Biochemistry

Role of Transcription Factors FoxO3 and Myogenin in Regulation of E3 Ligases MuRF-1 and MAFbx Expression in Rat Soleus at an Early Stage of Disuse Atrophy Abstract Hypokinesia and gravitational unloading lead to skeletal muscle atrophy resulting from alterations in the balance between anabolic and catabolic processes. Ubiquitin proteasome system plays a significant role in the process of protein breakdown in course of disuse atrophy. E3 ligases are essential components of this system. We analyzed changes in expression of E3 ligases MuRF-1 and MAFbx, as well as transcription factors FoxO3 and myogenin in rat soleus after 1 and 3 days of hindlimb unloading. We found that expression of E3 ligases was increased after 24 h of unloading, while phosphorylation of FoxO3 (S253) was decreased. However, the expression level of myogenin increased only 3 days after unloading. Our result suggests that earlier expression of MuRF-1 and MAFbx could be regulated by FoxO3, and that myogenin can also contribute to this process on the 3rd day of unloading

Inhibition of Proteasomes by Bortezomib Decreases MuRF-1 and MAFbx mRNA Expression in Rat Soleus during Hindlimb Unloading Abstract Skeletal muscle atrophy caused by unloading is accompanied by increased proteolysis and decreased protein synthesis. It is established that in conditions of muscle unloading, concentration of free amino acids is increased. We hypothesized that proteasome inhibition may decrease amino acid accumulation in skeletal muscle and prevent the atrophy. To test this hypothesis, we treated rats with bortezomib (a proteasome inhibitor) during 7-day hindlimb suspension. Content of key signaling proteins of various signaling pathways was measured by Western-blotting; mRNA level of E3 ligases by RT-PCR, rate of protein synthesis by SUnSET technique. Soleus muscle weight and intensity of the protein synthesis in the groups of "hindlimb suspension" (HS) and "HS + bortezomib" (HSB) were equally reduced as compared to control. Levels of MuRF-1 and MAFbx mRNAs, content of MuRF-1 and calpain-1 proteins, and level of the protein ubiquitination were increased only in the HS group and remained unchanged in the HSB group as compared to the control group. We conclude that inhibition of proteasomes during m. soleus unloading prevents increase in activity of some components of catabolic signaling pathways. However, this is not sufficient to reduce rate of the atrophic processes in skeletal muscle.

Effects of Abiotic Stresses on the Content of Glycoglycerolipids in the Vacuolar Membrane of Red Beetroot Abstract Glycoglycerolipids (GL) of the red beet vacuolar membrane under osmotic and oxidative stresses have been investigated. Variations of the GL content under stress conditions might be indicators of an important role of these compounds in protective mechanisms. Changes of the GL levels and the digalactosyldiacylglycerols/monogalactosyldiacylglycerol (DGDG/MGDG) ratio in the vacuolar membrane under hypoosmotic and oxidative stresses corresponded mainly to those observed in other cell membranes under majority of stresses studied, namely, the ratio increased that contributed to membrane stabilization. The changes of the GL content of vacuolar membrane caused by hyperosmotic stress notably differed. The DGDG content and DGDG/MGDG ratio significantly decreased. These alterations did not necessarily result in a decrease in the membrane stability under hyperosmotic stress, since the content of MGDG involved in the formation of a hexagonal structure and capable of destabilizing lipid bilayer also decreased. The changes of the GL level in the vacuolar membrane under hyperosmotic stress can be due to an increased degradation or reduced biosynthesis of GL; these changes may represent yet another defense mechanism of a plant cell against stresses.

Acetylcholinesterase Inhibitor Paraoxon Intensifies Oxidative Stress Induced in Rat Erythrocytes In Vitro Abstract Isoform H of acetylcholinesterase (AChE) is located on the outer surface of erythrocytes; its inhibition with organophosphorus compounds (OPC) may impair structural and functional properties of erythrocytes. The aim of this study was to elucidate the effects of paraoxon (POX) on the osmotic resistance, the level of reactive oxygen species (ROS), intracellular esterase activity, and externalization of phosphatidylserine in rat erythrocytes under oxidative stress induced by tert-butyl hydroperoxide (tBH). It has been found that POX did not affect the level of ROS and the activity of intracellular esterases; however, under conditions of induced oxidative stress, it potentiated the effects associated with impairment of the erythrocyte deformation characteristics and provoked cell death associated with externalization of phosphatidylserine. The effects of POX were not caused by the solvent DMSO and were observed only in the calcium containing medium. The results obtained with the in vitro model on the combined effects of the primary specific and secondary nonspecific factors on erythrocytes may be of help in the development or improvement of combined therapeutics for acute poisonings and the prevention of their consequences.

Extracellular Vesicles and Prospects of Their Use for Tissue Regeneration Abstract Extracellular vesicles are an important component of different cell secretomes that provide complex delivery of biologically active molecules and horizontal transfer of genetic information. They differ in their origin, composition, and functions. Selection of the vesicle isolation protocol, change in the cultivation conditions of the cells producing them and genetic modification influence the composition of the vesicles obtained. Stem cells produce vesicles carrying a wide range of growth factors, chemokines, cytokines, miRNAs that can affect the surrounding cells and have a therapeutic effect in various pathologies. The nature of biogenesis of extracellular vesicles, as well as their effects on target cells, is an important issue of fundamental biology, and improvement of methods for obtaining vesicles of the required composition opens broad prospects for their use in clinical practice.

Cationic Liposomes Cause ROS Generation and Release of Neutrophil Extracellular Traps Abstract Cationic liposomes are used as nanocarriers of drugs in chemotherapy, antibacterial therapy, and gene therapy. Positively charged stearylamine is commonly used in cationic liposome production. It has been shown that cationic liposomes, when applied intravenously, impact the functions and viability of blood cells, including neutrophils (innate immunity cells). In this work we studied the influence of phosphatidylcholine cationic liposomes containing stearylamine (SA liposomes) on the activity of human neutrophils–their ability of the formation of extracellular traps (NETs) and of the production of reactive oxygen species (ROS). The ratio of phosphatidylcholine to stearylamine in the liposomes was 9 : 1. Phosphatidylcholine liposomes (PC liposomes) were used as control. Liposomes were 140 ± 49 nm in diameter and differed in zeta-potential: PC liposomes had –1.74 ± 0.31 mV, while SA-liposomes had 11.40 ± 0.44 mV. NETs were visualized by light and fluorescent microscopy. It was found that the cells remained intact after a 90-min incubation with PC liposomes (control experiment), while incubation with SA liposomes caused an extrusion of thin DNA fibers into the extracellular space, i.e., the formation of NETs. ROS generation by neutrophils incubated with the liposomes and stimulated either with 0.5 mg/mL of zymozan, or with 40 nM of phorbol-12-miristate-13 acetate, or without stimulation was assessed by luminol-dependent chemiluminescence. It was shown that PC liposomes did not exert any significant effect on chemiluminescence of unstimulated and zymozan-stimulated neutrophils. PMA in the presence of liposomes did not exert any effect on the cells. SA liposomes had a complex impact on the activity of neutrophils: they inhibited the effects of zymozan and caused a prominent respiratory burst independently of the presence of the additional stimulation. The time before the maximal intensity of chemiluminescence peak after incubation with SA-liposomes ranged from 1 to 3 h and significantly differed from the time before the maximal intensity of chemoluminescence peak after incubation with zymozan or PMA (30–40 min). Apocynin, a NADPH-oxidase inhibitor, suppressed the respiratory burst induced by SA liposomes, indicating that NADPH oxidase has a role in ROS production caused by SA liposomes. To assess characteristics of the respiratory burst, lucigenin-dependent chemiluminescence sensitive to superoxide anion radical ( \({\text{O}}_{2}^{{ \bullet -}}\) ) was registered, and it was found that \({\text{O}}_{2}^{{ \bullet -}}\) is not produced after stimulation of neutrophils by SA liposomes. On the basis of these findings we conclude that SA liposomes are a polyfunctional factor that can affect the formation of NETs, suppress the action of zymozan, and stimulate ROS production by neutrophils.

Anticancer Activity of Spirocyclic Hydroxamic Acids (Derivatives of 1-Hydroxy-1,4,8-Triazaspiro[4,5]Decan-2-One), Histone Deacetylase Inhibitors Abstract The effect of 10 racemic spirocyclic hydroxamic acids (CHA 1–10, derivatives of 1-hydroxy-1,4,8-triazaspiro[4,5]decan-2-one), containing pharmacophore imidazolidinone and piperidine fragments with different substituents, on the activity of enzyme histone deacetylase (HDAC) was studied. It was shown that CHA (1–10) inhibit HDAC activity in cultured breast cancer cells. It was shown that CHA (1–10) as a part of polychemotherapy with cisplatin and cyclophosphane have a pronounced chemosensitizing antitumor activity in vivo. The results obtained on tumor models in vivo showed that CHA can be considered as potential medicinal components of tumor polychemotherapy.

Phosphorylation Level of Complex V Proteins of Higher Plant Mitochondria Correlates with Their Redox State Abstract— The effect of synthetic cytokinin BAP (6-benzylaminopurine) on protein phosphorylation in isolated maize mitochondria was investigated. The effect of BAP in comparison with classical inhibitors of the mitochondria respiratory complexes, such as rotenone, malonate, SHAM, and KCN on the level of mitochondrial protein phosphorylation was studied. Assessment of the activity of respiratory complexes I and II by the BN-PAGE (blue native polyacrylamide gel electrophoresis) in mitochondria untreated and treated with BAP has not revealed any difference in the activity of these complexes. Investigation of the mitochondrial complex V activity under BAP treatment has shown a significant decrease in the mitochondrial ATPase activity. The effect of BAP was similar to the effect of such a specific inhibitor of the membrane-bound mitochondrial ATPase as oligomycin and an inhibitor of protein tyrosine kinases genistein.

Analysis of Correlations between the Indexes of Light-Dependent Reactions of Photosynthesis and the Photochemical Reflectance Index (PRI) in Pea Leaves under Short-Term Illumination Abstract The photochemical reflectance index (PRI) is related to the conversion of violoxanthin into zeaxanthin in the xanthophyll cycle and thereby reflects the activity of electron-transport chain in chloroplasts. The PRI is recorded by a relatively simple and non-invasive technique; therefore it can be used in a perspective approach for the remote monitoring of photosynthetic processes and detection of photosynthetic stress. However, correlation coefficients between PRI and photosynthetic parameters vary widely in different studies; one of the possible causes of such variability under natural conditions may be the changes in illumination and the development of transient processes in the photosynthetic apparatus. In the present study, the influence of the duration of illumination (a minute interval) on the relationship between parameters of light-dependent reactions of photosynthesis and the PRI has been investigated. The photosynthetic parameters and PRI were determined in pea leaves. A high negative correlation has been found between changes of PRI induced by illumination and changes in the quantum yields of the photochemical reactions of photosystems I and II; the PRI changes did not correlate with changes of non-photochemical quenching. There was a positive correlation between the quantum yields of photochemical reactions and PRI averaged over the entire time range and a negative correlation in the case of non-photochemical quenching and PRI. The absolute value of the correlation coefficient of PRI with the parameters of the light-dependent reactions of photosynthesis increased with increasing duration of illumination. Thus, the correlation of PRI with the parameters of the light-dependent reactions of photosynthesis in a pea leaf may depend on the duration of illumination, at least in a minute interval.

Lateral Heterogeneity of Cholesterol Distribution in Cell Plasma Membrane: Investigation by Microfluorimetry, Immunofluorescence, and TOF-SIMS Abstract Lateral heterogeneity of cholesterol distribution in cell plasma membrane was revealed by a complex study using microfluorimetry, immunofluorescence microscopy, and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Glioblastoma cells U87MG exhibit a high motility, and cell movements are accompanied by redistribution of proteins along the membrane surface. The formation of the protein caveolin-1 clusters at one of the plasma membrane edges was detected by confocal microscopy on cells labeled with antibodies against caveolin 1. Using two-photon excitation fluorescence of a membrane probe 4-dimethylaminochalcone, membrane areas of about 200 × 200 nm were examined on glioblastoma cells. Simultaneous detection of the decay kinetics and the fluorescence spectrum revealed the presence of regions with an increased cholesterol concentration in the membrane at the poles of the live migrating cell. TOF-SIMS provided direct data with high spatial resolution indicating colocalization of cholesterol and caveolin 1 and confirmed previously published data on the association of caveolin 1 complexes with cholesterol clusters. Thus, three independent methods of cell membrane analysis testify that localization of cholesterol-enriched membrane regions correlates with morphological features of moving glioblastoma cells.