Dendrotoxin from the venom of the green mamba,Dendroaspis angusticeps

Summary The venom of the green mamba, Dendroaspis angusticeps has previously been shown to produce neuromuscular facilitation by increasing acetylcholine release. After gel filtration and ion-exchange chromatography of the whole venom, a basic polypeptide with facilitatory actions was isolated. This polypeptide, named dendrotoxin, has 59 amino acid residues, probably with only 3 disulphide bonds and a blocked N-terminus.When injected into conscious mice, dendrotoxin made the mice hypersensitive to external stimuli and subsequently produced respiratory paralysis. When tested on the isolated chick biventer cervicis nerve-muscle preparation, concentrations of dendrotoxin of 0.5 g/ml (7×10^–8 M) and greater, increased responses to indirect stimulation by 200–250%, without any increase in responses to submaximal concentrations of exogeneous acetylcholine, carbachol, KCl or direct stimulation. The augmentation was slow to develop, not reversed by washing, and could last several hours before slowly waning. Dendrotoxin did not produce spontaneous twitching or contractures.It is concluded that dendrotoxin is not an anticholinesterase, does not affect receptor sensitivity or muscle contractility, but produces twitch augmentation by increasing the amount of acetylcholine released by nerve stimulation. Thus, dendrotoxin appears to represent a snake venom neurotoxin with unusual chemical and pharmacological properties.     

Limbic epilepsy induced in the rat by dendrotoxin, a polypeptide isolated from the green mamba (Dendroaspis angusticeps) venom

Dendrotoxin was isolated from green mamba (Dendroaspis angusticeps) venom and its effects on motor behavior and cortical and subcortical bioelectrical activity were studied in the rat. In chronic experiments, free moving rats injected i.p. with dendrotoxin, presented motor behavior similar to that described in rat amygdaloid epilepsy and bioelectrical signs of epilepsy beginning at the amygdala were observed. In acute experiments, rats anaesthetized with urethane were intracerebrally or intracerebroventricularly injected with dendrotoxin, which produced bioelectrical signs of epilepsy. Following intracerebroventricular injection, signs of epileptic discharge were first observed at the dorsal hippocampus. When dendrotoxin was microinjected in the amygdala or the hippocampus, the seizures appeared at the injection sites with a shorter latency and the bioelectrical epileptic signs lasted longer than when injections were given in non-limbic structures, such as the globus pallidus or the mesencephalic reticular formation. Dendrotoxin is a very powerful toxin that acts effectively at the level of the limbic system.     

Effects of the venom of the green mamba, Dendroaspis angusticeps on skeletal muscle and neuromuscular transmission.

1 The venom of the green mamba, Dendroaspis angusticeps, was tested for effects on neuromuscular transmission and skeletal muscle contractility in isolated phrenic nerve-hemidiaphragm preparations of the rat and mouse, chick biventer cervicis muscle preparations and in aneural cultures of embryonic chick skeletal muscle. 2 The venom (10 to 40 micrograms/ml) augmented the responses to indirect but not direct stimulation. As the venom did not have anticholinesterase activity and did not increase receptor sensitivity, it is likely that the venom enhanced release of acetylcholine. 3 Higher concentrations of venom (40 to 80 micrograms/ml) inhibited acetylcholine receptor sensitivity. 4 Prolonged exposure to the higher concentrations of venom produced a failure of muscle contractility. Signs of muscle degeneration were seen in skeletal muscle cultures.     

Increase of evoked release of acetylcholine at the neuromuscular junction by a fraction from the venom of the eastern green mamba snake (Dendroaspis angusticeps)

The effects of a fraction (DA3) isolated from the venom of the Eastern green mamba, Dendroaspis angusticeps, were tested in the toad sartorius muscle-sciatic nerve preparation using intracellular recording techniques. In magnesium-paralysed preparations, DA3 (0·5μg/ml and above) caused a slow, progressive increase in the quantal content of the endplate potential (e.p.p.); the largest increase measuring 40 times the control value. DA3 also altered the spontaneous release of acetylcholine, firstly reducing the frequency of miniature e.p.p.s (m.e.p.p.s) to 20–50% of control and then increasing the frequency to about four times control. In the presence of the toxin, very large spontaneous e.p.p.s were seen. Such potentials could be 10 times the amplitude of control m.e.p.p.s although their time course was similar to that of control m.e.p.p.s. In the absence of external Ca²⁺ ions and in the presence of 1 mM EGTA, DA3 still produced the biphasic change in m.e.p.p. frequency and the appearance of large spontaneous e.p.p.s. We conclude that DA3 increases the amount of acetylcholine released by each nerve stimulation, possibly by inducing the clumping of synaptic vesicles.     

Kinobeon A as a potent tyrosinase inhibitor from cell culture of safflower: in vitro comparisons of kinobeon A with other putative inhibitors

Kinobeon A is produced from cell cultures of the medicinal plant, safflower. Mushroom tyrosinase activity was inhibited in a concentration-dependent manner when treated with kinobeon A using L-tyrosine or L-3,4-dihydroxyphenylalannine (L-DOPA) as substrates. IC50 values were 22 microM (substrate: L-tyrosine) and 27 microM (L-DOPA). Inhibition of human tyrosinase activity also increased with increasing concentrations of kinobeon A using L-DOPA as the substrate, with an IC50 value of 2.5 microM. Kinobeon A was a more potent competitive inhibitor than kojic acid, arbutin or L-ascorbic acid for both mushroom and human tyrosinase as determined from Lineweaver-Burk plots. These results suggested that kinobeon A could be a potent natural tyrosinase inhibitor.     

Molecular properties and structure-function relationships of lethal peptides from venom of Wagler's pit viper, Trimeresurus wagleri.

Two new lethal peptides (waglerins) were purified from the venom of Trimeresurus wagleri, and sequenced. We found them to be analogs of lethal peptides (waglerins) I and II reported previously (Weinstein et al., Toxicon 29, 227-236, 1991), with an additional Ser-Leu on the amino terminus. Three of the four waglerins were synthesized and the products were chemically and biologically equivalent to the naturally occurring counterparts in venom. Murine i.p. LD50 for synthetic waglerins I, SL-I and II were 0.33, 0.22, and 0.51 mg/kg, respectively. The single, intramolecular disulfide bond in each synthetic peptide formed rapidly in high yield. The reduced (cysteine-containing) forms of the peptides appeared to have significant toxicities, even without prior disulfide bond formation, but synthetic analogs with serine substituted for cysteine were not toxic. The synthetic dimer of waglerin I, formed by two intermolecular disulfide bonds, was not toxic, but rapidly rearranged to lethal, monomeric waglerin I at alkaline pH upon the addition of 5 mM beta-mercaptoethanol. Waglerin I was inactivated by cleavage at Tyr-15 with chymotrypsin.     

Oligomerization of trypsin and chymotrypsin inhibitor from chick pea. A kinetic study

A mathematical method for determining oligomerization of a reversible enzymatic inhibitor has been developed. The method was applied to chick pea trypsin and chymotrypsin inhibitor (CI) and to Bowman-Birk inhibitor (BBI) using trypsin and chymotrypsin with ester, amide and protein substrates. It has been shown that CI undergoes oligomerization whereas CNBr modified CI does not oligomerize. These results were substantiated by gel filtration on Sephadex G-75. Oligomerization of BBI has been shown earlier by different methods. This oligomerization is shown here by the mathematical method. The kinetic data can be useful in determining oligomerization when the available amounts of an inhibitor are too small for gel filtration or when the oligomers are present in very small, and thus hard to detect, amounts.     

血管紧张素转换酶抑制剂与四类药物的联用

目前临床联合用药治疗高血压病已成为趋势。作者根据国内外文献记载及中国人民解放军第454医院心血管病内科住院患者用药情况,观察常用的抗高血压药物——血管紧张素转换酶抑制剂在治疗高血压过程中与四类药物在联合用药时可能产生的相互作用,旨在探讨其联合用药的利弊,为临床安全、有效用药提供参考。     

Spatial structures of the bradykinin potentiating peptide F from Agkistrodon piscivorus piscivoris venom.

Bradykinin potentiating peptides usually show two different activities, potentiation of bradykinin and inhibition of angiotensin converting enzyme (ACE). Exceptions of this rule have been found suggesting that both effects occur independently. This study of peptide F by means of NMR spectroscopy shows clearly two different main conformations of the molecule. These different conformations may be the reason for the different activities.     

Control of pharmaceutical properties of soybean trypsin inhibitor by conjugation with dextran. I: Synthesis and characterization

The Kunitz-type soybean trypsin inhibitor (STI), a model protein, was conjugated with dextran (Mw, approximately 9900; STI-D), and its physicochemical and biochemical properties were studied to develop a novel delivery system for a protein drug. Conjugation was carried out using periodate oxidation, and cyanogen bromide, carbodiimide, cyanuric chloride, epichlorhydrin, and N-succiniimidyl-3-(2-pyridyldithio)propionate (SPDP) reagent methods. Dextran was conjugated to STI at a molar ratio of 1.5 to 4.6, but the degree of modification, as well as yield and contamination extent of unreacted STI and dextran, varied with the method of synthesis. Gel filtration and electrophoresis confirmed the covalent attachment of dextran to STI but also demonstrated the broad molecular weight distribution of the conjugates. The STI-D conjugate retained satisfactory activity, although the attachment partially reduced its inhibitory activity against trypsin. The periodate oxidation method seemed to be the best for the preparation of STI-D since it gave the conjugate with a high modification ratio (4.6 molecules per STI), high yield (95%), and satisfactory activity recovery (63%). Chemical modification of STI was also carried out with activated polyethylene glycol (PEG) for comparison. The STI-PEG conjugate was obtained in a satisfactory yield (96%) and modification degree (5.8 molecules per STI), but the remaining activity was considerably lower (34%). Thus, conjugation of protein with dextran by the periodate oxidation method is suggested to be preferable for preparing a protein-carrier system without significant diminution of its biological activity.     

Primary structure of gamma-bungarotoxin, a new postsynaptic neurotoxin from venom of Bungarus multicinctus.

The primary structure of gamma-bungarotoxin, a new toxin from Bungarus multicinctus venom, was determined using mass spectrometry and Edman degradation. The toxin has a mass of 7524.7 D and consists of 68 residues having the following sequence: MQCKTCSFYT CPNSETCPDG KNICVKRSWT AVRGDGPKRE IRRECAATCP PSKLGLTVFC CTTDNCNH. Gamma-bungarotoxin is structurally similar to both kappa-bungarotoxin and elapid long postsynaptic neurotoxins. Its C-terminal nine residues are identical to those of the kappa-toxins. Its disulfide bond locations appear identical to those of several elapid toxins of unknown pharmacology and its hydrophobicity profile is also strikingly similar. However, with an LD50 of 0.15 microg/g i.v. in mice, gamma-bungarotoxin is 30-150-fold more toxic than other members of this latter class. Its toxicity is comparable to those of alpha-nicotinic acetylcholine receptor antagonists.     

Control of pharmaceutical properties of soybean trypsin inhibitor by conjugation with dextran. II: Biopharmaceutical and pharmacological properties

Biopharmaceutical and pharmacological properties of the Kunitz-type soybean trypsin inhibitor (STI)-dextran conjugate (STI-D) were studied. Dextran having a molecular weight of approximately 10,000 was covalently attached to the STI molecule by periodate oxidation. The STI-polyethylene glycol (PEG) conjugate (STI-PEG) was also tested for comparison. After iv injection to mice, native STI showed rapid elimination of activity from plasma (t 1/2 = 2 min), and approximately 60% of the dose was excreted in urine within 1 h after injection. On the other hand, STI-D was slowly cleared from plasma and its urinary excretion was restricted. The STI-PEG conjugate showed a pharmacokinetic behavior similar to that of STI-D. Pharmacological activities of native and modified STI were evaluated by two animal experimental models; that is, trypsin-induced shock in mice and acute pancreatitis in rats. In mice, shock induced by iv injection of trypsin was inhibited by the iv pretreatment with native STI, but the effect was observed for only 1 h. The STI-D conjugate showed a superior inhibitory effect on trypsin-induced shock to that of STI alone at the same dose, and this effect continued for 5 h. A similar effect was also observed in mice given an iv injection of STI-PEG. In rats with acute pancreatitis, no significant therapeutic effect was shown by the iv treatment with native STI, as well as saline treatment. On the other hand, the iv treatment with STI-D at the same dose as STI lowered the mortality of the rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the effects of four dendrotoxin peptides, 4-aminopyridine and tetraethylammonium on the electrically evoked [3H]noradrenaline release from rat hippocampus.

We have examined the effects of four dendrotoxin (DaTX) peptides, alpha-, beta-, gamma- and delta-DaTX, separated from the venom of the green mamba (Dendroaspis angusticeps), on field stimulation-evoked [3H]noradrenaline (NA) release from rat hippocampus and compared their effects with those of two other inhibitors of K+ channels, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP (10-300 microM) and TEA (0.1-5 mM) facilitated the evoked [3H]NA release in a concentration-dependent manner. The evoked [3H]NA release was reduced to about half by alpha 2-adrenoceptor stimulation (UK 14,304; 100 nM) and this reduction was antagonized by 4-AP (10-100 microM), whereas TEA even at 5 mM was a poor inhibitor of alpha 2-effects. alpha-DaTX (10-200 nM) mimicked 4-AP in increasing the electrically evoked [3H]NA release and diminishing the inhibitory effects of UK 14,304 in a concentration-dependent manner. delta-DaTX did not itself alter the electrically evoked [3H]NA release, but at 200 nM, it reduced the effects of alpha 2-receptor stimulation. beta- and gamma-DaTX (up to 200 nM) had no significant effects. 4-AP, 3,4-diaminopyridine (3,4-DAP), TEA and the four dendrotoxins displaced the binding of [3H]p-aminoclonidine ([3H]PAC) from alpha 2-receptors. The IC50 values were 6.6 x 10(-4), 1.42 x 10(-3), 5.6 x 10(-2) for 4-AP, 3,4-DAP and TEA, respectively, and 3.19 x 10(-6) M for alpha-DaTX. Thus, their potency as inhibitors of alpha 2-receptors is apparently too low to account alone for the antagonism by K+ channel inhibitors of alpha 2-effects on NA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of an acidic phospholipase A(2) from the venom of Naja Kaouthia.

A phospholipase A(2) purified from the venom of Naja kaouthia (Guangxi cobra) exhibits anticoagulant activities. The structures of two crystal forms were determined by X-ray crystallography at 2.8A resolution with the Naja naja (India cobra) PLA(2) as an initial model. The enzyme exhibits a trimer structure, which is similar to that of India cobra PLA(2). This reinforces the physiological relevance of the oligomer. The trimer has a wide cavity, which allows the substrate to enter and interact with the catalytic site. The formation of the trimer may serve as a storage method to improve the solubility at high concentration in the venom gland. The Ca2+ binding loop in the absence of the cation can exist in different conformations depending on its surroundings.     

Two crystal structures of human neutrophil collagenase, one complexed with a primed- and the other with an unprimed-side inhibitor: implications for drug design

Two crystal structures of human neutrophil collagenase (HNC, MMP-8), one complexed with a primed- and the other with an unprimed-side inhibitor, were determined using synchrotron radiation at 100 K. Both inhibitors contain non-hydroxamate zinc-binding functions. The Pro-Leu-L-Trp(P)(OH)(2) occupies the unprimed region of the active site, furnishes new structural information regarding interaction between the catalytic zinc ion and the phosphonate group, and is the only example of occupation of the S(1) subsite of MMP-8 by the bulky tryptophan side chain. The (R)-2-(biphenyl-4-ylsulfonyl)-1,2,3, 4-tetrahydroisochinolin-3-carboxylic acid, a conformationally constrained D-Tic derivative, accommodates its biphenyl substituent into the deep primary specificity S(1)' subsite, inducing a widening of the entrance to this pocket; this modification of the protein, mainly consisting in a shift of the segment centered at Pro217, is observed for the first time in MMP-8 complexes. Cation-aromatic interactions can stabilize the formation of both complexes, and the beneficial effect of aromatic substituents in proximity of the catalytic zinc ion is discussed. The phosphonate group bound to either a primed- or unprimed-side inhibitor maintains the same relative position with respect to the catalytic zinc ion, suggesting that this binding function can be exploited for the design of combined inhibitors assembled to interact with both primed and unprimed regions of the active cleft.     

Comparison of kinin-forming and amidolytic activities of four trimucases, oedema-producing and kinin-releasing enzymes, from Trimeresurus mucrosquamatus venom.

Four kinin-releasing enzymes, trimucase I, II, III and IV, isolated from Trimeresurus mucrosquamatus venom (TMV) caused rat hind-paw swelling. Trimucase I and III were less potent than trimucase II and IV in this effect. Pretreatment with diphenhydramine or methysergide significantly reduced trimucase-induced paw swelling, while aspirin had no effect. Cellulose sulphate pretreatment suppressed the oedematous responses elicited by trimucases. The residual response was further depressed by diphenhydramine and methysergide. Trimucases also caused kinin generation in-vitro from rat plasma. This kinin-forming activity was in the order of trimucase II greater than IV greater than or equal to III greater than I greater than TMV. All trimucases hydrolysed chromogenic peptides N-benzoyl-Pro-Phe-Arg p-nitroanilide, N-benzoyl-Phe-Val-Arg p-nitroanilide and DL-Val-Leu-Arg p-nitroanilide; the order of this amidolytic activity was trimucase I greater than II greater than III greater than or equal to IV. These data indicate that the effects of venom kinin-releasing enzymes on plasma kininogen are not parallel to their amidolytic effects.     

Neuromuscular effects of four phospholipases A2 from the venom of Pseudechis australis, the Australian king brown snake.

Four homologous single chain phospholipases A2 (Pa-1G, Pa-5, Pa-12C and Pa-15) were tested for neuromuscular effects on chick biventer cervicis and mouse hemidiaphragm nerve-muscle preparations. The four isozymes blocked directly elicited (mouse hemidiaphragm) and indirectly elicited (mouse and chick nerve-muscle preparations) twitch responses in concentrations of 1-30 micrograms/ml. The order of potency seen in both types of preparations was Pa-1G = Pa-5 greater than Pa-12C much greater than Pa-15. All four isozymes caused slow-onset, sustained contractures and reduction of muscle membrane potentials. In the chick preparation, responses to acetylcholine, carbachol and KCl were reduced by exposure to the toxins. It is concluded that the toxins act primarily postsynaptically to depress muscle contractility, perhaps by directly damaging muscle fibres. The order of potency agrees with their phospholipase A2 activity. Pa-1G is unusual because it is an acidic molecule, most toxic phospholipases being basic.