase. The attenuation of the [Ca2+]i increase by l-cis-diltiazem was not dependent on inhibition of Ca2+ channels, but was partly due to inhibition of excessive Na+ entry via veratridine-modified Na+ channels.     

Protective effect of melatonin in a non-septic shock model induced by zymosan in the rat

N-acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N-acetyl dopa formed is oxidized by the enzyme to N-acetyl dopaquinone (lambda max = 390 +/- 10 nm). H2O2 and NH2OH each shortened the lag period of NAT hydroxylation by the enzyme. H2O2 had an effect on the changes with time in the spectrum of product(s) formed and on the spectrum of the final product(s) obtained when NAT was hydroxylated by mushroom tyrosinase, in a manner suggesting that H2O2 converts N-acetyl dopaquinone to a pink-violet product(s) (lambda max = 490 nm), whereas such a product(s) was not formed in the absence of H2O2. A pink-violet product(s) (lambda max 490 +/- 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH2OH or para amino benzoic acid (PABA), probably as a result of an interaction between N-acetyl dopaquinone and NH2OH or PABA forming mono- or di-oximes. Kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) inhibited effectively the rate of NAT hydroxylation by mushroom tyrosinase in the absence or presence of H2O2. When NAT was oxidized by the enzyme in the absence of kojic acid, N-acetyl dopaquinone was formed at once and a shoulder at 490-530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a peak at 320 +/- 10 nm, was detected, suggesting that N-acetyl dopaquinone oxidizes kojic acid to the yellow product(s). Maltol (3-hydroxy-2-methyl-4H-pyran-4-one), a gamma-pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroom tyrosinase. The addition of maltol at the plateau phase of the reaction resulted in an immediate decline in absorbance at 400 nm, suggesting that maltol conjugates with N-acetyl dopaquinone, yielding a product(s) characterized by a lower extinction coefficient at 400 nm than that of N-acetyl dopaquinone alone. The final brown-red product(s) formed when NAT was hydroxylated by mushroom tyrosinase was bleached in the presence of ascorbic acid or H2O2.     

Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist

kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.