Using produced polyclonal antibodies specific to the N-terminal sequence (residues 61-298) of rat obscurin, we investigated the isoform composition of this protein in 4 striated muscles: myocardium of the left ventricle, diaphragm, skeletal m. gastrocnemius (containing mainly fast fibers), and m. soleus (containing mainly slow fibers). The m. gastrocnemius, m. soleus, and diaphragm were found to have 2 giant isoforms of obscurin: a smaller A-isoform and a larger B-isoform. Their molecular weights were ~870 and ~1150 kDa in the diaphragm and m. gastrocnemius and ~880 and ~1130 kDa in m. soleus, respectively. The B-isoform to A-isoform ratio was 1:3 in the diaphragm and m. soleus and 1:4 in the m. gastrocnemius. In the left-ventricular myocardium, A-isoform of obscurin with a molecular weight of ~880 kDa was found. No other obscurin isoforms or their fragments within the molecular weight range of 10 up to ~800 kDa were revealed in the investigated rat striated muscles. The antibodies produced are recommended for research into qualitative and quantitative changes of giant obscurin isoforms in rat striated muscles in the norm and during the development of pathological processes.

A mesenchymal stem cell (MSC)-derived conditioned medium (CM) was tested for effects on the severity of the systemic inflammatory response induced by acetaminophen (APAP), including its long-term influence on hepatic tissue. A CM fraction of <30 kDa showed a maximum protective effect. Proteins of the fraction reduced the systemic inflammatory response and liver tissue fibrosis long after the toxin administration. Liver tissues from experimental animals were examined, and reduction of granulocytic infiltration was found to correlate with the degree of hepatic parenchymal necrosis, which was confirmed by the cytolysis enzyme level. The <30-kDa fraction increased the spontaneous and decreased the phorbol 12-myristate 13-acetate (PMA)-induced intracellular production of reactive oxygen species (ROS). Expression of the macrophage migration inhibitory factor (MIF) and thioredoxin significantly increased as compared to controls during increased spontaneous ROS production in neutrophils.

Turpentine oil, owing to the presence of 7–50 terpenes, has analgesic, anti-inflammatory, immunomodulatory, antibacterial, anticoagulant, antioxidant, and antitumor properties, which are important for medical emulsion preparation. The addition of turpentine oil to squalene emulsions can increase their effectiveness, thereby reducing the concentration of expensive and possibly deficient squalene, and increasing its stability and shelf life. In this study, squalene emulsions were obtained by adding various concentrations of turpentine oil via high-pressure homogenization, and the safety and effectiveness of the obtained emulsions were studied in vitro and in vivo. All emulsions showed high safety profiles, regardless of the concentration of turpentine oil used. However, these emulsions exhibited dose-dependent effects in terms of both efficiency and storage stability, and the squalene emulsion with 1.0% turpentine oil had the most pronounced adjuvant and cytokine-stimulating activity as well as the most pronounced stability indicators when stored at room temperature. Thus, it can be concluded that the squalene emulsion with 1% turpentine oil is a stable, monomodal, and reliably safe ultradispersed emulsion and may have pleiotropic effects with pronounced immunopotentiating properties.

Mouse monoclonal antibodies to Hsp90β (β isoform of heat shock protein 90) have been shown to bind specifically to Hsp90β localized on the surface of tumor and nontransformed cells. After binding to the membrane-associated Hsp90β, the antibodies actively dissociated into the culture medium and were also internalized by the cells. An immunoconjugate based on the Hsp90β-specific antibody and the cytotoxic agent mertansine did not have high cytotoxic activity for tumor cells in vitro. Administration of Hsp90β-specific antibodies in mice did not affect the growth of the primary Lewis lung carcinoma, while tumor metastasis to the lungs decreased and the average lifespan of mice increased. The results indicate a certain therapeutic potential of antibodies to Hsp90β for the treatment of tumor diseases.

It is known that the alpha isoform of heat shock protein 90 (HSP90α) plays an important role in wound healing. HSP90α has a stimulating effect on migration and invasion of cells in the wound area. The role of beta isoform of HSP90 (HSP90β) in wound healing has not been fully determined. Recombinant human HSP90β expressed in E. coli has been obtained and characterized in the present work. By using an excision model of wound healing in mice, it was shown that purified recombinant human HSP90β introduced into the wound area increases the rate of wound healing by 28% compared to the control. This fact allows HSP90β to be considered a promising drug for wound healing.

We showed that the mouse monoclonal antibodies directed against the beta isoform of heat shock protein 90 (Hsp90beta) bind specifically to Hsp90beta found on the surface of tumor and untransformed cells. After binding to membrane-associated Hsp90beta, antibodies actively dissociated into the culture medium and were internalized by cells. An immunoconjugate prepared on the basis of the Hsp90beta-specific antibody and the cytotoxic agent mertansine did not exhibit high cytotoxic activity against tumor cells in vitro. The injection of the Hsp90beta-specific antibody into mice did not influence the growth of primary tumor of epidermoid Lewis lung carcinoma but suppressed the metastasis of tumor in the lungs and increased the mean life span of mice. The results suggest a certain therapeutic potential of antibodies directed against Hsp90beta for tumor therapy.

The effect of HDACs 4 and 5 on the level of atrophy, calpain-1 and titin content, and TTN gene expression in rat soleus after 7-day gravitational unloading (hindlimb suspension model) was studied. The development of atrophic changes induced by gravitational unloading in rat soleus was accompanied by an increase in the calpain-1 content, an increase in titin proteolysis, and a decrease in the mRNA content of the protein. Inhibition of HDACs 4 and 5 did not eliminate the development of unloading-induced atrophy but significantly prevented proteolysis of titin and the decrease in the TTN gene expression.

We studied the effect of histone deacetylase 1 (HDAC1) inhibition on titin content and expression of TTN gene in rat m. soleus after 3-day gravitational unloading. Male Wistar rats weighing 210±10 g were randomly divided into 3 groups: control, 3-day hindlimb suspension, and 3-day hindlimb suspension and injection of HDAC1 inhibitor CI-994 (1 mg/kg/day). In hindlimb-suspended rats, the muscle weight/animal body weight ratio was reduced by 13.8% (p<0.05) in comparison with the control, which attested to the development of atrophic changes in the soleus muscle. This was associated with a decrease in the content of NT-isoform of intact titin-1 by 28.6% (p˂0.05) and an increase in TTN gene expression by 1.81 times (p˂0.05) in the soleus muscle. Inhibition of HDAC1 by CI-994 during 3-day hindlimb suspension prevented the decrease in titin content and development of atrophy in rat soleus muscle. No significant differences in the TTN gene expression from the control were found. These results can be used when finding the ways of preventing or reducing the negative changes in the muscle caused by gravitational unloading.

Abstract—Changes in the expression of the titin gene and alternative splicing of titin pre-mRNA from exon 50 to exon 111 in rat soleus muscle were analyzed following 3-day functional unloading (the HS group). Using real-time RT-PCR, it was found that the expression level of the titin gene in the rat soleus muscle from the HS group was higher by 1.81 times (p ≤ 0.01, n = 6) than the control level (n = 7). It was shown that all studied exons of titin mRNA are present in rats soleus muscle from the two groups. Our results demonstrate that 3-day functional unloading is accompanied by an increase in the expression of the titin gene in rat soleus without changes in alternative splicing from exon 50 to exon 111.

This work studied the changes in the levels of the main proteins of the calpain system (μ-calpain, Са2+-dependent protease, and fragments of its autolysis, inhibitor calpastatin) and μ-calpain substrates (giant proteins of the sarcomere cytoskeleton, titin and nebulin) in skeletal muscle (m. gastrocnemius, m. soleus, m. longissimus dorsi) of rats alcoholized for three months by different methods using agar containing 30% ethanol and nutrient-balanced liquid feed containing 5% ethanol using gel electrophoresis methods under denaturing conditions and immunoblotting. No decrease in the muscle mass/body weight ratio, indicating the development of atrophy, no increase in autolysis of μ-calpain, indicating an increase in the activity of this enzyme, no changes in the content of intact titin (T1), nebulin, μ-calpain and calpastatin, as well as the total calpain activity measured using Calpain Activity Assay Kit were detected in alcoholized rats of both groups. No changes in the total level of titin phosphorylation in the rat muscles of alcoholized groups were detected using Pro-Q Diamond fluorescent dye for phosphate groups of proteins. No statistically significant differences in the content of titin and nebulin mRNA in skeletal muscles of control rats and rats alcoholized using agar were detected. In rats, alcoholized by the method of liquid feed, the levels of titin and nebulin mRNA were increased 1.5–2.5 times possibly due to a higher fat content in such a diet. The presented data may be useful for choosing a chronic alcoholization model for animals.

Abstract—Changes in the fatty acid levels in the cardiac and gastrocnemius muscles of rats that were chronically alcoholized for 3 and 6 months were studied using two methods of alcoholization: 30% ethanol-containing agar (method I) and a 5% ethanol-containing liquid diet with a balanced nutritional status (method II). In the control group, the fatty acid level in the cardiac muscle was considerably higher than that in the gastrocnemius muscle. In the animals that were alcoholized over a 3-month period using method I, a considerable increase in the levels of myristic, pentadecanoic, palmitic, stearic, and dihomo-γ-linolenic acids and the total amount of fatty acids and a decrease in ω-3 docosapentaenoic acid level were found in the cardiac muscle. After a 6-month period, during which rats were alcoholized using method I, an increase in the levels of palmitoleic and ω-6 docosapentaenoic acids and a decrease in the levels of stearic, eicosadienoic, and arachidonic acids were found. The amount of ω-3 docosapentaenoic acid in the myocardium, compared to that observed after a 3-month period during which rats were alcoholized, remained reduced compared to the control. In the gastrocnemius muscle of rats alcoholized for 3 months using method I, the amounts of myristic, vaccenic, dihomo-γ-linolenic, and ω-6 docosapentaenoic acid increased. Simultaneously, there was a tendency for the total amount of saturated, monounsaturated, and ω-6 polyunsaturated fatty acids and the total amount of fatty acids to rise. After a 6-month alcoholization, a decrease in the levels of myristic, oleic, linoleic, α- and γ-linolenic, and eicosadienoic acids, as well as the total amount of saturated, ω-6 polyunsaturated fatty acids and the total amount of all fatty acids was found. When animals were alcoholized over a 3-month period using method II, a significant increase in the amount of dihomo-γ-linolenic acid was detected in the cardiac and gastrocnemius muscles. The role of these changes in the muscle pathologies is discussed.

The composition of plasma proteins adsorbed on the surface of perfluorocarbon emulsions stabilized by different triblock copolymers and their quantitative ratio were analyzed. The results allowed us to describe three types of protein adsorption on the surface of emulsion droplets. Opsonin proteins prevailed during the first type of adsorption. Their adsorption occurred on a dense and inactive layer of triblock copolymers. The second type of adsorption occurred due to the hydrophobic effect on a dense and mobile layer, with low-molecular-weight proteins being predominantly adsorbed (monoadsorption). The third type of adsorption occurred on a loose layer of triblock copolymers. In this case, the adsorption of a large amount of proteins with a molecular weight of 10–500 kDa was observed, while the total molecular weight was distributed over a large number of proteins.

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion stabilized with proxanol 268 is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to an increase in the total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and fibrinogen is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been set that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicate considerable conformational rearrangements of this protein during adsorption. It was shown that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction is completely or partially quenched.

The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 mL of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 h or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorbed new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5–5 mg of proteins per 1 mL of the emulsion) after 24 h.