Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus. Ts3 binds to the domain IV voltage sensor of voltage‐gated sodium channels (Na_v) and slows down their fast inactivation. The covalent structure of the Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Herein, we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure–activity studies that enabled us to establish the covalent structure of biologically active Ts3 toxin. We also report the synthesis of the mirror image form of the Ts3 protein molecule, and the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X‐ray diffraction.     

Photoactivable neurotoxins in studying membrane receptors

Seven monomodified photoactivable derivatives of neurotoxin M₁₀ from the scorpion Buthus eupeus venom and neurotoxin II from the cobra Naja naja oxiana venom have been obtained and characterized. By means of these toxin analogs fast sodium channels and the acetylcholine receptor have been studied.     

Tx1, from Phoneutria nigriventer spidervenom, interacts with dihydropyridine sensitive-calcium channels in GH3 cells

Summary The aim of this work was to use the binding assay of tritiated-dihydropyridine and radioiodinated Tx1, isolated from the Phoneutria nigriventer venom, in order to show the presence of Ca_v1 calcium channels on pituitary tumour cell (GH3). We showed that GH3 cells have specific sites for ¹²⁵I-Tx1, which are sensitive to nifedipine (~20%). Reverse competition assay with ³H-PN200-110 (40% inhibition) and electrophysiological data (50% inhibition) suggest that Ca_v1 calcium channels are target sites for this toxin. To summarize, Tx1 binds to specific sites on GH3 cells and this interaction results in Ca_v1 calcium channel blockade. ³H-PN200-110 and ¹²⁵I-Tx1 binding assays proved to be useful tools to show the presence of calcium channels on GH3 cells.     

Structure and enantioselective synthesis of polyamine toxin MG30 from the venom of the spider Macrothele gigas

A novel polyamine toxin, named MG30, was isolated from the venom of the spider, Macrothele gigas, and its structure was elucidated by two-dimensional NMR and mass analysis. In addition, the enantioselective synthesis of MG30 was achieved to assign its absolute stereochemistry.     

Structure Determination of Hydroxytrypargine: A New Tetrahydro‐β‐Carboline Toxin from the Venom of the Spider Parawixia bistriata

A new, highly active tetrahydro‐β‐carboline toxin from the spider Parawixia bistriata, the most‐common species of social spider occurring in Brazil, was isolated. The new toxin was identified as 1,2,3,4‐tetrahydro‐6‐hydroxy‐β‐carboline (=N‐[3‐(2,3,4,9‐tetrahydro‐6‐hydroxy‐1H‐pyrido[3,4‐b]indol‐1‐yl)propyl]guanidine; 3 ). This type of alkaloid, not common among spider toxins, was found to be the most‐potent constituent of the spider's chemical weaponry to kill prey. When P. bistriata catch arthropods in their web, they apparently attack their prey in groups of many individuals injecting their venoms. In vivo toxicity assays with 3 demonstrated a potent lethal effect to honeybees, giving rise to clear neurotoxic effects (paralysis) before death. The compound's toxicity (LD₅₀ value) was determined to be ca. 8 ng/g of honeybee. The investigation of the pharmacological properties and neurotoxic actions of 3 may be used in the future for the development of new drugs to be applied for pest control in agriculture.     

Observation of noncovalent complexes between margatoxin and the Kv1.3 peptide ligands: A model investigation using ion-spray mass spectrometry

Margatoxin (MgTX), a 39 amino acid peptide present in the venom of the new world scorpion Centruroides margaritatus, is a potent inhibitor of the voltage-gated potassium channel (K_v1.3) in human peripheral T lymphocytes. Peptide analogs corresponding to the amino acid segments that are located at the rat K_v1.3 putative binding site for the ion channel blockers were synthesized. Gas phase noncovalent complexes of the synthetic analogs of the rat K_v1.3 peptide ligands with MgTX were detected using ion-spray mass spectrometry.     

Venomics: unravelling the complexity of animal venoms with mass spectrometry

Animal venoms and toxins are now recognized as major sources of bioactive molecules that may be tomorrow's new drug leads. Their complexity and their potential as drug sources have been demonstrated by application of modern analytical technologies, which have revealed venoms to be vast peptide combinatorial libraries. Structural as well as pharmacological diversity is immense, and mass spectrometry is now one of the major investigative tools for the structural investigation of venom components. Recent advances in its use in the study of venom and toxins are reviewed. The application of mass spectrometry techniques to peptide toxin sequence determination by de novo sequencing is discussed in detail, in the light of the search for novel analgesic drugs. We also present the combined application of LC-MALDI separation with mass fingerprinting and ISD fragmentation for the determination of structural and pharmacological classes of peptides in complex spider venoms. This approach now serves as the basis for the full investigation of complex spider venom proteomes, in combination with cDNA analysis.     

Strategies to Overcome Oxygen Transfer Limitations During Hairy Root Cultivation of Azadiracta indica for Enhanced Azadirachtin Production

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O₂ in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20–100% (v/v) O₂ in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O₂ in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3–2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1–4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day).     

Mode-to-mode vibrational energy transfer in D2CO: Evidence of coriolis-enhanced rotational selectivity

Infrared—ultraviolet double resonance spectroscopy is used to demonstrate rapid collision-induced V-V transfer between the v₆ and v₄ vibrational manifolds of D₂CO. The rate of transfer is at least gas-kinetic and is explained in terms of Coriolis coupling and rotationally specific, quasi-resonant relaxation channels     

Biochemistry and Molecular Biology of Snake Neurotoxin

A review of research on snake neurotoxin is presented, with emphasis on the chemical modification studies and molecular cloning of postsynaptic and presynaptic neurotoxins from Naja naja atra (Taiwan cobra) (Fig. 1a) and Bungarus multicinctus (Taiwan banded krait) (Fig. 1b). Cobrotoxin and α-bungarotoxin are the primary postsynaptic neurotoxins isolated from the venom of N. naja atra and B. multicinctus, respectively. Although they share a common three-dimensional structure, the functional elements essential for the manifestation of their toxicity are different. Selective and stepwise chemical modification of cobrotoxin indicate that at least two cationic groups, an ?-amino group of Lys-47 and a guanidino group of Arg-33 common to all known postsynaptic neurotoxins, are functionally important for its neuromuscular blocking activity. However, for α-bungarotoxin, the side chains of several basic amino acid residues are involved in the multipoint contact between the toxin and acetylcholine receptor. Moreover, the conserved Trp residue is essential for the neurotoxicity of cobrotoxin, but not for α-bungarotoxin. The cDNAs encoding cobrotoxin and α-bungarotoxin was constructed from the cellular RNA isolated from the venom glands of N. naja atra and B. multicinctus by polymerase chain reaction. The sequence of their 3′-untranslational region, signal peptide and 5′-untranslational region share a high degree of homology, suggesting that they are evolutionarily related. Expression of both neurotoxic protein in E. coli generated polypeptide chains for reactions with the antisera against the native neurotoxins. Presynaptic neurotoxins constitute a different group of neurotoxic proteins in snake venom proteins. These presynaptic neurotoxins are either basic phospholipase A₂ (PLA₂) per se or contain basic PLA₂ as an indispensible part of their structures. Thus, the presynaptic neurotoxins usually show both PLA₂ activity and presynaptic neurotoxicity. β-Bungarotoxin (β-Bgt), the main presynaptic PLA₂ neurotoxin isolated from the venom of B. multicinctus (Taiwan banded krait), consists of two dissimilar polypeptide chains, a PLA₂ subunit (A chain) and potassium channel recognition subunit (B chain). Chemical modification studies show that the toxin might possess two functional sites, one responsible for the catalytic activity and the other for its pharmacological properties. Molecular cloning and expression of the A chain and B chain of β-Bgt reveal that the A chain of β-Bgt is an active subunit with PLA₂ activity, and that the B chain is involved in voltage-gated potassium channel blocking action observed with β-Bgt.     

Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus

Background

Scorpion venom contains insect and mammal selective toxins. We investigated the venom of the South Indian red scorpion, Mesobuthus tamulus for the purpose of identifying potent insecticidal peptide toxins.

Results

A lepidopteran-selective toxin (Buthus tamulus insect toxin; ButaIT) has been isolated from this venom. The primary structure analysis reveals that it is a single polypeptide composed of 37 amino acids cross-linked by four disulfide bridges with high sequence homology to other short toxins such as Peptide I, neurotoxin P2, Lqh-8/6, chlorotoxin, insectotoxin I5A, insect toxin 15 and insectotoxin I1. Three dimensional modeling using Swiss automated protein modeling server reveals that this toxin contains a short α-helix and three antiparallel β-strands, similar to other short scorpion toxins. This toxin is selectively active on Heliothis virescens causing flaccid paralysis but was non-toxic to blowfly larvae and mice.

Conclusion

This is the first report of a Heliothine selective peptide toxin. Identification of diverse insect selective toxins offer advantages in employing these peptides selectively for pest control.     

In situ construction of ligand nano-network to integrin αvβ3 for angiogenesis inhibition

Angiogenesis occurs during the process of tumor growth, invasion and metastasis, and is essential for the survival of solid tumors. As an integrin significantly overexpressed in human tumor vascular endothelial cells, α_vβ₃ is a suitable targeting site for anti-angiogenesis of tumor. We designed and prepared a self-assembling peptide (SAP) with the ability to targeting α_vβ₃ and self-assembly. SAP formed nanoparticles in solution and transformed into nanofibrous network once specifically binding to integrin α_vβ₃ on the surface of human umbilical vein endothelial cells (HUVECs). The SAP network stably anchored on HUVECs over 24 h, which consequently resulted in high-efficient inhibition of vascularization. In vitro anti-angiogenesis experiment displayed that the inhibition rate of tube-formation reached 94.9%. In vivo anti-angiogenesis array based on chick chorioallantoic membrane (CAM) model exhibited that the SAP had an inhibition rate up to 63.1%. These results indicated the outstanding anti-angiogenic ability of SAP, potentially for tumor therapy.     

Development of environmentally friendly biological algicide and biochemical analysis of inhibitory effect of diatom Skeletonema costatum

Skeletonema costatum is a diatom widely distributed in red tide microalgae blooms and as one of the main algae causing harmful algal blooms, because of their rapid reproduction and production of toxic and harmful substances, often play a negative role in aquatic ecosystems, and human health and wellbeing. Bacillomycin D is a nonribosomal cyclic antifungal lipopeptide in the iturins family. In this study,Bacillomycin D was tested for its ability to inhibit the growth of S. costatum. The EC_50...     

Infrared spectra of bis(aniline) and bis (p-toluidine) metal(II) isothiocyanate complexes

The infrared spectra of eight complexes of general formula [ML₂(NCS)₂] (M = Co, Ni, Cu, Zn; L = aniline or p-toluidine) have been determined over the range 4000–4150 cm^?1. Colour, magnetic moments and IR spectra are consistent with polymeric octahedral coordination in the Co(II) and Ni(II) complexes and polymeric tetragonal coordination in the Cu(II) complexes, while the Zn(II) complexes are assigned polymeric octahedral (L = aniline) and tetrahedral (L = p-toluidine) structure on the basis of their IR spectra. Independent ¹⁵N-labelling of the nitrogen atoms of the amino and isothiocyanate groups yields assignments for the internal vibrations of both groups and enables the metal-amine and metal—isothiocyanate stretching vibrations (vM-NH₂ and vM-NCS) to be distinguished. Both vM-NH₂ and vM-NCS are metal ion dependent in the Irving-Williams sequence (Co < Ni < Cu > Zn) expected from their proposed structures while the vN-H and vN-CS vibrations are inversely related to the masses of the coordinated metal ions.     

The total synthesis of argiotoxins 636, 659 and 673

Practical syntheses of three polyamine spider toxins isolated from the venom of Argiope aurantia are described.     

A laser induced fluorescence study of vibrational energy transfer processes in cyclopropane

Results of infrared laser induced fluorescence studies on cyclopropane are presented. Molecules were excited from the ground state to the v₁₀ level of cyclopropane using a Q-switched CO₂ laser operating on either the P(14) or P(20) transition of the 9.6 μ branch. Fluorescence was observed from the v₆, v₈, v₁₀ + v₁₁ and v₅ + v₁₀ levels of cyclopropane. The self-deactivation of vibrationally excited cyclopropane through V → T/R processes was found to have a rate of 8.0 ± 1.5 ms^?1 torr^?1. Deactivation by rare gas collisions was also studied with comparison to simple V → T and V → R theories. V → V equilibration processes are discussed involving the v₆, v₈, v₁₀, v₁₁, and v₁₀ + v₁₁ levels.     

A simplified method to replace the Westcott formalism in k 0-NAA using non-1/v nuclides

The Westcott formalism was developed to accurately describe the activation of nuclides whose neutron capture cross-sections do not follow the 1/v-law and it was incorporated in the k ₀-method about 20 years ago. However, its implementation in the original fashion is not easy, requiring the use of a different formalism for the non-1/v nuclides, with a g(T _n) factor for the thermal neutron activation and an s ₀ factor for epithermal activation, the measurement of the neutron spectral index of the irradiation channel and the irradiation of a temperature monitor with each batch of samples as well. In this work we investigated the accuracy of maintaining the simpler formula of the Høgdahl convention for all nuclides and introducing the g(T _n) factor for the thermal neutron activation for non-1/v nuclides. The epithermal activation is calculated using a Q ₀-value estimated from the measured s ₀-value for non-1/v nuclides. This new approach has been shown, by calculations with the parameters of the neutron spectra of five irradiation channels, not to give a significant loss of accuracy relative to the Westcott formalism.     

Photofragment translational spectroscopy at 304 nm from C-H symmetric stretch excited CH3I [v 1 = 1]

The 304 nm photodissociation of the C-H symmetric stretch excited CH3I[v1=1,v2=0](v1 denotes the C-H symmetric stretch mode,and v2 denotes the umbrella mode)is studied with our simple photofragment translational spectrometer.An IR laser is used to excite the ground state CH3I[0,0]to the C-H symmetric stretch excited CH3I[1,0].With IR laser OFF and ON,the fractions of photofragments CH3(ν1,ν2)from the 304 nm photodissociation of CH3I[1,0]have been determined through the photofragment translational spectra(PTS)from measuring I and I*and also through the PTS from measuring CH3(0,0)(1,0)(0,1)and(1,1).The experimental results show that the C-H symmetric stretch vibration(v1=1)in parent molecules is about 66%retained in the photofragments in the I channel,but only 24%in the I*channel.The populations of photofragments CH3(0,2)and(0,3)are higher than CH3(0,0)and(0,1),showing strong inverted population both in I and I*channels.     

Ligand isotope studies of Zeise's salt derivatives (and their CO analogues) with some aza-heterocycles and their N-oxides. II : Their preparation,characterisation, and use in developing 1H nmr and infrared spectra as a diagnostic tool

Forty-one π-acid complexes of the type cis-[Pt(bipyO₂H)(A)X₂]X and trans-[PtL(A)X₂] (A = C₂H₄, CO; X = Cl, Br; L = pyridazine {pdz}, pyrazine N-oxide {pzO}, quinoline {quin}, quinoline N-oxide {quinO}, 2,2′-bipyridine {bipy}, 1,10-phenanthroline {phen}) and their deuterated L and C₂D₄ analogues have been characterised employing infrared and ¹H nmr spectroscopy. The employment of v₁₂ (CH₂ scissors ∼1460 cm⁻¹) of η²-ethene as a diagnostic probe in distinguishing between 4- and 5-coordination is proposed, while the summed percentage decrease of v₂+v₃ (vC=C/δCH₂) may be used to distinguish between N- and O-coordination. The use of chemical shifts (¹H nmr spectroscopy) is confirmed as a suitable means to distinguish between 4- and 5-coordination and is also shown to be suitable for distinction between N- and O-coordination in four coordinate Pt(II) η²-ethene complexes. In contradiction of previous reports, it is found that J_Pt-H cannot be employed to determine the coordination number.     

Mineral Composition of Seawater Bittern Nigari Products and Their Effects on Changing of Browning and Antioxidant Activity in the Glucose/Lysine Maillard Reaction

Seawater bittern (nigari) is a concentrated solution remaining after the crystallization process of salt that has been used as a coagulant for tofu. Recently, various nigari products are distributed in the East Asia. To clarify the properties of nigari products, major mineral composition of six nigari products was determined. Then, effects of the nigari on the browning and antioxidant activity during the glucose/lysine Maillard reaction were investigated. Though the predominant cation was Mg²⁺, the content was varied by each product (0.88–6.49 mol/L). The other major ion contents were also varied. Each 0.5 mol/L of d-glucose and l-lysine were incubated with the nigari (5–50 % (v/v)) or nigari-related salts (1 or 2 mol/L). The browning (OD at 465 nm) and antioxidant activity (1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity and ferrous-reducing power) were increased remarkably by the nigari containing high Mg²⁺ content. The browning tended to be high with sulfates (Na₂SO₄, (NH₄)₂SO₄). On the other hand, high content of MgCl₂ decreased slightly the browning and antioxidant activity. These results suggest that the reaction and antioxidant activities were affected not only by salinity and cations but also by anions and other elements in the nigari.