Protection of actin from heat denaturation by various phallotoxins

Phallotoxins protect F-actin from heat denaturation at a temperature of 70 degrees C for 3 min. This has been shown by difference spectroscopy. G-actin is not protected from heat denaturation by phalloidin. Among the various phallopeptides investigated, 4 toxic ones showed the same protecting ability, whereas non-toxic seco-compounds has no effect. The non-toxic (S)-phalloidinsulfoxide A exhibited only a partial protecting activity, corresponding to an affinity for F-actin of about one tenth of that of the toxic peptides. Evidence for this was obtained from the easy displacement of the sulfoxide A from F-actin by phalloidin as well as by a spectroscopic dilution titration.     

Heat denaturation of hemocytocardiotoxin from the venom of the Central Asian cobra

It has been stated that boiled for three hours haemocytocardiotoxin (HT) from cobra poison loses "direct" hemolytic activity and is unable to potentiate a haemolytic effect of phospholipase A. Surface activity of HT does not change. It is shown that in the course of heat denaturation the aggregation of toxin molecules to dimers and trimers takes place and electrophoretic mobility is decreased. The fluorescence of HT tyroxin residues supported the fact of its irreversible heat denaturation.     

Structure-function properties of venom components from Australian elapids

A comprehensive review of venom components isolated thus far from Australian elapids. Illustrated is that a tremendous structural homology exists among the components but this homology is not representative of the functional diversity. Further, the review illuminates the overlooked species and areas of research.     

Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw

A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya (1974) Biochim. Biophys. Acta 342, 272-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, Which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantium yield value, the shortened phosphorescence lifetime (80% of the emission has tau p less than 0.5 s, 20% has tau p = 4.8 s, comparing with usual tau p = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I- (0.455) is much higher than that for the positively charged Cs+ (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive quenchers is even more pronounced in the case of the neurotoxin I (1.04 and 0 +/- 0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together with the analysis of the amino acid sequence, suggest that the space folding of this polypeptide results in the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).     

Phospholipase A2 from Naja naja sputatrix venom is a muscarinic acetylcholine receptor inhibitor

DNA topoisomerase I (topo I) is the molecular target for the camptothecin group of anticancer drugs. These drugs are showing activity against a wide array of human tumors. Many data have indicated that the sensitivity of a tumor cell to the camptothecins is dependent on tumor topo I levels. Drug-sensitive cells have high levels of topo I. Unfortunately, there is still a relative lack of information on topo I levels in human malignancies. Because of this, we investigated topo I activity and immunoprotein levels in a variety of normal murine and human tissues, as well as tissues obtained from several carcinomas, lymphomas, and sarcomas. Flow cytometric analysis was also performed on the neoplastic specimens to determine the percentage of cycling cells. Topo I catalytic activity was detected in all normal tissues at a fairly constant level. The average topo I catalytic activity in normal mammalian tissues was 2.7 +/- 1.3 x 10(4) units/mg protein (range 1.1 to 5.0 x 10(4)). Topo I catalytic activity was much more variable in human malignancies and ranged from a low of 1.4 x 10(4) units/mg protein in a rhabdomyosarcoma to a high of 160 x 10(4) units/mg protein in a poorly differentiated ovarian carcinoma. Western blot analysis with either a mouse monoclonal antibody or scleroderma antibodies directed against topo I revealed that the elevated topo I catalytic activity levels in the malignant tissues are due to elevated amounts of topo I immunoprotein. It is possible that the high topo I levels that characterize several different types of human malignancies might indicate that these tumors would be sensitive to many of the new drugs that target topo I.     

Nerve growth factor from the venom of the Chinese cobra Naja naja atra: purification and description of non-neuronal activities

Nerve growth factor (NGF) was separated from crude Naja naja atra venom by using weak cation-exchange chromatography, followed by reversed-phase liquid chromatography. The yield of the purification was 0.2-0.5% (w/w). The mol. wt was determined to be 13,600 and the protein still induced the typical fibre outgrowth of cultured PC-12 cells in a concentration range of 5-10 ng/ml. Beside this neuronal effect we demonstrated non-neuronal effects of cobra venom NGF, such as induction of plasma extravasation and histamine release from whole blood cells. With human leucocyte preparations, including enriched basophils, there was an increase in C5a-induced histamine release, whereas NGF alone was inactive. Cobra NGF was one-tenth as potent as human recombinant NGF, with a half-maximal stimulation occurring at 10 ng/ml. Cobra NGF and human recombinant NGF showed a modulatory effect on histamine release comparable to the haematopoietic growth factor IL-3. Thus, the non-neuronal effects of cobra NGF may account for immunomodulatory activities during inflammatory events.     

Isolation and characterization of an endogenous inhibitor of phospholipase A2 from Indian cobra (Naja naja naja) venom

PURPOSE: To evaluate the efficacy of stent deployment in the treatment of recurrent stenosis of transplant renal arteries (TRAs). PATIENTS AND METHODS: This retrospective study includes six consecutive patients who underwent a mean of 3.66 previous treatments of TRA stenosis per patient before stent implantation (20 angioplasties and two surgical procedures). The endoprostheses were a Wallstent in four patients and a Palmaz stent in two patients. Clinical, laboratory, and duplex scanning follow-up was performed every 6 months after stent placement in all patients. RESULTS: The procedure was a technical success in all patients. At 6 months, mean systolic blood pressure decreased from 179 to 152 mm Hg (P = .018) and mean diastolic blood pressure decreased from 102 to 90 mm Hg (P = .09). Mean serum creatinine level dropped from 269 to 182 mmol/L (P = .03) and the number of antihypertensive drugs per patient decreased from 2.5 to 1.6. At a mean follow-up of 34 months (range, 7-60 months), all TRAs were patent, with a stenosis less than 50% without clinical consequences in one patient. No secondary procedure was necessary. CONCLUSION: Stent placement seems to be an effective treatment of TRA recurrent stenosis. Midterm follow-up shows satisfactory clinical results and TRA patency rates. This technique might be considered as a valuable therapeutic option for the treatment of TRA recurrent stenosis.     

Purification and characterization of three acidic, cytotoxic phospholipases A2 from Indian cobra (Naja naja naja) venom

Three acidic phospholipases A2 (NN-I2c-PLA2, NN-I2d-PLA2 and NN-I2c-PLA2) were purified by successive chromatography of Indian cobra (Naja naja naja) venom on CM-Sephadex C-25, Sephadex G-50 and QAE Sephadex A-25 columns. They have molecular weights of 13,000-14,500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis. They showed tryptophan specific fluorescence emission spectra (approximately 345 nm). All the three phospholipases A2 were enzymatically highly active with specific activities 9-17 micromol min(-1) mg(-1). They were non-lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight. They induced edema in mouse foot pads and were cytotoxic to Ehrlich ascites tumour cells. They did not exhibit direct haemolytic and anticoagulant activities.     

Effects of additives on heat denaturation of rhDNase in solutions

PURPOSE: To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions. METHODS: Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T(m)) and enthalpy (H(m)) of rhDNase. The effects of denaturants (guanidine HCl and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6-7. RESULTS: The T(m) and H(m) of rhDNase in pure water were measured as 67.4 degrees C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca(2+) stabilizes rhDNase against thermal denaturation and elevates T(m) and H(m) while Mg(2+), Mn(2+) and Zn(2+) destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T(m) and H(m). All destabilization of rhDNase can be prevented by adding Ca(2+) to the solutions. CONCLUSIONS: CaCl(2) and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HCl destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca(2+) the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.     

Depressant insect selective neurotoxins from scorpion venom: chemistry, action, and gene cloning

The present study examines the similarity in the symptoms and binding properties between the depressant and excitatory insect-selective neurotoxins, derived from scorpion venom. A comparison of their primary structures and neuromuscular effects is presented. A new depressant toxin (LqhIT2) was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic its effects on the intact insect, i.e, a brief period of repetitive bursts of regular junction potentials (JPs) is followed by reduced amplitude JPs ending with a block of the neuromuscular transmission. "Loose" patch clamp recordings indicate that the repetitive activity has a presynaptic origin (the motor nerve) and resembles the effect of the excitatory toxin AaIT. The final synaptic block is supposed to be the end result of neuronal membrane depolarization. Such an effect is not caused by an excitatory toxin, which induces long "trains" of repetitive firing. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation indicating a high degree of sequence homology. This conservation differs from those of other groups of scorpion toxins. The opposing pharmacological effects of depressant toxins are discussed in light of the above neuromuscular effects and sequence analysis. A genetic approach in the study of the structure-function relationships of the depressant toxins was initiated by isolating cDNA clones encoding the LqhIT2 and BjIT2 toxins. Their sequence analysis revealed the precursor form of these toxins: A 21 amino acid residue signal peptide followed by a 61 amino acid region of the mature toxin, and three additional amino acids at the carboxy terminus.     

Acanthoxin, a toxic phospholipase A2 from the venom of the common death adder (Acanthophis antarcticus)

This is the first report of a phospholipase A2 (PLA2) from the venom of the common death adder, Acanthophis antarcticus. Acanthoxin is a basic, monomeric PLA2 of mol. wt 13,000, consistent with the weight of neurotoxic PLA2s from other Australian elapids. However, preliminary ultracentrifugation experimentation has shown that it is able to undergo concentration-dependent aggregation to form dimers. It has a relatively high degree of enzymatic activity (23.93 +/- 1.18 mumoles of phospholipid hydrolysed/min/mg protein), but a low level of toxicity (3.2 mg/kg, s.c.). Acanthoxin is known to exist as two isoforms (A1 and A2), both of which show a high degree of homology with numerous elapid PLA2 neurotoxins, in particular pseudexin A from the red-bellied black snake (Pseudechis porphyriacus).     

Semantic and spatial components of selective attention.

Semantic and spatial effects on selective processing were examined. A prime presented 250 ms or 1,000 ms before a pair of masked words was related to 1 of the words on 1/3 of the trials. Although Experiments 1 and 2 required report of both words, processing was selective; typically, just 1 word could be reported. In Experiment 1, reported words were more often top words, showing spatial selectivity, and more often words related to the prime, showing semantic selectivity. Experiment 2 included to-be-ignored peripheral cues 50 ms before the pair. Related words were reported more often at both delays, and cuing produced an additive benefit at 1,000 ms. Experiment 3 required report of just the cued word and included 100-ms cues. Cued words were reported more often, especially at 1,000 ms and with 100-ms cues, but semantic selectivity also occurred. Selectivity via semantic priming and via location reflect separate attentional mechanisms. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Reversible denaturation of cyclosporin synthetase by urea

The reversible denaturation of the multifunctional polypeptide, cyclosporin synthetase, by urea was analyzed. It is possible to discriminate between at least two stages of enzyme denaturation. While at low urea concentration (up to 0.8M) cyclosporin A formation is inhibited, synthesis of the diketopiperazine cyclo-(D-alanyl-N-methylleucyl), a molecule representing a partial sequence of cyclosporin A is still detectable. At higher concentrations of urea the enzyme preparation is totally inactive. This inactivation is a consequence of conformational change(s) of cyclosporin synthetase as shown by fluorescence emission spectra of native and denatured enzyme. These data imply a consecutive folding/defolding mechanism for the different domains forming the multifuntional polypeptide.