Development of recombinant scFv-antibodies against diphtheria toxin using phage display system

Phage display technology is an effective approach to the development of the next generation of immunodiagnostic reagents. Naive murine phage display a library of single-chain variable antibodies (scFv) was used to isolate scFv recognizing the diphtheria toxin, an important diagnostic antigen of diphtheria. The diphtheria toxin B subunit-binding clone with affinity constant of 1.13 x 10(7) M(-1) was selected. scFv preserved activity on storage in the course of 8 months.     

New delta8(14)-ketosterols with an oxygenated side chain

New analogues of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3beta-hydroxy-24-methyl-22,23-oxido-5alpha-cholest-8(14)-en-15-ones and (22RS,23xi,24S)-24-methyl-5alpha-cholesta-3beta,22,23-triol-15-one] were synthesized from (22E,24S)-3beta-acetoxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC50 0.9 +/- 0.2 and 0.7 +/- 0.2 microM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC50 4.0 +/- 0.5 microM), (22E,24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one (IC50 3.1 +/- 0.4 microM), and the 3beta,22,23-triol synthesized (IC50 6.0 +/- 1.0 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

The in vitro motility assay to study the calcium-mechanical relationship in skeletal and cardiac muscles

In a series of experiments on regulated contractile systems (i.e., in vitro mobile systems with reconstructed thin filaments), the velocities of the movement of a thin filament on the surface covered by either rabbit skeletal or rat cardiac myosin at various concentrations of calcium ions in solution (in the pCa range from 4 to 8) were assessed. The corresponding "pCa-velocity" relationships were plotted, which proved to be of the sigmoid form. It was found that, at a saturating calcium concentration (pCa 4), the velocity of regulated thin filaments was 65% higher than for unregulated ones in the case of skeletal myosin and 87% higher than for unregulated thin filaments in the case of cardiac myosin. It was also found that the Hill coefficient was 1.95 and 2.5 for skeletal and cardiac myosins, respectively. The difference in the Hill coefficients for skeletal and cardiac myosins is discussed in terms of the difference in contribution of cooperativity mechanisms of contractile and regulatory proteins in the regulation of contraction in these types of muscles.     

The identification of potential radiation risk groups in case of chronical occupational exposure: individual cancer risks among the industrial firm "Maiak" personnel

The draft new recommendations of the ICRP emphasize the need for transition from the collective dose to the dose matrix concept with optimization of the radiation protection system. In fact, this means assessment of the attributive (radiation-induced) risk at individual level with allowance for the dynamics of dose accumulation during the whole length of professional experience. The work provides assessments for high potential risk group from the "Mayak" personnel based on the dose matrix and using the UNSCEAR technique for assessing the attributive risk. It was found that about 2% of "Mayak" personnel subject to individual dosimetric monitoring in 2005 can be attributed to this group.     

Beginning and duration of expression of photoperiodic response genes in winter common wheat

The influence of various Ppd genes on the beginning and duration of photoperiodic responce has been investigated in near isogenic lines of winter bread wheat Mironovskaya 808. During ontogenesis the photoperiodic responce is ascertained from the middle 2nd stage of ontogenesis according to Kuperman (usually a week later after vernalization completion in winter genotypes) and it is completed to the late 5th stage (2-3 weeks before heading). Different Ppd alleles do not affect the photoreaction intensity however they have an influence on its duration through the rate of development. Ppd-A1a and Ppd-B1a genes manifest shorter duration of expression when compared to the recessive alleles in the initial Mironovskaya 808 cultivar. Effect of the Ppd-B1a gene is stronger and of the Ppd-A1a is weaker.     

Functional activity of mouse peritoneal macrophages contaminated with gram-positive bacteria

The analysis of the functional and enzymatic activity of mouse peritoneal macrophages contaminated with Staphylococcus aureus and Listeria monocytogenes virulent strains is presented. The low bactericidal and digestive activity of these cells with respect to the above-mentioned microorganisms was determined. In this study a decrease in the activity of plasmatic membrane enzymes (5'-nucleotidase and ATP-ase) of macrophages contaminated with S. aureus and L. monocytogenes was observed, which was indicative of the stimulation of phagocytes. A rise in the activity of the oxygen-dependent system of macrophages contaminated with S. aureus and L. monocytogenes was detected by means of the nitro blue tetrazolium test. At the same time a decrease in the intracellular content of nitrogen oxide end metabolites in macrophages was detected with a rise in content of nitrogen oxide in the supernatants.     

Characteristics of some stages of energy metabolism and antioxidant system in bone marrow myeloid cells and leukocytes from piglets

The age changes of enzymes of activity catalyzing several links of energy metabolism (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase, cytochrome c-oxidase) and antioxidant system (superoxide dismutase and glutathione reductase) in bone marrow myeloid cells and blood leukocytes of pig in the 10-day period after birth were investigated. The bone marrow cells and leukocytes of the new born piglets were characterized by low intensity of oxidative steps of energy metabolism as well as by low activity of antioxidant enzymes. In the period of neonatal adaptation reorganization of energy metabolism, particularly, intensification of oxidative processes in the investigated cells occurred. It included the pentose phosphate way and cytochrome c-oxidase activation. During the neonatal period of development the functional activity of antioxidant enzymes in the investigated cells of piglets increased.     

Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers

The molecule of photoreceptor Ca(2+)-binding protein recoverin contains four potential Ca(2+)-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the interaction of Ca2+ with recoverin and its mutant forms containing point amino acid substitutions at the working Ca(2+)-binding sites by measuring the intrinsic protein fluorescence and found that the substitution of Gln for Glu residues chelating Ca2+ in one (the second or the third) or simultaneously in both (the second and the third) Ca(2+)-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (for Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca(2+)-ions. On the other hand, the Gln for Glu85 substitution only in the second site moderately affects its affinity to the cation. Hence, we assumed that recoverin successively binds Ca(2+)-ions: the second site is filled with the cation only after the third site has been filled. The binding constants for the third and the second Ca(2+)-binding sites of recoverin determined by spectrofluorimetric titration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.     

Disruptions in the blood system upon exposure to low-dose ionizing radiation depending on duration of emotional stress

In experiments on rats, disturbances in the development of blood system adaptive reactions on emotional stress were found. Under the combined effect of preliminary (before stress) exposure to 0.8 Gy of gamma-radiation and the emotional stress of various duration (2, 4 and 8 days), a compensatory capability of the blood system decreased. The degree of the disturbances directly depended on the duration of the emotional stress.