Kinetic investigation of cooperativity in coenzyme binding by transketolase active sites

The two-step mechanism of coenzyme (thiamine diphosphate, ThDP) binding with two initially identical active sites of apotransketolase has been examined with a kinetic model. Cooperativity between sites in the primary ThDP binding and in the following conformational transition has been analyzed. The only reliable difference between sites is shown to be the tenfold difference in the backward rate constants of the conformational transition; this means that the cooperative interaction between sites takes place only after termination of both steps of ThDP binding in both sites.     

Circadian changes in levels of free amino acids in rat retina, and regulation of them by melatonin

Alpha-tocopherol (vitamin E) is an important fat-soluble antioxidant in biological systems and, as a result of scavenging reactive oxygen, it is converted to alpha-tocopherylquinone. Alpha-tocopherol binds to alpha-tocopherol transfer protein (alphaTTP) in the liver cytosol, whereas alpha-tocopherylquinone does not. We found that alpha-tocopherylquinone binds to a liver protein with a molecular mass of about 40 kDa that is distinct from alphaTTP. This alpha-tocopherylquinone binding protein was purified further by multiple-step column chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the final preparation yielded a single band with an apparent molecular mass of 25 kDa, which microsequencing revealed was identical to glutathione-S-transferase (GST). The GST activity was inhibited in the presence of alpha-tocopherylquinone, as it is by other non-substrate ligands for GST, confirming that GST and alpha-tocopherylquinone interact directly. Alpha-tocopherylquinone binds to GST and may be transported to the site of metabolism or excreted in the bile as other non-substrate ligands for GST.     

Development of a method for creating a colony of Macaca mulatta, free of herpes B virus infection

To form the colony of monkeys, free of herpes B virus, the serological study was made with the aim of finding out the carriers of this virus. 482 rhesus macaques (Macaca mulatta) from the Adler monkey house were examined for the presence of antibodies to herpes B virus by the method of point immunoblotting with the use of Herpes virus simiae as antigen. The contamination of monkeys in different open-air cages varied from 12.5% to 92%. In different age groups, it was 27% in nonpubescent monkeys (49 out of 182 animals), 55% in adolescent monkeys (55 out of 99), 73% in pubescent monkeys (131 out of 179) and 95% in monkeys over 15 years (21 out of 22 animals). 9 groups of rhesus macaques (comprising altogether 81 animals), free of herpes B virus, were selected. The monkeys were repeatedly tested within a year; after that 10-17% of formerly seronegative monkeys were rejected and removed from the selected group. After the third testing 2.5% more of the animals were found to have seroconversion. The colony of rhesus macaques thus created exists at present. The animals are subjected to constant serological observation.     

Langmuir films from antibodies based on amphiphilic polyelectrolytes

A technique for forming Langmuir films from antibodies based on an amphiphilic polyelectrolyte was developed. The physicochemical and immunochemical properties of the Langmuir films obtained were studied. The interaction of HBsAg with the films was found to be described by a model with one binding site, whereas that of HBsAg with antibodies adsorbed on a polystyrene plate, by a model with a positive cooperativity. The use of the novel Langmuir films from antibodies increases the sensitivity of the immunoenzyme assay.     

New data on the etiological factors in acute pyelonephritis

Spectrum of microbe agents of acute pyelonephritis has been studied. It is shown that Mycoplasma hominis plays an important role in etiology of acute pyelonephritis besides the already known enterobacteria and Gram-positive cocci. It is also established that mycoplasmal pyelonephritis more often develops in patients with the diseases of urinary blade (cystitis) and genital organs. Results of serological investigations which confirm etiological role of isolated microorganisms in the initiation of acute pyelonephritis are presented.     

Study of sialated neoglycoconjugates by surface enhanced Raman scattering spectroscopy

Neoglycoconjugates based on polyacrylamide and sialic acid with N-acetylneuraminic acid or sialooligosaccharides as side chains were studied by surface-enhanced Raman scattering (SERS) spectroscopy. It had previously been found that these polymers can effectively inhibit influenza virus adhesion. This study revealed the possibility to evaluate, based on the intensity of SERS signals, the overall availability for interaction and the conformational freedom of sialic acid residues in glycoconjugates. The dependence of these two factors on the structure and density of sialylated side chains was studied. The uniformity of distribution of sialylated side chains in conjugates was shown. Comparison of the results of the SERS spectroscopic study of the conjugates and the data on their inhibitory effect on the adhesion of specific strains of influenza virus allowed the identification of the conjugates for which the availability and conformational freedom of sialic acid are the main factors determining their inhibitory properties. A conclusion was also reached about the predominance of one of the mechanisms (competitive inhibition or steric stabilization) in the inhibitory properties of the specific conjugates.