Storage of industrial microorganisms entrapped into polymer matrices

Our study of the techniques of long-term storage of the biomass of various strains of microorganisms, which cause breakdown or transformation of synthetic organic compounds, demonstrates that desiccated agar beads with immobilized microbial cells can be used for this purpose. In addition, the cells can be stored in desiccated matrices of agar or polyvinyl alcohol, coating synthetic cords. Such dry biocatalysts may be used for quick starting of bioreactors and in other biotechnological processes. The technique is applicable to storage of various strains of Pseudomonas, Corynebacterium, Rhodococcus, and, to a lesser extent, Enterobacteriaceae.     

Microbial degradation of components of waste water from phenol-producing industry

Processes of aerobic biodegradation of components of phenol production sewage (phenol, acetophenone, dimethylphenylcarbinol, cumene hydroperoxide, alpha-methylstyrene, benzoate, and p-hydroxybenzoate) by bacterial strains obtained from the collection of Saratov Institute of Biocatalysis were studied. The metabolic reactions were shown to be oxidative and have a common catabolic sequence (cumene hydroperoxide-dimethylphenyl-carbinol alpha-methylstyrene-acetophenone-phenyl acetate-phenol-pyrocatechol-aromatic ring breakage). Benzoate and p-hydroxybenzoate were degraded through the formation of pyrocatechol and protocatechuate, respectively. Metabolic pathways were similar in model mixtures of components and sewage samples.     

Radionuclide accumulation in fruit bodies of macromycetes

Coefficients of 137Cs accumulation and 90Sr were determined in macromycetes of different trophic groups (137Cs in 43 species and 90Sr in 19 species) in the conditions of droughty year (1992). Their variability in forest formations was determined in the period from 1992 to 1998. In the year with increased atmospheric humidity (1998), two-fold rise of 137Cs accumulation in fruit bodies was registered on average. The pollution of Boletus edulis correlates with photosynthetically active part of Betula pendula and Pinus silvestris closer than with soil pollution. This shows the possibility to indicate the pollution of short-living fruit bodies of fungi by the pollution of plants-symbiotrophs.     

Influence of Exon Duplication on Intron and Exon Phase Distribution

Nonrandomness in the intron and exon phase distributions in a sample of 305 human genes has been found and analyzed. It was shown that exon duplications had a significant effect on the exon phase nonrandomness. All of the nonrandomness is probably due to both the processes of exon duplication and shuffling. A quantitative estimation of exon duplications in the human genome and their influence on the intron and exon phase distributions has been analyzed. According to our estimation, the proportion of duplicated exons in the human genome constitutes at least 6% of the total. Generalizing the particular case of exon duplication to the more common event of exon shuffling, we modeled and analyzed the influence of exon shuffling on intron phase distribution. Received: 28 March 1997 / Accepted: 9 July 1997     

Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins

Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Gα or Gβγ subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gβγ subunits are likely to be involved since the expression of the C terminus of β-adrenergic receptor kinase 1, a Gβγ subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gβγ dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gβγ subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gβγ subunits and activation of MAPKs to repress skeletal muscle differentiation.     

Dynamics of the pathomorphological changes in the rat brain in relation to the dose of gamma irradiation

Neurohistological, histochemical, electron-microscopic and biometric techniques were used to study the response of rat brain to irradiation within a wide range of doses. Nerve cells were shown to be highly radioresistant. At the same time, synapses and blood-brain barrier structures were highly radiosensitive. The pathomorphologic changes in different brain areas followed a dose-time function.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

Synthesis of bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis> by the purple nonsulfur bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h^?1 were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h^?1.     

Evolution of enzymatic activities in the enolase superfamily: L-talarate/galactarate dehydratase from Salmonella typhimurium LT2

We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of l-talarate is accompanied by competing epimerization to galactarate; little epimerization to l-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the l-talarate-specific base. The epimerization of l-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.