New paradoxical three-finger toxin from the cobra <Emphasis Type="Italic">Naja kaouthia</Emphasis> venom: Isolation and characterization

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of “orphan” toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.     

Meiotic analysis of four cross-pollinated generations in a synthetic autotetraploid population of husk tomato (<i>Physalis ixocarpa</i>)

The cultivated husk tomato (Physalis ixocarpa) (2n = 2x = 24) is native from Mexico and Central America and shows a wide genetic variation. Presently, it is the fourth horticultural crop in cultivation surface in Mexico. The working team of this research previously developed an autotetraploid population by using colchicine. The objectives of the present work were to analyze the ploidy level and meiotic behavior of the subsequent generations (C3, C4, C5, C6) from the original (C2) composed only by plants with the duplicated genome from the Rendidora cultivar, and to determine pollen viability. As a diploid control the cultivar Rendidora of P. ixocarpa was used. Ploidy level was determined by flow citometry and meiotic analysis. For the meiotic study, the microsporocytes were prepared by the squash method, stained with carmin and analyzed in diakinesis. Pollen viability was evaluated through 0.01% Buffalo Black staining. The tetraploid condition prevailed through four cross-pollinating generations, maintaining a constant chromosome number 2n = 4x = 48. In diakinesis, the chromosomes of the diploid cultivar were associated into bivalents, whereas in tetraploid plants the chromosomes associated into univalents, bivalents and trivalents. Highly significant differences in bivalent pairing were detected between autotetraploid plants and between generations. Pollen viability did not show significant differences between generations and allowed reproduction. These results indicate that it is possible to develop an autotetraploid cultivar, because the polyploid state is naturally maintained and the plants are fertile. Furthermore, given the differences in bivalent pairing between plants and generations, a response to selection toward meiotic stability is expected.     

Novel non-Mendelian determinant involved in the control of translation accuracy in Saccharomyces cerevisiae.

Two cytoplasmically inherited determinants related by their manifestation to the control of translation accuracy were previously described in yeast. Cells carrying one of them, [PSI(+)], display a nonsense suppressor phenotype and contain a prion form of the Sup35 protein. Another element, [PIN(+)], determines the probability of de novo generation of [PSI(+)] and results from a prion form of several proteins, which can be functionally unrelated to Sup35p. Here we describe a novel nonchromosomal determinant related to the SUP35 gene. This determinant, designated [ISP(+)], was identified as an antisuppressor of certain sup35 mutations. We observed its loss upon growth on guanidine hydrochloride and subsequent spontaneous reappearance with high frequency. The reversible curability of [ISP(+)] resembles the behavior of yeast prions. However, in contrast to known prions, [ISP(+)] does not depend on the chaperone protein Hsp104. Though manifestation of both [ISP(+)] and [PSI(+)] is related to the SUP35 gene, the maintenance of [ISP(+)] does not depend on the prionogenic N-terminal domain of Sup35p and Sup35p is not aggregated in [ISP(+)] cells, thus ruling out the possibility that [ISP(+)] is a specific form of [PSI(+)]. We hypothesize that [ISP(+)] is a novel prion involved in the control of translation accuracy in yeast.     

Geobacillus uralicus,a New Species of Thermophilic Bacteria

The K^T ₂ strain of thermophilic spore-forming bacteria was isolated from a biofilm on the surface of a corroded pipeline in an extremely deep well (4680 m, 40–72°C) in the Urals. The cells are rod-shaped, motile, gram-variable. They grow on a complex medium with tryptone and yeast extract and on a synthetic medium with glucose and mineral salts without additional growth factors. The cells use a wide range of organic substances as carbon and energy sources. They exhibit a respiratory metabolism but are also capable of anaerobic growth on a nitrate-containing medium. Growth occurs within the 40–75°C temperature range (with an optimum of 65°C) and at pH 5–9. The minimum generation time (15 min) was observed at pH 7.5. Ammonium salts, nitrates, and arginine are used as nitrogen sources. The G+C content of the DNA is 54.5 mol %. From the morphological, physiological, and biochemical properties and the nucleotide sequence of the 16S rRNA gene, it was concluded that the isolate K^T ₂ represents a new species of the genus Geobacillus, Geobacillus uralicus.     

Interaction of hypochlorite with oxyhemoglobin leads to liberation of iron in a catalytically active form]

Interaction of hemoglobin with hypochlorite (OCI-) induces changes in hemoglobin absorption spectra resulting in Soret band decrease and shift similar to those observed under the action of hydrogen peroxide (H2O2). Hemoglobin decomposition is accompanied by free iron release, as estimated by coloured iron-phenanthroline complex formation. The released iron is catalytically active: the incubation of hemoglobin with H2O2, OCl- or activated neutrophils increases the intensity of H2O2-dependent chemiluminescence of hemoglobin. In both reactions OCl- was more efficient than H2O2. These results show that hemoglobin can serve as a source of catalytically active ("free") iron in the reaction with OCl- and with H2O2.     

Effect of local UV microirradiation on the nuclear apparatus and cytoplasm of the infusorian Paramecium caudatum]

The work is the first attempt to study nuclear dualism of ciliates with ultraviolet microbeam (UV-beam), which was not applied earlier for these purposes. The special advantage of the UV-beam is that it allow to inactivate selectively of the particular elements of nuclear apparatus of living ciliates is to observe consequences of operation on distant descendants of irradiated cell. On Paramecium caudatum the ways of UV-beam have been devised specifically. The original rotocompression chamber was employed for immobilization of cell in space of irradiation. The effective UV-doses were selected, the relative resistance of cytoplasm, macro-, micronucleus (Mi), and symbiotic bacteria of Mi -- omega particles -- was detected. The "pure" and infected cells were irradiated, in some different variants. The effect of UV-beam depends on dose and consists in a reduction the effectiveness of cell cloning, the loss of omega-particles from Mi, the appearance of the cells with 2--3 Mi, and, possibly, of amicronucleate cells. The injuries due to UV-beam are restored by photoreactivation of visible light.     

Genetic effects induced by nickel sulfate in germline and somatic cells of WR mice

We examined the effects of nickel sulfate at doses 0.5 to 5.0 mg/kg (LD50) on the frequency of dominant lethal mutations and two-strand DNA breaks (TSBs) in germline cells and on an increase in frequency in gene mutations W(y) in pigment cells of first-generation mice. The results indicated that spermatogenesis stages most sensitive to nickel sulfate (at a dose of 1.0 mg/kg) are spermatozoids, early spermatids, late spermatocytes, and stem spermatogonia. No statistically significant increase in the total TSB level was detected in spermatozoids 4 weeks after exposure. At the same time, a significant (P < 0.05) increase in percentage of cells with an extremely high level of DNA fragmentation (supposedly apoptotic cells) was observed upon exposure at a dose of 0.5 mg/kg. Nickel sulfate at doses of 5.0 and 1.0 mg/kg induced a marked increase in the c-kit gene expression in pigment cells of heterozygous first-generation WR mice as compared to control (P < 0.001). It was shown that the nonobservable adverse effect level (NOAEL) of nickel sulfate on the dominant lethal mutation frequency and gene mutations was 1/200 LD50, while the lowest observable adverse effect level (LOAEL) was 1/100 LD50.     

Generation of Free Radicals during Decomposition of Hydroperoxide in the Presence of Myeloperoxidase or Activated Neutrophils

It was shown with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone that myeloperoxidase (MPO) in the presence of its substrates H2O2 and Cl- as well as activated neutrophils destroy tert-butyl hydroperoxide producing two adducts of O-centered radicals which were identified as peroxyl and alcoxyl radicals. Inhibitory analysis performed with traps of hypochlorite (taurine and methionine), free radical scavengers (2,6-di-tret-butyl-4-methylphenol and mannitol), and MPO inhibitors (salicylhydroxamic acid and 4-aminobenzoic acid hydrazide) revealed that the destruction of the hydroperoxide group in the presence of isolated MPO or activated neutrophils was directly caused by the activity of MPO: some radical intermediates appeared as a result of the chlorination cycle of MPO at the stage of hypochlorite generation, whereas the other radicals were produced independently of hypochlorite, presumably with involvement of the peroxidase cycle of MPO. The data suggest that the activated neutrophils located in the inflammatory foci and secreting MPO into the extracellular space can convert hydroperoxides into free radicals initiating lipid peroxidation and other free radical reactions and, thus, promoting destruction of protein-lipid complexes (biological membranes, blood lipoproteins, etc.).     

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and O-12, growing in the temperature range from 30 to 61°C with a optimum at 55°C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes and genes of membrane-bound methane monooxygenase, as well as on phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulosephosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55°C than in cells grown at 37°C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH₄ ⁺ assimilation occurred through reductive amination of -ketoglutarate and via the glutamate cycle. The relationship between the physiological and biochemical peculiarities of the isolates and their thermophilic nature is discussed.