Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol

In a crystal structure of the amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG), the biologically active estrogen metabolite, 2-methoxyestradiol (2-MeOE2), binds in the same orientation as estradiol. The high affinity of SHBG for 2-MeOE2 relies primarily on hydrogen bonding between the hydroxyl at C-3 of 2-MeOE2 and Asp(65) and an interaction between the methoxy group at C-2 and the amido group of Asn(82). Accommodation of the 2-MeOE2 methoxy group causes an outward displacement of residues Ser(128)-Pro(130), which appears to disorder and displace the loop region (Leu(131)-His(136)) that covers the steroid-binding site. This could influence the binding kinetics of 2-MeOE2 and/or facilitate ligand-dependent interactions between SHBG and other proteins. Occupancy of a zinc-binding site reduces the affinity of SHBG for 2-MeOE2 and estradiol in the same way. The higher affinity of SHBG for estradiol derivatives with a halogen atom at C-2 is due to either enhanced hydrogen bonding between the hydroxyl at C-3 and Asp(65) (2-fluoroestradiol) or accommodation of the functional group at C-2 (2-bromoestradiol), rather than an interaction with Asn(82). By contrast, the low affinity of SHBG for 2-hydroxyestradiol can be attributed to intra-molecular hydrogen bonding between the hydroxyls in the aromatic steroid ring A, which generates a steric clash with the amido group of Asn(82). Understanding how C-2 derivatives of estradiol interact with SHBG could facilitate the design of biologically active synthetic estrogens.     

Recombinant human insulin IX. Investigation of factors,influencing the folding of fusion protein-S-sulfonates,biotechnological precursors of human insulin

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.     

Influence of helio-geophysical factors (HGF) on environments, on health state and reliability of man's professional functioning, on processes of society development

The present work reveals the helio-geophysical factors HGF) influence on natural surroundings, human and society, health state and professional reliability of human functioning in technogenic and social systems of extreme risk. The interdisciplinary complex investigations were performed on the social-population, organismal, organ cell and molecular levels. The HGF synergistic influence on a human state and functioning and the society especially within megalopolises' non-equilibrium geo-dynamic zones was studied in details. We analyzed the HGF influence on the causes and development of instabilities in ecology-social processes and the formation of intellectual and social climate. We also worked out some recommendations on stabilizing the society development concerning the geo-space and ecology-humanitarian imperative on the threshold of the current maximum of solar activity in 2000 - 2003.     

Oxygen transport to skeletal muscle working at VO(2)max in acute hypoxia: theoretical predictions

The influence of acute hypoxia (30 < or = PaO(2) < or = 100 mmHg) on the values of VO(2)max and parameters of oxygen transport in muscle working at VO(2)max was studied. We investigated muscle working under different values of blood flow F (60 < or = F < or = 120 ml/min per 100 g), blood pH (7.0-7.6), and different diffusion conditions. Investigations were performed on a computer model of O(2) delivery to and O(2) consumption in the working muscle. VO(2)max, PvO(2), pO(2)- and VO(2)-distribution in muscle fiber were calculated. It was shown that the greater the degree of arterial hypoxemia, the lower the muscle VO(2)max and blood pO(2) values. When working at VO(2)max, the average and minimal values of tissue pO(2) depend on PaO(2). The greater the blood flow through muscle, the greater the VO(2)max. However, with an increasing degree of arterial hypoxemia, the effect of F and blood pH on the value of VO(2)max is weakened. The diffusion conditions produced a powerful influence on the VO(2)max value. At reduced PaO(2) they are the most important limiting factors of O(2) supply to muscle working at maximal effort.     

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F₀F₁-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca²⁺-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (_M) and the Ca²⁺-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca²⁺ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.     

Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH)

3D-FISH has become a major tool for studying the higher order chromatin organization in the cell nucleus. It is not clear, however, to what extent chromatin arrangement in the nucleus after fixation and 3D-FISH still reflects the order in living cells. To study this question, we compared higher order chromatin arrangements in living cells with those found after the 3D-FISH procedure. For in vivo studies we employed replication labeling of DNA with Cy3-conjugated nucleotides and/or chromatin labeling by GFP-tagged histone 2B. At the light microscope level, we compared the intranuclear distribution of H2B-GFP-tagged chromatin and the positions of replication-labeled chromatin domains in the same individual cells in vivo, after fixation with 4% paraformaldehyde, and after 3D-FISH. Light microscope data demonstrate a high degree of preservation of the spatial arrangement of approximately 1-Mb chromatin domains. Subsequent electron microscope investigations of chromatin structure showed strong alterations in the ultrastructure of the nucleus caused mainly by the heat denaturation step. Through this step chromatin acquires the appearance of a net with mesh size of 50-200 nm roughly corresponding to the average displacement of the chromatin domains observed at light microscope level. We conclude that 3D-FISH is a useful tool to study chromosome territory structure and arrangements down to the level of approximately 1-Mb chromatin domain positions. However, important ultrastructural details of the chromatin architecture are destroyed by the heat denaturation step, thus putting a limit to the usefulness of 3D-FISH analyses at nanometer scales.