Comparative structural analysis of the calcium free and bound states of the calcium regulatory protein calbindin D9K

The solution structure of apo calbindin D9K, a member of the calmodulin superfamily of calcium-binding regulatory proteins, has been investigated by 1H nuclear magnetic resonance spectroscopy and the results compared with a corresponding study of the calcium-loaded protein. On the basis of complete sequence-specific assignments, characteristic patterns of short proton-proton distances have been identified in two-dimensional nuclear Overhauser effect spectra, allowing the elements of secondary structure to be determined. It is found that four helices and a short section of antiparallel beta-sheet are present regardless of the calcium content of the protein. In addition, a preliminary analysis of the long-range nuclear Overhauser effects shows that the global folding patterns are the same and that the tertiary structures of the apo protein is very similar to that of the calcium-loaded protein. These results are in stark contrast to a number of very substantial changes in 1H chemical shift. Preliminary studies of protein dynamics show some very large differences in flexibility and internal mobility. This suggests that protein dynamics may play a role more important than was initially realized in the function of calbindin D9K and other homologous calcium-binding regulatory proteins.     

Antibodies to Streptococcus pneumoniae antigens in newborn infants

The levels of antibodies to capsular polysaccharide antigens of pneumococci (serotypes 1, 3, 6B, 8, 9N, 15F, 23F), C-polysaccharide and protein antigen of pneumococci in the blood sera of 38 newborn infants at the moment of their birth (umbilical blood) and on the 5th or 6th day of their life, in their mothers' blood sera, as well as in the colostrum and milk of 48 nursing women, have been studied by means of the enzyme immunoassay. The study showed that in the normal course of pregnancy antibodies to pneumococci were transferred transplacentally from the mother to the fetus. Though in most cases their content in the blood of newborn infants was lower than that in maternal blood, it exceeded the average level of antipneumococcal antibodies in children aged 3-12 months. In the milk of nursing mothers considerable amount of IgA antibodies to pneumococci was detected, which might be an additional protective factor with respect to pneumococcal infection in infants.     

Activity of ectoenzymes of the heart breaking up ATP in the period of myocardial reperfusion after ischemia

Nowadays the activity of heart ectoenzymes, breaking up ATP, are determined in the undamaged tissue to eliminate the effect to the ATP of intracellular enzymes. Our aim is to study the activity of ecto-adenosine triphosphatase in the heart damaged by ischemia. It was established that the activity of ectoenzymes, breaking up ATP, may be measured in the tissue damaged by ischemia only in case when the cellular membrane becomes impermeable for the intracellular enzymes. The activity of adenosine triphosphatase is significantly reduced during the myocardial reperfusion after ischemia and appeared as one of the criteria by which we can assume the degree of the heart reperfusion damage.     

Activation of endogenous lipid peroxidation in the brain in oxidation stress caused by administration of iron and its prevention by vitamin E

Looking for an appropriate model of accelerated aging in vivo we investigated the content of endogenous products of lipid peroxidation (LP) in the rat brain after single or 4 day-lasting intramuscular injection of complex-bind iron (ferum Hausman, 50 mg/kg body weight) like promoter of LP. We found that the single administration of this iron complex fails to induce endogenous LP; after 4 day-application of iron we observed significant increase in content of primary (lipid peroxides) and final (fluorescent) products of LP. Iron-promoted activation of endogenous LP could be abolished by animal pretreatment with the natural antioxidant alpha-tocopherol. The calcium antagonist nifedipine didn't affect the content of endogenous LP products neither alone nor in combination with alpha-tocopherol.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.     

Primary structure of NADP-dependent malic enzyme in the dicotyledonous C4 plant Flaveria trinervia

The primary structure of NADP-dependent malic enzyme (NADP-ME) of the dicotyledonous C4 plant Flaveria trinervia was determined from sequence analysis of a cDNA clone containing the complete coding region. Comparison of the mature F. trinervia NADP-ME with the maize enzyme reveals extensive sequence similarity. In contrast, no significant similarity can be detected between the putative transit peptides of the two enzymes. This suggests that the corresponding parts of the genes arose independently from each other during evolution of mono- and dicotyledonous C4 plants.     

Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques

We have investigated the protein-protein cross-links formed within the 50 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified: L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L30, L17-L32, L19-L25, L20-L21, L22-L32, and L23-L34. Our results are compared with those of Traut and coworkers (Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds) pp. 286-308, Springer-Verlag, New York). Our cross-linking data allow us to propose the approximate locations of eight proteins of the 50 S ribosomal subunit that so far have not been localized by immunoelectron microscopy and they thus contribute considerably to our knowledge of ribosome structure.     

Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains

The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.