Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant.

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments.

Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

[X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase].

The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 A resolution. The kinetic parameters of the hydrolysis of substrates of different lengths (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease in the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.

[Complex influence of chemo- and ozonotherapy on the immunity of patients with destructive pulmonary tuberculosis].

The impact of standardized antimycobacterial treatment versus a complex combination of chemo- and ozonotherapy on the T and B links and specific antituberculous immunity was studied in patients with destructive pulmonary tuberculosis. A course of ozonotherapy was performed in the early intensive phase of antimycobacterial therapy via endovascular injection of ozonized 0.9% NaCl solution. Ozonotherapy used in the early intensive phase of the standardized treatment in patients with destructive pulmonary tuberculosis results in normalization of the count of tuberculin-sensitized T cells and the number of PPD-L proliferating lymphocytes, promotes decreased tuberculin sensitivity, accelerates normalization of humoral immunity (IgM, IgE), and activates the proliferative capacity of T cells in the early periods.

[Telangiectasis: arterial or venous?]. / Telangiectasias: arteriales o venosas?

In this work we have tried to elucidate whether telangiectases are dilatations of the arteriolar or the venular sections of the circulation system. To this end, we have made use of resources which have allowed us to study in the first place, the wall of these small vascular dilatations and, in the second place, their content. Through our studies we could determine that telangiectases are originated by dilatation of the venular sector of the circulatory system.

[Characteristics of the effect of a constant electromagnetic field on reparative processes in spinal cord injuries]. / Osobennosti vozdeistviia postoiannogo élektromagnitnogo polia na vosstanovite'lnye protsessy pri travmakh spinnogo mozga.

In 171 white rats the spinal cord was completely sectioned in the upper thoracic region. In 46 control rats an early crude spinal cord scar was identified which precluded the growth of nervous fibers. On the contrary, in animals exposed to constant magnetic field a smooth spinal cord scar was found with collaterals of anterior spinal arteria, recrease of the destruction zone and nervous fiber growth across the scar, This resulted in restored hind paw motility by 56 +/- 4.8% as related to control rats in which motility did not restore. Magnet therapy applied as a part in combined restitutional therapy in 104 patients in early restitution period yielded a 46.15 +/- 4.89% to 76.32 +/- 4.87% increase in the rate of restoration of motor and sensory functions. With the therapy applied in late periods, the restitution was 48.1 +/- 5.6% and 78.1 +/- 3.9%, respectively. The magnetic field is considered as a reparation booster in spinal cord trauma applicable in any stage of the disease.

[Interrelationships between the tissue link of the hemostatic system and the sympatho-adrenal system following transfusion of heterogenous blood]. / Vzaimootnosheniia mezhdu tkanevym zvenom sistemy gemostaza i simpato-adrenalovoi sistemoi pri transfuzii geterogennoi krovi

In an experiment using 86 white rats it was found that during incompatible hemotransfusion shifts take place not only in the humoral but also in the tissue link of the hemostatic system. It was also seen that the rise of the functional condition of sympatho-adrenal system precedes those changes of hemostatic figures of the tissues. The interrelationships between a tissue link of the hemostatic system and sympatho-adrenal system under the incompatible hemotransfusion were demonstrated.