A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

Molecule Modeling of Human Pentameric alpha(7) Neuronal Nicotinic Acetylcholine Receptor and Its Interaction with its Agonist and Competitive Antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved i interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

金环蛇蛇毒中的一种新型胰蛋白酶抑制剂

目的:从金环蛇蛇毒中分离纯化名为bungaruskunin 1的一种新型胰蛋白酶抑制剂,并从其毒腺的cDNA文库中克隆出该胰蛋白酶抑制剂的cDNA全序列.方法:通过Sephadex G-50, CM-Sephadex C-25, HPLC, RP-HPLC (C4 column)方法分离纯化bungaruskunin 1.样品的丝氨酸蛋白酶抑制剂活性则是在室温条件下50mmol·L-1 Tris-HCl, pH 7.8的缓冲液中通过对显色底物的水解抑制作用来检测的.金环蛇毒腺RNA用TRIZOL提取,并用SMARTM PCR cDNA synthesis kit (Clontech)建成cDNA文库.根据其信号肽的保守区域合成引物从该文库中扩增出bungaruskunin 1的cDNA全序列,进行胶回收,酶连到pMDl8-T载体中转化测序.结果:bungaruskunin 1的前体由83个氨基酸组成,其中信号肽含有24个氨基酸,成熟肽即:bungaruskunin 1合有59个氨基酸.bungaruskunin 1的cDNA序列与从红腹伊澳蛇Pseudechis porphyriacus中分离纯化得到的丝氨酸蛋白酶抑制剂blackelin的cDNA序列的相似性高达64%.bungaruskunin 1是一种含有保守Kunitz端的Kuntiz蛋白酶抑制剂家族的一员,从而能够抑制蛋白酶和弹性酶的活性.在cDNA文库中,我们同时还筛选到了2种新的β-bungarotoxin B链的序列.结论:这些发现很好地证明了蛇中Kunitz/BPTI胰蛋白酶抑制剂和毒性神经的家族可能起源于共同的祖先.

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta -bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor. (c) 2007 Elsevier Inc. All rights reserved.

Interaction of corticosterone and nicotine in regulation of prepulse inhibition in mice

The aim of the present Study was to investigate if different levels of circulating corticosterone (CORT) modulate the effect of nicotine on prepulse inhibition (PPI), a measure of sensorimotor gating that is disrupted in schizophrenia and other mental illnesses. Four groups of mice were investigated: sham-operated, adrenalectomized (ADX) and implanted with a cholesterol pellet, ADX and implanted with a 10 mg CORT pellet, or ADX and 50 mg, of CORT. Different CORT levels or doses of nicotine did not significantly affect startle responses. Baseline PPI was significantly reduced in mice implanted with the highest dose of CORT. In ADX mice implanted with cholesterol, nicotine treatment influenced PPI depending on the prepulse intensity. In ADX mice implanted with 50 mg of CORT, treatment with 10 mg/kg of nicotine caused a significant increase in PPI at all prepulse intensities. Binding studies showed that corticosterone treatment had significantly affected nicotinic acetylcholine receptor (nAChR) density in the mouse brain. Treatment with 50 mg CORT decreased I-125-epibatidine binding in the globus pallidus and I-125-alpha-bungarotoxin binding in the claustrum. These results suggest a possible interaction of corticosterone and nicotine at the level of the alpha4- and alpha7-type nAChR in the regulation of PPI. In situations of high circulating levels of corticosterone, nicotine may be beneficial to restore disruption of PPI. (C) 2004 Elsevier Ltd. All rights reserved.

金环蛇蛇毒丝氨酸蛋白酶抑制剂和三种胡蜂蜂毒的研究

蛇毒和蜂毒是提供药理学活性分子的丰富来源，它们富含肽和蛋白，包括一 些酶类和毒素。 丝氨酸蛋白酶抑制剂广泛存在于动物、植物和微生物体内，参与许多重要的 生理过程，如血液凝集、纤维蛋白溶解、细胞凋亡、发育以及炎症反应和补体活 化等（van Gent D. et al., 2003）。通过凝胶过滤、离子交换和反向高压液相色谱， 我们从金环蛇毒液中纯化得到一种天然的丝氨酸蛋白酶抑制剂，命名为 bungaruskunin。并且从该蛇的毒腺cDNA 文库中克隆到了它的核苷酸序列。 bungaruskunin 预测的前体由83 个氨基酸组成，包括含有24 个氨基酸的信号肽 和含有59 个氨基酸的成熟肽。它与一种由红腹伊澳蛇（Pseudechis porphyriacus） 的cDNA 预测到的丝氨酸蛋白酶抑制剂blackelin 具有最大相似性，达64％。 Bungaruskunin 是一种Kunitz 型的蛋白酶抑制剂，具有一个保守的Kunitz 结构域， 能够抑制胰蛋白酶、胰凝乳蛋白酶和弹性蛋白酶。通过对金环蛇毒腺cDNA 文库 的筛选，我们还得到了另外两条β-bungarotoxin B 链，Bungaruskunin 的整体结 构与β-bungarotoxin B 链相似，特别是它们都具有高度保守的信号肽序列。这些 发现强烈地表明蛇毒Kunitz/BPTI 蛋白酶抑制剂与神经毒性的类似物可能起源于 共同的祖先。 肥大细胞脱粒肽是从膜翅目昆虫的毒液中鉴别出的一个小肽家族，是一种具 有潜在的药物治疗作用的诱导活性分子（Xueqing Xu et al., 2006）。来源于蜂类的 缓激肽样的类似物vespakinin 家族是一种具有调节和激素功能的活性成分，与哺 乳动物和两栖动物的缓激肽类似（Nakajima T., 1984）。本研究对三种胡蜂的 毒液进行了一系列的活性检测，发现黑尾胡蜂的蜂毒对白色念珠菌Candida albicans 和金黄色葡萄球菌 Staphylococcus aureus 有抑制作用。凹纹胡蜂和黑尾 胡蜂的蜂毒具有微弱的磷酯酶A2 活性。通过凝胶过滤和反向高压液相色谱，没 有得到相关的活性组分。通过对三种胡蜂毒腺cDNA 文库的筛选，我们得到了2 条来源于黑尾胡蜂的核苷酸序列，Blast 分析表明，其中一条编码类似肥大细胞 脱粒肽，但未克隆到全长，序列比对结果显示其与来源于大胡蜂（Vespa magnifica） 的Mastoparan-like peptide 12c precursor（GenBank accession A0SPI0）的核苷酸序 列相似性达98％（Xueqing Xu et al., 2006）；另一条编码缓激肽类似物，命名为 Hw-bradykinin，序列比对结果显示其与来源于大胡蜂（Vespa magnifica）的 vespakinin-M precursor（GenBank accessionABG75944）的核苷酸相似率达96% (Zouhong Zhou et al., 2006)。

The initial mode of action of copper on the cardiac physiology of the blue mussel, Mytilus edulis

Previous studies have shown that low levels of copper (down to 0.8 muM) induce bradycardia in the blue mussel (Mytilus edulis) and that this is not caused by prolonged Valve closure. The aim of this study was to determine the precise mechanism responsible. To establish if copper was directly affecting heart cell physiology, recordings of contractions from isolated ventricular strips were made using an isometric force transducer, in response to copper concentrations (as CuCl2) ranging between 1 muM and 1 mM. Inhibition of mechanical activity only occurred at 1 mM copper, suggesting that the copper-induced bradycardia observed in whole animals cannot be attributed to direct cardiotoxicity. Effects of copper on the cardiac nerves were subsequently examined. Following removal of visceral ganglia (from where the cardiac nerves originate), exposure to 12.5 muM copper had no effect on the heart rate of whole animals. The effect of copper on the heart rate of mussels could not be abolished by depletion of the monoamine content of the animal using reserpine. However, pre-treatment of the animals with alpha -bungarotoxin considerably reduced the sensitivity of the heart to copper. These results indicated that the influence of copper on the heart of M. edulis might be mediated by a change in the activity of cholinergic nerves to heart. In the final experiments, mussels were injected with either benzoquinonium or D-tubocurarine, prior to copper exposure, in an attempt to selectively block the inhibitory or excitatory cholinoreceptors of the heart. Only benzoquinonium decreased the susceptibility of the heart to copper, suggesting that copper affects the cardiac activity of blue mussels by stimulating inhibitory cholinergic nerves to the heart. (C) 2001 Elsevier Science B.V. All rights reserved.

Molecule modeling of human pentameric alpha(7) neuronal nicotinic acetylcholine receptor and its interaction with its agonist and competitive antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved in interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Lymph heart in chick - somitic origin, development and embryonic oedema

The lymph heart is a sac-like structure on either side of avian tail. In some adult birds, it empties the lymph from the copulatory organ; however, during embryonic development, it is thought to circulate extra-embryonic lymph. Very little is known about the origin, innervation and the cellular changes it undergoes during development. Using immunohistochemistry and gene expression profiling we show that the musculature of the lymph heart is initially composed solely of striated skeletal muscle but later develops an additional layer composed of smooth myofibroblasts. Chick-quail fate-mapping demonstrates that the lymph heart originates from the hypaxial compartments of somites 34-41. The embryonic lymph heart is transiently innervated by somatic motoneurons with no autonomic input. In comparison to body muscles, the lymph heart has different sensitivity to neuromuscular junction blockers (sensitive only to decamethonium). Furthermore, its abundant bungarotoxin-positive acetylcholinesterase receptors are unique as they completely lack specific acetylcholinesterase activity. Several lines of evidence suggest that the lymph heart may possess an intrinsic pacing mechanism. Finally, we assessed the function of the lymph heart during embryogenesis and demonstrate that it is responsible for preventing embryonic oedema in birds, a role previously thought to be played by body skeletal muscle contractions.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

Melatonin and the time window for the expression of the alpha 8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of alpha 8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha 8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional alpha 8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha 8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha 8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha 8 nAChR subtype in full functional form. (C) 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

Actions of octocoral and tobacco cembranoids on nicotinic receptors

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies. (C) 2009 Elsevier Ltd. All rights reserved.

The neurotoxicological effects of mastoparan Polybia-MPII at the murine neuromuscular junction: an ultrastructural and immunocytochemical study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for 125I-a-bungarotoxin was reduced. Despite the presence of a8-like immunoreactivity at DIV4, functional responses mediated by a-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the a-bungarotoxin-sensitive response at DIV5. Therefore, a-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an a-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by a-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express a-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.