Bacterial expression, NMR, and electrophysiology analysis of chimeric short/long-chain alpha-neurotoxins acting on neuronal nicotinic receptors

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.     

Formation of nitric oxide in animal tissues during inflammatory process

EPR evidence was obtained that more intensive formation of mononitrosyl non-heme iron complexes with diethyl-dithiocarbamate (DETC) took place in mouse liver when inflammation process was initiated in mice by the lipopolysaccharide isolated from Salmonella typhimurium bacterium wall DETC intraperitoneally injected bound with endogenous non-heme iron resulted with DETC-Fe complex formation. These complexes were as a traps of nitric oxide appeared in animal tissues, and NO-Fe-DETC complexes were observed. Phenazone known as a free radical process inhibitor lowered NO production in animal organism. The free radical processes were suggested to intensify under inflammation reactions and to cause the various amino groups oxidation to nitroso groups which were capable to release free nitric oxide.     

Seed dormancy release and germination characteristics of <i>Corispermum lehmannianum</i> Bunge,an endemic species in the Gurbantunggut desert of China

Seed dormancy release and germination of Corispermum lehmannianum Bunge were tested using various treatments: temperature, cold stratification, gibberelins (GA3), dry storage and sand burial. Results showed that temperature and light did not affect the germination of fresh seeds, cold stratification and GA3 could improve seed germination, whereas dry storage and sand burial did not. The germination percentage was highest at 35/20 °C after the cold stratification and GA3 treatments. Corispermum lehmannianum seeds were classified as non-deep, Type-2, physiological dormancy (PD), whose seed dormancy could be released by cold stratification and GA3.     

Movement of a female polar bear (<Emphasis Type="Italic">Ursus maritimus</Emphasis>) in the Kara Sea during the summer sea-ice break-up

The polar bear movement trajectory in relation to onset date of the sea-ice break-up was studied in the coastal zone of the Taimyr Peninsula, eastern part of the Kara Sea, using as an example a female polar bear tagged by a radio collar with an Argos satellite transmitter. Analysis of the long-term pattern of ice melting and tracking, by means of satellite telemetry, of the female polar bear who followed the ice-edge outgoing in the north-eastern direction (in summer 2012) suggests that direction of the polar bear movement depends precisely on the direction of the sea-ice cover break-up.     

Effect of physico-chemical factors and species specificity on the structure of dinitrosyl iron complexes

Formation in mouse, rat and man's blood of iron nitrosyl complexes with pair thiol groups of proteins (complexes 2.03) was shown by ESR method. This formation was initiated by the introduction in blood in vitro or in vivo of low molecular dinitrosyl complexes of iron with phosphate, thiosulphate, cysteine or reduced gluthatione. Three forms of these complexes were found. They were characterized by ESR signals with rhombic or axial symmetry of g-factor tensor. These forms pass into one another under the effect of a number of thiol-containing compounds or at blood freezing. The life time of the complexes 2.03 in the blood in vivo is several hours.     

Nitroprusside ions as a liposome label

It has been shown that nitroprusside ions incorporated into bilayer liposomes may be used as a label to form an opinion as to the liposome intactness in the blood and distribution in animal organ tissues in vivo.     

Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.     

Vasopressin receptors: structure and stimulation by 1-deamino-arginine-vasotocin in rats

In experiments on non-anesthetized female Wistar rats, it has been shown that injection of 1-deamino-arginine-vasotocin (1dAVT) increases sodium excretion and solute-free water reabsorption. Antagonists of V1-receptors (OPC-31260, Otsuka Pharmaceutical Co., Ltd., Japan) eliminates the effect water reabsorption whereas antagonist of V1-receptors (OPC-21268, Otsuka Pharmaceutical Co., Ltd., Japan) decreases the 1dAVT-dependent increase of Na+ and water excretion. A model of V1-receptors an a supposed topography of their interaction with 1dAVT are constructed.