Glycation and post-translational processing of human interferon-gamma expressed in Escherichia coli

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-gamma during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140/Arg141, Phe137/Arg138, Met135/Leu136, and Lys131/Arg132) and two minor (Lys109/Ala110 and Arg90/Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-gamma originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-gamma, whereas covalent dimerization completely abolished it.     

Polymorphism of the SUP35 gene and its product in the Saccharomyces cerevisiae yeasts

The product of the SUP35 gene of the saccharomycete yeast, the translation termination eRF3 factor, can be converted in prion, the heritable determinant of protein nature. The nucleotide sequence of this gene from the strain belonging to Peterhof genetic lines of the yeast Saccharomyces cerevisiae was determined. A comparison of the identified sequence with SUP35 sequences in the database of GenBank allowed the detection of polymorphic sites both in the SUP35 gene and its product. The location of polymorphic sites in the evolutionarily nonconserved N-terminal protein region confirmed that this eRF3 fragment lacks functions vital to life activity. Nevertheless, these sites are located in the vicinity of sites, whose role in the prion conversion of eRF3 has been established. Based on this, natural polymorphism of the primary eRF3 structure is assumed to be connected with the existence of different variants (strains) of its prion analog.     

Microbiological degradation of asymmetrical dimethylhydrazine--a toxic component of rocket fuel

A possibility of microbiological cleaning of water and soil polluted with asymmetric dimethylhydrazine (ADMH), a highly toxic rocket fuel ingredient (RFI), was studied. Several isolates (bacteria, yeast, and micromycetes) capable of utilizing ADMH as the only source of nitrogen, carbon, and energy were isolated from RFI-polluted tundra soil. Acceleration of RFI biodegradation was achieved using a biosorbent that involved cells of the degrader strain immobilized on granulated activated carbon. Biological testing in Escherichia coli and cereals (wheat and barley) demonstrated that biodegradation significantly decreased the integral toxicity of solutions containing ADMH, suggesting its utility for microbiological cleaning of polluted territories.     

Catalytic reduction of acetylene in the presence of molybdenum and iron clusters, including FeMo cofactor of nitrogenase

Acetylene was reduced by zinc amalgam in the presence of three synthetic polynuclear complexes: {[Mg₂Mo₈O₂₂(OMe)₆(MeOH)₄]⁻²·[Mg(MeOH)₆]²⁺}6MeOH (I), (Bu₄N)₂[Fe₄S₄(SPh)₄] (II), [Me₄N][VFe₃S₄Cl₃(DMF)₃]·2DMF (III) and the iron-molybdenum cofactor of nitrogenase Azotobacter vinelandii MoFe₇(S²⁻)₉·homocitrate, FeMo-co (IV). Thiophenol was found to greatly facilitate the reaction in the presence of complexes I, II, IV. The reaction is catalytic and for I and IV proceeds at the amalgam surface. Thiophenol seems to increase the adsorption of the complexes, serving as an electron bridge to transfer electrons to the catalyst. In the case of II a homogeneous reduction of the substrate occurs presumably after the cluster reduction at the surface and with III the catalytic reduction proceeds only under the action of sodium amalgam; no thiophenol cocatalytic action is observed. Relevance to N₂ enzymatic reduction is discussed.     

A cellular automaton model of morphogenesis in Arabidopsis thaliana

Development of organisms is a very complex process in that a lot of gene networks of different cell types are to be integrated. Development of cellular automata that model the morphodynamics of different cell types is the first step in understanding and analyzing the regulatory mechanisms that underlie the developmental gene networks. We have developed a model of a cellular automaton that simulates the embryonic development of the shoot meristem in Arabidopsis thaliana. The model adequately describes the basic stages in the development of this organ in wild type and mutants.

     

A plausible mechanism for auxin patterning along the developing root

Background

Recent experimental work has uncovered some of the genetic components required to maintain the Arabidopsis thaliana root stem cell niche (SCN) and its structure. Two main pathways are involved. One pathway depends on the genes SHORTROOT and SCARECROW and the other depends on the PLETHORA genes, which have been proposed to constitute the auxin readouts. Recent evidence suggests that a regulatory circuit, composed of WOX5 and CLE40, also contributes to the SCN maintenance. Yet, we still do not understand how the niche is dynamically maintained and patterned or if the uncovered molecular components are sufficient to recover the observed gene expression configurations that characterize the cell types within the root SCN. Mathematical and computational tools have proven useful in understanding the dynamics of cell differentiation. Hence, to further explore root SCN patterning, we integrated available experimental data into dynamic Gene Regulatory Network (GRN) models and addressed if these are sufficient to attain observed gene expression configurations in the root SCN in a robust and autonomous manner.

Results

We found that an SCN GRN model based only on experimental data did not reproduce the configurations observed within the root SCN. We developed several alternative GRN models that recover these expected stable gene configurations. Such models incorporate a few additional components and interactions in addition to those that have been uncovered. The recovered configurations are stable to perturbations, and the models are able to recover the observed gene expression profiles of almost all the mutants described so far. However, the robustness of the postulated GRNs is not as high as that of other previously studied networks.

Conclusions

These models are the first published approximations for a dynamic mechanism of the A. thaliana root SCN cellular pattering. Our model is useful to formally show that the data now available are not sufficient to fully reproduce root SCN organization and genetic profiles. We then highlight some experimental holes that remain to be studied and postulate some novel gene interactions. Finally, we suggest the existence of a generic dynamical motif that can be involved in both plant and animal SCN maintenance.     

Mathematical model of auxin distribution in the plant root

Regulation of plant growth and development by auxin is mediated by the hormone controlled distribution and dose-dependent mechanisms of its action. A mathematical model is proposed, which described the distribution of auxin in the cells extending along the central axis of the Arabidopsis thaliana root. This model reproduces qualitatively both auxin distribution in cells of the root central axis under the normal conditions and under the conditions of decreased active transport and the recovery of auxin distribution and related meristem restoration during root regeneration after ablation of its tip. Different types of distribution of the auxin concentration over the vertical root axis are described, possible variants of root growth and lateral roots formation are proposed, and biological interpretation is given to different regimes of model behavior.     

Survival of civilian and prisoner drug-sensitive, multi- and extensive drug- resistant tuberculosis cohorts prospectively followed in Russia

Objective and Methods

A long-term observational study was conducted in Samara, Russia to assess the survival and risk factors for death of a cohort of non-multidrug resistant tuberculosis (non-MDRTB) and multidrug resistant tuberculosis (MDRTB) civilian and prison patients and a civilian extensive drug-resistant tuberculosis (XDRTB) cohort.

Results

MDRTB and XDRTB rates of 54.8% and 11.1% were identified in the region. Half (50%) of MDRTB patients and the majority of non-MDRTB patients (71%) were still alive at 5 years. Over half (58%) of the patients died within two years of establishing a diagnosis of XDRTB. In the multivariate analysis, retreatment (HR = 1.61, 95%CI 1.04, 2.49) and MDRTB (HR = 1.67, 95%CI 1.17, 2.39) were significantly associated with death within the non-MDR/MDRTB cohort. The effect of age on survival was relatively small (HR = 1.01, 95%CI 1.00, 1.02). No specific factor affected survival of XDRTB patients although median survival time for HIV-infected versus HIV-negative patients from this group was shorter (185 versus 496 days). The majority of MDRTB and XDRTB strains (84% and 92% respectively) strains belonged to the Beijing family. Mutations in the rpoB (codon 531 in 81/92; 88.8%), katG (mutation S315T in 91/92, 98.9%) and inhA genes accounted for most rifampin and isoniazid resistance respectively, mutations in the QRDR region of gyrA for most fluroquinolone resistance (68/92; 73.5%).

Conclusions

Alarmingly high rates of XDRTB exist. Previous TB treatment cycles and MDR were significant risk factors for mortality. XDRTB patients'' survival is short especially for HIV-infected patients. Beijing family strains comprise the majority of drug-resistant strains.     

Dynamics of the reversible transition of Vibrio cholerae into the uncultivable state in the presence of organic and inorganic microcosmic components

The dynamics of the transition of V. cholerae into the uncultivable state in distilled, river and tap water, containing organic and inorganic components added, was studied. As additives, potassium nitrate, potassium phosphate, magnesium sulfate, ammonium chloride, lysine, alpha-ketoglutarate, succinic acid, catalase were used. The study of the influence of biotic factors on transition into the uncultivable state was carried out in the presence of one-celled green algae Scenedesmus quadricauda or infusoria Paramecium caudatum. The linear dependence of speed of transition into the uncultivable form on the concentration of cells was noted. The composition of the microcosmic medium was also found to have some influence on the speed of transition into the uncultivable form and on the reversibility of this process. The presence of organic substances, such as peptone solution or destroyed cells of phyto- and zooplankton, in the microcosmic medium prolonged the time of transition into the uncultivable form and produced a positive effect on the capacity of the population to reversion. In respect of live biotic components, no such dependence was found. Inorganic additives prolonged the time of transition into the uncultivable state, but did not promote reversion.