The Relationship between the Structure of the Tick-Borne Encephalitis Virus Strains and Their Pathogenic Properties

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.     

Investigating Models of Protein Function and Allostery With a Widespread Mutational Analysis of a Light-Activated Protein

To investigate the relationship between a protein’s sequence and its biophysical properties, we studied the effects of more than 100 mutations in Avena sativa light-oxygen-voltage domain 2, a model protein of the Per-Arnt-Sim family. The A. sativa light–oxygen–voltage domain 2 undergoes a photocycle with a conformational change involving the unfolding of the terminal helices. Whereas selection studies typically search for winners in a large population and fail to characterize many sites, we characterized the biophysical consequences of mutations throughout the protein using NMR, circular dichroism, and ultraviolet/visible spectroscopy. Despite our intention to introduce highly disruptive substitutions, most had modest or no effect on function, and many could even be considered to be more photoactive. Substitutions at evolutionarily conserved sites can have minimal effect, whereas those at nonconserved positions can have large effects, contrary to the view that the effects of mutations, especially at conserved positions, are predictable. Using predictive models, we found that the effects of mutations on biophysical function and allostery reflect a complex mixture of multiple characteristics including location, character, electrostatics, and chemistry.     

Characterization of transcriptional and posttranscriptional properties of native and cultured phenotypically modulated vascular smooth muscle cells

Various in vitro models are used for studying phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of vascular smooth muscle cells (cVSMCs) is most often used for this purpose. On the other hand, vascular interstitial cells (VICs) are native phenotypically modulated VSMCs present in blood vessels under normal physiological conditions. The aim of this work has been to compare the difference in expression of a number of VSMC-specific markers, which are commonly used for the characterisation of phenotypic modulation of VSMCs, between freshly dispersed VSMCs, VICs and cVSMCs from rat abdominal aorta. Our experiments show that VICs are present in the rat aorta and express markers of VSMCs. Both VICs and cVSMCs display the presence of sparse individual stress fibres enriched in alpha smooth muscle actin (αSM-actin), whereas in VSMCs, this protein is more densely packed. Compared with contractile VSMCs, both VICs and cVSMCs display decreased expression of VSMC-specific markers such as smoothelin, myosin light chain kinase and SM22α; however, the expression of two major cytoskeletal and contractile proteins (smooth muscle myosin heavy chain and αSM-actin) was downregulated in cVSMCs but not in VICs compared with contractile VSMCs. These results suggest different mechanisms for the phenotypic modulation of cVSMCs and VICs. VICs might therefore represent a novel convenient model for studying molecular mechanisms that govern the phenotypic modulation of VSMCs.     

Cloning ofCandida boidinii DNA fragments promoting autonomous replication of plasmids inSaccharomyces cerevisiae

Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from the C. boidinii chromosome. Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S. cerevisiae.     

Patterns of energy metabolism and growth kinetics of Kluyveromyces marxianus in whey chemostat culture

The influence of physiological parameters such as carbon substrate flux and O₂ uptake rates on energy metabolism are reported with reference to biomass productivity in whey chemostat culture. The combined results show that oxidoreductive energy metabolism may be attained independently of the yeast reaching its maximum respiratory capacity. A novel metabolic interpretation is presented proposing that a relative imbalance between glycolysis and subsequent oxidative steps alone is sufficient to account for the observed results. By means of a mathematical model the results could be reproduced under all experimental conditions. The new interpretation provides an insight into the manner in which energy mettbolism is regulated and influences growth-related process Kluyveromyces marxianus, as well as other yeasts with similar physiological characteristics. Correspondence to: J. I. Castrillo     

Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.     

Individual variability of pathological parameters in chemically induced rat colon tumors.

The development of tumorigenic conditions in the carcinogen-exposed rat colon was studied using selected morphological, histochemical, immunohistochemical and biochemical methods of analysis. Rats were treated with two carcinogens: 1,2-dimethylhydrazine and N-methyl-N'-nitro-N-nitrosoguanidine alone or with deoxycholic acid as a tumor promoter. It was found that 3 months after treatment of animals with the carcinogens the following changes were developed in colonic tissue: infiltration of lymphocytes in the mucous membrane, high increase in mitotic index among epithelial cells, negative reactions of colonic cells for neutral mucopolysaccharides and sulfomucins and positive reactions to carboxyl groups, nonsulfated acid mucosubstances and tissue polypeptide antigens. An increase in the activity of ornithine decarboxylase in colonic tissue was developed within the same time period and has been seen only in those tissues which were characterized by the development of precancerous conditions. Individual variations were observed in the manifestation of the studied parameters in rat neoplastic colonic tissues. It is suggested that these differences reflect an individual sensitivity of animals to carcinogens and the magnitude of the dysplastic processes induced in the colon.