[Structure of the cortical cytoskeleton of the left cardiac ventricle hystiocytes and of soleus muscle fibers in rats following short-time suspension].

Purpose of the investigation was to examine structural rearrangements in the cortical cytoskeleton as a result of changes in external mechanic conditions. Objects of the investigation were Wistar rat's m. soleus fibers and left ventricle hystiocytes. Suspension for 6, 12, 18, 24 and 72 hours was performed according to the Ilyin-Novikov procedure modified by Morey-Holton. Cell stiffness, non-muscle actin and actin-binding proteins in the protein membrane and cytoplasmic fractions, and respective gene expression were evaluated. In addition, corticosterone levels were measured in blood serum. After 6 hours of suspension, actin-binding proteins went down in the membrane fraction, i.e. alpha-actinin-1 decreased in hystiocytes and alpha-actinin-4 in m. soleus fibers. On the contrary, their content in the cytoplasmatic fraction increased. Expression of genes coding beta- and gamma-actin, alpha-actinin-1 and alpha-actinin-4 in m. soleus fibers showed a decrease. However, the apha-actinin-1 gene regained its baseline expression rate in 72 hours. Following 18 to 24 hours of suspension, expression of the beta-actin and alpha-actinin-4 genes in hystiocytes grew in comparison with baseline values, while expression of alpha-actinin-1 gene decreased. After 12 hours of suspension, beta- and gamma-actin levels were reduced in membrane proteins and increased in cytoplasmatic proteins in hystiocytes and m. soleus fibers. Stiffness showed a decline in each type of cell. Further into suspension, membrane proteins of hystiocytes increased levels of non-muscle actin and actin-binding proteins, and stiffness of these cells. Levels of these proteins decreased in m. soleus fibers, as also did cell stiffness. Serum corticosterone hardly altered but was slightly increased after 6 hours of suspension.

[Insulin-like growth factor-I splice-variants expression and myosatellite cells in the rat m. soleus under unloading and passive stretch].

Hindlimb unloading produces atrophy and suppresses proliferative processes in postural muscles. Muscle stretch applied simultaneously with hindlimb suspension is known to prevent soleus muscle atrophy. In the rat experiments, we assessed the number of M-cadherin (satellite cell marker molecule)-labeled cells per one myofiber cross-section. After 2-week hindlimb suspension the number of labeled cells decreases by 33% as compared to the control group. The amount of labeled cells was 2.5-fold greater in passive stretch group in comparison with the hindlimb suspended animals and 1.7-fold greater as compared to the control group. We suppose that that proliferation of satellite cells with subsequent incorporation of their nuclei in myofiber is sufficient for increasing the protein synthesis. The insulin-like growth factor (IGF-I) is involved in regulation of protein turnover and exerts potent mitogenic and differentiating effects. We investigated the level of IGF-I expression in soleus muscle tissue after 14 day hindlimb suspension with stretch and observed no changes as compared to the control or 14 day hindlimb suspended group. We conclude that the muscle IGF-I and satellite cells incorporation do not make essential contribution to passive stretch preventive action under the conditions of simulated weightlessness.

[Insulin-like growth factor 1 and the key markers of proteolysis during the acute period of readaptation of the muscle atrophied as a result of unloading].

It has been shown that, after prolonged disuse, the accumulation of muscle mass and the recovery of soleus fibers volume are caused by water accumulation rather than protein synthesis intensification. At the same time, expression rate of the main markers of the activity of ubiquitin-proteasome system remained increased on the 3rd day of reloading and decreased to the control by the 7th day. Both the quantity of the insulin-like growth factor 1 and the number of satellite cells fused with muscle fibers and of myonuclei began to increase only on the 7th day of reloading. The data obtained evidenced a significant inertness of the postural muscle during its adaptation to the load (normal gravity) after prolonged disuse.

Na-K-ATPase in rat cerebellar granule cells is redox sensitive.

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.

Design of sequence-specific DNA binding ligands that use a two-stranded peptide motif for DNA sequence recognition.

The design and DNA binding activity of beta-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides-S,S'-bis(Lys-Gly-Val-Cys-Val-NH-NH-Dns)-bridged by an S-S bond binds at least 10 times more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT). This peptide can also discriminate between 5'-GpG-3' and 5'-GpC-3' steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides-Gly-Cys-Gly- or Val-Cys-Val-bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5'-GpG-3' steps on DNA, whereas the netropsin-like fragments bind preferentially to runs of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5'-GpG-3'-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.

[Synthetic DNA-bindings ligands containing reaction centers specific for AT- and GC-pairs in DNA]. / Sinteticheskie DNK-sviazyvaiushchie ligandy, soderzhashchie reaktsionnye tsentry, spetsifichnye k AT- i GC-param osnovanii DNK.

In the present communication design, synthesis and DNA binding activities of three bis-netropsins and two netropsin analogs containing two N-propylpyrrolecarboxamide fragments linked covalently to peptides Gly-Gly-(analog I) and Val-Val-Val-Gly-Gly-(analog II) are reported. Each bis-netropsin consists of two netropsin-like fragments attached to peptides -Gly-Cys-Gly-NH2 (compound IIIa), H-Gly-Cys-Gly-Gly-Gly-(compound IV) or Gly-Cys-Sar-NH2 (compound IIIb) which are linked symmetrically via S-S bonds. Physico-chemical studies show that each bis-netropsin carries 6 AT-specific reaction centers and covers approximately 10 base pairs upon binding to poly(dA).poly(dT). This indicates that two netropsin-like fragments of the bis-netropsin molecule are implicated in specific interaction with DNA base pairs. The peptide fragments of bis-netropsins IIIa and IV form small beta-sheets containing two-GC-specific reaction centers. The DNase I cleavage patterns of bis-netropsin-DNA complexes visualized by high resolution gel electrophoresis show that the preferred binding sites for bis-netropsins IIIa and IV are identical and contain two runs of three or more AT pairs separated by two GC pairs. Specificity determinants of netropsin analog II binding in the beta-associated dimeric form are identical to those of bis-netropsin IIIa thereby indicating that there is a similarity in the structure of complexes formed by these ligands with DNA. In the monomeric form analog II exhibits binding specificity identical to that of analog I. Replacement of C-terminal glycine residues by sarcosines in the peptide fragments of bis-netropsin IIIa leads to a decrease in the affinity of ligand for DNA.

[Attachment of trivaline changes the specificity of binding of netropsin analogs with DNA]. / Prisoedinenie trivalina izmeniaet spetsifichnost' sviazyvaniia analoga netropsina s DNK.

In the present communication, synthesis and DNA binding activities of three analogs of the antibiotic netropsin are reported. Each analog contains two N-propylpyrrolecarboxamide units linked covalently to either Dns-Gly-Val-Val-Val-Gly-Gly- (I), Val-Val-Val-Gly-Gly (II) or Gly-Gly (III). It is shown that analogs I and II can self-associate in aqueous solution and methanol as revealed from the fact that UV absorbance and circular dichroism spectra obtained for these analogs are concentration-dependent. By contrast, analogs III exists as a monomer, even at concentration levels of the order of 1.10(-3) M. Determination of the apparent sizes of intramolecular aggregates by gel-filtration shows that analog I in aqueous solution at concentration levels of the order of 1.10(-3) M forms a series of aggregates containing from 2 to 12 monomers. Analog II exhibits a lower tendency to form intermolecular aggregates as compared with that of analog I. Dimerization constants are determined for analogs I and II in aqueous solution and methanol. The binding of N-propylpyrrolecarboxamide units and peptide fragments of analog I to DNA can be independently monitored by circular dichroism and fluorescence methods. If self-associated species of analog I (or II) are present in solution, the ligand exhibits a markedly different order of base pair sequence preferencies as compared with that of analog III. The results obtained are consistent with the inference that analogs I and II in a beta-associated form recognizes base pair sequences containing two runs of 3 AT pairs separated by two GC pairs.