Fractionation and purification of DNA-methylases from Shigella sonnei 47

Possible applications of various column chromatography techniques and isoelectrofocusing for the study of DNA-methylases of Shigella sonnei 47 cells were analyzed. A simple, rapid and convenient procedure based on the use of cation-exchange chromatography was developed for obtaining a highly active total preparation of methylases. Affinity chromatography on heparin-Sepharose was shown to be a promising approach for separating methylases according to their specificity towards nitrous bases. Isoelectrofocusing was used to identify in Shigella sonnei 47 cells six individual methylating enzymes differing in their pI values. Under the stipulation that Shigella sonnei 47 DNA-methylases show a tendency to aggregate in the course of fractionation, column chromatography is of little or no use in isolating and purifying individual methylating enzymes of the given strain. The advantages of the isoelectrofocusing technique and its utility in the study of different molecular forms of site-specific enzymes are discussed.     

Structuro-functional characteristics of aldolase from rabbit muscles in diabetes

The structural peculiarities of rabbit muscle aldolase accompanying enhancement of the aldolase activity in diabetes are described from the data of tryptophan phosphorescence at the room temperature and fluorescence polarization. It is shown that the pathology-concomitant conformational changes occur in both the hydrophobic part and NAD-binding site of the enzyme. The character of the structural changes in the hydrophobic part of the protein in diabetes and an increase in the enzymic activity are similar to that observed in normal aldolase after its interaction with NADH and are believed to be associated with the enhancement of the rigidity in the Trp-147 environment.     

Effect of salt composition of the medium and ethanol on cholinesterase hydrolysis of various substrates

Sodium chloride, phosphate buffer and ethanol were studied for their effect on butyryl cholinesterase hydrolysis rate of acetylcholine, acetylthiocholine, butyrylthiocholine and nonion substrate of indophenylacetate. The concentrations of 1.10(-2) = 1.10(-1) M of sodium chloride activated enzymatic hydrolysis of ion substrates at the concentrations lower than 1.10(-4) M but sodium chloride is a competitive inhibitor at higher concentrations. Phosphate buffer also activates substrates enzyme hydrolysis at the concentrations of 2.10(-4) M and lower, but it inhibits incompetitively the nonion substrate indophenylacetate hydrolysis. Ethanol activates butyrylthiocholine hydrolysis and is a competitive inhibitor in acetylthiocholine and indophenylacetate hydrolysis. The observed effects are discussed on the assumption of two forms of butyrylcholinesterase E' and E" existence. These two forms are determined by different kinetic parameters and are in equilibrium.     

Interaction of bromophenol blue with serum albumin: criteria for using dyes for assessing the state and quantity of protein

The optical properties of the complexes of the pH-dependent dye bromophenol blue (BPB) with human serum albumin were investigated by the spectrophotometric method. The solvatochromic longwave displacement of bound BPB-2 absorption and BPB-1/BPB-2 redistribution were shown to form the optical signal of complexes. Because of the distortion of the bound BPB-2 signal its quantity was determined as delta A630 = A630 - A660 and the use of lambda max as structural parameter was limited to low pH less than or equal to 3. The conclusion was made that BPB is inapplicable as a structural probe on account of low structural dependence of delta A630 and pH-limitation of lambda max used. The maximal absorption delta Amax = Amax - A660 and its structural independence were obtained in the region of 70-100% occupation of the dye-binding centers of the protein. It is the optimal conditions for the quantitative determination of protein. After maximal dye binding (15-16 molecules of BPB per 1 molecule of albumin) the aggregation and precipitation of the complexes occurred.     

Use of the immunofluorescence reaction for evaluating the immunological transformation in those inoculated with a chemical typhus vaccine

The results of the serological examination of persons immunized with chemical typhus vaccine (CTV) are presented. The examination was carried out by means of the complement fixation test (CFT), the passive hemagglutination test (PHAT), the toxin neutralization test (TNT) and the immunofluorescence test (IFT). The acetone-fixed live culture of Rickettsia prowazekii, strain Breinl, served as antigen in IFT. If persons immunized with CTV showed positive titers in CFT, TNT and PHAT, the results of IFT were highly correlated with the CFT titers. In 6-12 months after immunization with CTV the titers of CFT, TNT and PHAT became negative, while the IFT titers remained positive for several subsequent years.     

Stabilization of fly head cholinesterase immobilized on a gelatin membrane

Immobilization of meat fly head cholinesterase in gelatin membrane essentially increases the enzyme stability and changes its kinetic characteristics: the Michaelis constant and maximum velocity of acetylthiocholine hydrolysis and also biomolecular velocity constant of phosphorylation by DDVP.     

Study of the methylation process and DNA methylase specificity in Shigella]

The nature and content of minor bases in DNA of 3 Shigella strains are investigated. DNAs from Shigella stutzeri 2, Sh. sonnei 1188 and Sh. sonnei 311 are found to contain 0.43, 0.56 and 0.45 mol.% of N6-methyladenine respectively. 5-methylcytosine (0.16 mol.%) is discovered in Sh. sonnei 311. Substrate specificity of adenine methylase from Sh. sonnei 1188 with respect to phage DNAs of different host modification is investigated. Recognition sites for guanine methylase of DDVI phage and for adenine methylase of Sh. sonnei 1188 turned to be different. DNA of DDII phage grown in Sh. stutzeri 2 cells does not accept methyl groups under the treatment with Sh. sonnei 1188 extracts, but it is methylated by Escherichia coli extract. Adenine methylases of Sh. sonnei 1188 and Sh. stutzeri 2 are suggested to be either the same enzyme, or enzymes, which recognition sites are partially overlapped.     

Spontaneous death of mononuclear cells, obtained from health donors and patients with systemic lupus erythematosus]

Unstimulated human peripheral blood mononuclear cells (MNC) underwent death during incubation in vitro. According to morphological criteria the type of death was identified as apoptosis. There was a good correlation between a fraction of apoptotic cells qualified morphologically, and fraction of "apoptotic" comets. The use of DNA-comets for studying the spontaneous death in vitro of MNC from patients with systemic lupus erythematosus (SLE) showed that 24 h after isolation cells from SLE patients demonstrated a higher level of apoptosis than MNC from normal donors. It is likely that the increased apoptosis of SLE-MNC in vitro may reflect changes occurring in those cells in vivo which are bound with pathogenesis of disease. In this context the comet assay may be promising in diagnostics and monitoring of therapeutic treatments.     

Psychostimulation of Wistar rats after intraperitoneal administration of high-molecular heparin

The influence of repeated administration of heparin in learning was studied in Wistar rats (n = 20). High-molecular heparin (Serva, Germany, 10 kDa, activity of 180 IU/mg) was intraperitoneally injected daily in the dose of 64 IU/kg in a volume of 0.3 ml during 5 days prior to 4-link freechoice operant conditioning in a complex maze. As distinct from 40% of control rats, practically all heparin-treated rats were successful in conditioning. Parameters such as rate and efficiency of learning, behavior organization, habit stability, and extinction of errors were significantly better than in control animals. Anxiety was decreased, but sensitivity to external factors was increased in comparison with the control rats. The data obtained suggest that prior treatment with high-molecular heparin has a strong psychostimulant effect through activation of biogenic amines and formation of complexes with them.     

Magnetic field and alcohol addiction

The effect of weak disturbance (up to 300 microT) of natural magnetic field on the development of alcohol addiction in rats exposed to information load (training in a complex maze) was studied. It was found that learning against the background of inhomogenous magnetic field led to 3-13-fold increase of alcohol intake in 100% of rats. At the same time, under conditions of the same information load against the background of natural magnetic field, this phenomenon was observed in 45% of animals and was less expressed (a 2.4-fold increase). Unlike the control groups, the effect of magnetic field was independent neither on the level of initial alcohol preference, nor on the exploratory ability of animals. The increased alcohol uptake persisted for two months after the termination of the information load. It is suggested that the higher energetic level is needed in the presence of the weak inhomogenous magnetic field for normal exploratory activity. In this case, ethanol can serve as an additional source of energy.