Enrichment of functional redox reactive proteins and identification by mass spectrometry results in several terminal Fe(III)-reducing candidate proteins in Shewanella oneidensis MR-1

Identification of the proteins directly involved in microbial metal-reduction is important to understanding the biochemistry involved in heavy metal-reduction/immobilization and the ultimate cleanup of DOE contaminated sites. Although previous strategies for the identification of these proteins have traditionally required laborious protein purification/characterization of metal-reducing capability, activity is often lost before the final purification step, thus creating a significant knowledge gap. In the current study, subcellular fractions of Shewanella oneidensis MR-1 were enriched for Fe(III)-NTA reducing proteins in a single step using several orthogonal column matrices. The protein content of eluted fractions that demonstrated activity was determined by ultra-high pressure liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A comparison of the proteins identified from active fractions in all separations produced 30 proteins that may act as the terminal electron-accepting protein for Fe(III)-reduction. These include MtrA, MtrB, MtrC and OmcA as well as a number of other proteins not previously associated with Fe(III)-reduction. This is the first report of such an approach where the laborious procedures for protein purification are not required for identification of metal-reducing proteins. Such work provides the basis for a similar approach with other cultured organisms as well as analysis of sediment and groundwater samples from biostimulation efforts at contaminated sites.     

Mapping the amino acid properties of constituent nucleoporins onto the yeast nuclear pore complex

Visualization of molecular structures aids in the understanding of structural and functional roles of biological macromolecules. Macromolecular transport between the cell nucleus and cytoplasm is facilitated by the nuclear pore complex (NPC). The ring structure of the NPC is large and contains several distinct proteins (nucleoporins) which function as a selective gate for the passage of certain molecules into and out of the nucleus. In this note we demonstrate the utility of a python code that allows direct mapping of the physiochemical properties of the constituent nucleoporins on the scaffold of the yeast NPC׳s cytoplasmic view. We expect this tool to be useful for researchers to visualize the NPC based on their physiochemical properties and how it alters when specific mutations are introduced in one or more of the nucleoporins. The code developed using Python is available freely from the authors.     

Distribution and activity of microorganisms in the deep repository for liquid radioactive waste at the Siberian Chemical Combine

The physicochemical conditions, composition of microbial communities, and the rates of anaerobic processes in the deep sandy horizons used as a repository for liquid radioactive wastes (LRW) at the Siberian Chemical Combine (Seversk, Tomsk oblast), were studied. Formation waters from the observation wells drilled into the production horizons of the radioactive waste disposal site were found to be inhabited by microorganisms of different physiological groups, including aerobic organotrophs, anaerobic fermentative, denitrifying, sulfate-reducing, and methanogenic bacteria. The density of microbial population, as determined by cultural methods, was low and usually did not exceed 10(4) cells/ml. Enrichment cultures of microorganisms producing gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) and capable of participation in the precipitation of metal sulfides were obtained from the waters of production horizons. The contemporary processes of sulfate reduction and methanogenesis were assayed; the rates of these terminal processes of organic matter destruction were found to be low. The denitrifying bacteria from the underground repository were capable of reducing the nitrates contained in the wastes, provided sources of energy and biogenic elements were available. Biosorption of radionuclides by the biomass of aerobic bacteria isolated from groundwater was demonstrated. The results obtained give us insight into the functional structure of the microbial community inhabiting the waters of repository production horizons. This study indicates that the numbers and activity of microbial cells are low both inside and outside the zone of radioactive waste dispersion, in spite of the long period of waste discharge.     

Efficacy of polymeric 239Pu removal by liposome-encapsulated pentacin

Liposome-incapsulated pentacine (DTPA) removes intracellular polimeric 239Pu and colloidal hydroxide of polymeric 239Pu from a rat body in the amounts 2- and 4 times, respectively, exceeding those eliminated by free DTPA. However the amount of 239Pu removed decreases sharply with increasing 239Pu hydrolysation and polymerization. It is concluded that the effectiveness of 239Pu removal depends on the physico-chemical status of the radionuclide and its interaction with the biosubstrate rather than on the amount of DTPA entering the cells.     

Leukinferon in immunological correction of acute inflammatory diseases of the abdominal organs]

Addition of (intramuscular+intravenous) leukinferon (LF) to the schemes for the treatment of acute peritonitis promoted a more rapid positive development of the time course of clinical signs and decreasing of leukocytosis in the presence of a pronounced tendency to normalization of the main immunological indices i. e. the counts of differential T-lymphocytes and T-helper cells. There was also activation of neutrophil phagocytic function. A rapid decrease in objective signs of endotoxicosis was recorded: the intoxication leukocytic index and the level of medium-mass molecules. In parallel with the decrease in the intoxication leukocytic index, there was a decrease in cytosis of the peritoneal exudate. The use of LF in the treatment of elderly patients with acute cholecystitis eliminated the clinical signs and normalized the main laboratory indices without surgical interventions which allowed one to make a planned operation with the minimum risk.     

Effect of t12 haplotype on the manifestation and inheritance of fused and kinky genes in house mice

The influence of some t-haplotypes on the phenotypic manifestation of fused and kinky genes located on chromosome 17 of the house mouse was studied. It was shown that t12-haplotype decreases the penetrance of these genes to 59-70%. The effect was observed when the Fu gene (or Ki) is transmitted from the females heterozygous for t12-haplotype. This haplotype only affects manifestation of the Fu and Ki genes in the F1.     

Localization of basal cell antigen in the epithelial tissues of animals and humans detected by means of natural antibodies

By means of the immunofluorescent method using rabbit serum that contains natural antibodies against the basal cell antigen of epidermis, the distribution of the antigen has been demonstrated in cells of the basal layer of all types of the stratified epithelium. The reaction is also noted in cytoplasm of the epithelial cells in the thymus and the tracheal mucous membrane. This demonstrates their histogenic affinity to stratified epithelii. The antigen studied is not species-specific, since it is revealed in the stratified epithelium of all species examined (human being, mouse, rat, guinea pig, rabbit). It is possible to use the basal cell antigen as a marker for immunomorphological reveal of epithelial cells in the thymus in the process of its physiological and pathological involution.     

Shift in the allele frequencies of the Ld locus in mink stock infected with Aleutian disease

An idea on organization of the Ld-system of low density lipoprotein in the domestic mink as on a "closed" immunogenetic system with two codominant alleles Ld1 and Ld2 was confirmed. Significant differences in frequencies of genes and genotypes of the Ld-system between state farm "populations" unaffected with Aleutian disease and those which were centres of this epizootic were established. The results obtained confirm the assumption made earlier on subvitality of the Ld2 gene.     

Genetic control over 7 Lpm-system allotypes of very high-density serum alpha2-lipoprotein the mink]

Population and hybridological analyses have demonstrated that the alloantigenic markers of very high density alpha2-lipoprotein of mink serum, Lpm7 and Lpm8, along with Lpm1, Lpm2, Lpm3, Lpm4, Lpm5, belong to a common immunogenetic system. 21 Lpm-phenotypes are determined at least by 36 genotypes; each of the 11 phenotypes is conditioned by a single genotype, each of the remaining 10 are conditioned by 2, 3 or 4 genotypes. The Lpm allotypes and allogroups are coded by the genetic units Lpm8, Lpm4, Lpm4,8, Lpm4,7, Lpm3,4,8, Lpm1,8, Lpm1,2,7, Lpm2,4,5,7, which behave as alleles. The last, six seem to be haploid sets of closely linked genes.     

Proteins of bacterial membranes. H+-adenosinetriphosphatase from Acholeplasma laidlawii cells

The cytoplasmic membrane of micoplasmic cells, in particular of A. laidlawii cells, contains a proton-carrier Mg2+ -activated ATPase. A whole H+ -ATPase complex (F0-F1) was isolated from these cells and characterized. The isolation procedure included solubilization of the enzyme with Triton X-100 followed by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The enzyme was inhibited by dicyclohexylcarbodiimide (10(-4) M). The Km value for ATP hydrolysis and Ki for ADP hydrolysis were determined. The order of the constants did not differ from those measured earlier for factor F1 of the complex. The purified enzyme, similar to its hydrophylic moiety is sensitive to the action of bivalent cations. The subunit composition of the whole complex and of its water-soluble part was investigated. The complex was found to contain 11 polypeptides, five of which belong to factor F1. The molecular weights of these polypeptides were determined.