Molecular Serum Albumin Unmask Nanobio Properties of Molecular Graphenes in Shungite Carbon Nanoparticles.

Serum albumin is a popular macromolecule for studying the effect of proteins on the colloidal stability of nanoparticle (NP) dispersions, as well as the protein-nanoparticle interaction and protein corona formation. In this work, we analyze the specific conformation-dependent phase, redox, and fatty acid delivery properties of bovine albumin in the presence of shungite carbon (ShC) molecular graphenes stabilized in aqueous dispersions in the form of NPs in order to reveal the features of NP bioactivity. The formation of NP complexes with proteins (protein corona around NP) affects the transport properties of albumin for the delivery of fatty acids. Being acceptors of electrons and ligands, ShC NPs are capable of exhibiting both their own biological activity and significantly affecting conformational and phase transformations in protein systems.

Metformin Pre-Treatment as a Means of Mitigating Disuse-Induced Rat Soleus Muscle Wasting.

Currently, no ideal treatment exists to combat skeletal muscle disuse-induced atrophy and loss of strength. Because the activity of AMP-activated protein kinase (AMPK) in rat soleus muscle is suppressed at the early stages of disuse, we hypothesized that pre-treatment of rats with metformin (an AMPK activator) would exert beneficial effects on skeletal muscle during disuse. Muscle disuse was performed via hindlimb suspension (HS). Wistar rats were divided into four groups: (1) control (C), (2) control + metformin for 10 days (C+Met), (3) HS for 7 days (HS), (4) metformin treatment for 7 days before HS and during the first 3 days of 1-week HS (HS+Met). Anabolic and catabolic markers were assessed using WB and RT-PCR. Treatment with metformin partly prevented an HS-induced decrease in rat soleus weight and size of slow-twitch fibers. Metformin prevented HS-related slow-to-fast fiber transformation. Absolute soleus muscle force in the HS+Met group was increased vs. the HS group. GSK-3ß (Ser9) phosphorylation was significantly increased in the HS+Met group vs. the HS group. Metformin pre-treatment partly prevented HS-induced decrease in 18S+28S rRNA content and attenuated upregulation of calpain-1 and ubiquitin. Thus, pre-treatment of rats with metformin can ameliorate disuse-induced reductions in soleus muscle weight, the diameter of slow-type fibers, and absolute muscle strength.

AMPK Phosphorylation Impacts Apoptosis in Differentiating Myoblasts Isolated from Atrophied Rat Soleus Muscle.

Regrowth of atrophied myofibers depends on muscle satellite cells (SCs) that exist outside the plasma membrane. Muscle atrophy appears to result in reduced number of SCs due to apoptosis. Given reduced AMP-activated protein kinase (AMPK) activity during differentiation of primary myoblasts derived from atrophic muscle, we hypothesized that there may be a potential link between AMPK and susceptibility of differentiating myoblasts to apoptosis. The aim of this study was to estimate the effect of AMPK activation (via AICAR treatment) on apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle. Thirty rats were randomly assigned to the following two groups: control (C, n = 10) and 7-day hindlimb suspension (HS, n = 20). Myoblasts derived from the soleus muscles of HS rats were divided into two parts: AICAR-treated cells and non-treated cells. Apoptotic processes were evaluated by using TUNEL assay, RT-PCR and WB. In differentiating myoblasts derived from the atrophied soleus, there was a significant decrease (p < 0.05) in AMPK and ACC phosphorylation in parallel with increased number of apoptotic nuclei and a significant upregulation of pro-apoptotic markers (caspase-3, -9, BAX, p53) compared to the cells derived from control muscles. AICAR treatment of atrophic muscle-derived myoblasts during differentiation prevented reductions in AMPK and ACC phosphorylation as well as maintained the number of apoptotic nuclei and the expression of pro-apoptotic markers at the control levels. Thus, the maintenance of AMPK activity can suppress enhanced apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle.

Inhibition of mTORC1 differentially affects ribosome biogenesis in rat soleus muscle at the early and later stages of hindlimb unloading.

Prolonged inactivity of skeletal muscles due to limb immobilization, bedrest, and exposure to microgravity results in a significant muscle atrophy. Inactivity-induced muscle atrophy is caused by a downregulation of protein synthesis (PS) and increased proteolysis. Mechanistic target of rapamycin complex 1 (mTORC1) is considered to be one of the main regulators of translational capacity (quantity of ribosomes), a key determinant of PS. Using a specific mTORC1 inhibitor (rapamycin) we aimed to determine if mTORC1 activity would influence ribosome biogenesis in rat soleus muscle at both early and later stages of mechanical unloading. Wistar rats were subjected to 1- and 7-day hindlimb suspension (HS) with and without rapamycin injections (1.5 mg/kg) and compared to weight-bearing control animals. The key markers of ribosome biogenesis were assessed by RT-PCR or agarose gel electrophoresis. The rate of PS was measured by SUnSET method. Both 1-day and 7-day HS resulted in a significant downregulation of ribosome biogenesis markers (c-Myc, 47S pre-rRNA, 18S + 28S rRNAs) and the rate of PS. Rapamycin administration during 1-day HS fully prevented a decrease in 47S pre-rRNA expression and amount of 18S + 28S rRNAs (without affecting c-Myc mRNA expression) and partially attenuated a decline in PS. Rapamycin treatment during 7-day HS significantly decreased p70S6K phosphorylation but failed to rescue a reduction in both the markers of ribosome biogenesis and the rate of PS. All together, our results suggest that mTORC1 inhibition at the initial (1 day), but not later (7 days) stage of HS can be beneficial for the maintenance of translational capacity (ribosome biogenesis) and the rate of PS in rat soleus muscle.

Cultured Myoblasts Derived from Rat Soleus Muscle Show Altered Regulation of Proliferation and Myogenesis during the Course of Mechanical Unloading.

The structure and function of soleus muscle fibers undergo substantial remodeling under real or simulated microgravity conditions. However, unloading-induced changes in the functional activity of skeletal muscle primary myoblasts remain poorly studied. The purpose of our study was to investigate how short-term and long-term mechanical unloading would affect cultured myoblasts derived from rat soleus muscle. Mechanical unloading was simulated by rat hindlimb suspension model (HS). Myoblasts were purified from rat soleus at basal conditions and after 1, 3, 7, and 14 days of HS. Myoblasts were expanded in vitro, and the myogenic nature was confirmed by their ability to differentiate as well as by immunostaining/mRNA expression of myogenic markers. The proliferation activity at different time points after HS was analyzed, and transcriptome analysis was performed. We have shown that soleus-derived myoblasts differently respond to an early and later stage of HS. At the early stage of HS, the proliferative activity of myoblasts was slightly decreased, and processes related to myogenesis activation were downregulated. At the later stage of HS, we observed a decrease in myoblast proliferative potential and spontaneous upregulation of the pro-myogenic program.

The Role of Glycogen Synthase Kinase-3 in the Regulation of Ribosome Biogenesis in Rat Soleus Muscle under Disuse Conditions.

It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.

Plantar mechanical stimulation attenuates protein synthesis decline in disused skeletal muscle via modulation of nitric oxide level.

Both research conducted under microgravity conditions and ground-based space analog studies have shown that air pump-based plantar mechanical stimulation (PMS) of cutaneous mechanoreceptors of the sole of the foot is able to increase neuromuscular activity in the musculature of the lower limbs. This type of stimulation is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. The aim of the present study was to evaluate the effects of PMS on anabolic signaling pathways in rat soleus muscle following 7-day hindlimb suspension (HS) and to elucidate if the effects of PMS on anabolic processes would be NO-dependent. The soles of the feet were stimulated with a frequency of 1-s inflation/1-s deflation with a total of 20 min followed by 10 min rest. This cycle was repeated for 4 h each day. We observed a decrease in the soleus muscle mass after 7-day HS, which was not prevented by PMS. We also observed a decrease in slow-type fiber cross-sectional area (CSA) by 56%, which significantly exceeded a decrease (-22%) in fast-type fiber CSA. PMS prevented a reduction in slow-twitch fiber CSA, but had no effect on fast-twitch fiber CSA. PMS prevented a 63% decrease in protein synthesis after 7-day HS as well as changes in several key anabolic signaling regulators, such as p70S6k, 4E-BP1, GSK3ß, eEF-2, p90RSK. PMS also prevented a decrease in the markers of translational capacity (18S and 28S rRNA, c-myc, 45S pre-rRNA). Some effects of PMS on anabolic signaling were altered due to NO-synthase inhibitor (L-NAME) administration. Thus, PMS is able to partially prevent atrophic processes in rat soleus muscle during 7-day HS, affecting slow-type muscle fibers. This effect is mediated by alterations in anabolic signaling pathways and may depend on NO-synthase activity.

Effects of Plantar Mechanical Stimulation on Anabolic and Catabolic Signaling in Rat Postural Muscle Under Short-Term Simulated Gravitational Unloading.

It is known that plantar mechanical stimulation (PMS) is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. However, molecular mechanisms underlying the effect of PMS on skeletal muscle during unloading remain undefined. The aim of the study was to evaluate the effects of PMS on anabolic and catabolic signaling pathways in rat soleus at the early stages of mechanical unloading. Wistar rats were randomly assigned to ambulatory control, hindlimb suspension (HS) for 1 or 3 days, and HS for 1 or 3 days with PMS. The key anabolic and catabolic markers were assessed by western blotting and RT-PCR. Protein synthesis (PS) rate was estimated using SUnSET technique. PMS attenuated a 1-day HS-induced decrease in 4E-BP1, GSK-3ß, and AMPK phosphorylation. PMS also partially prevented a decrease in PS, phosphorylation of GSK-3ß, nNOS, and an increase in eEF2 phosphorylation after 3-day HS. PMS during 1- and 3-day HS prevented MuRF-1, but not MAFbx, upregulation but did not affect markers of ribosome biogenesis (18S + 28S rRNA, c-myc) as well as AKT phosphorylation. Thus, PMS during 3-day HS partially prevented a decrease in the global rate of PS in rat soleus muscle, which was accompanied by attenuation of MuRF-1 mRNA expression as well as changes in GSK-3ß, nNOS, and eEF2 phosphorylation.

Mechanism of aggregation of UV-irradiated ß(L)-crystallin.

Thermal denaturation and aggregation of UV-irradiated ß(L)-crystallin from eye lenses of steers have been studied. The data on size-exclusion chromatography and SDS-PAGE indicated that UV irradiation of ß(L)-crystallin at 10 °Ð¡ resulted in fragmentation of the protein molecule and formation of cross-linked aggregates. Fluorescence data showed that tryptophan fluorescence in the irradiated protein decreased exponentially with the UV dose. Decrease in tryptophan fluorescence is a result of photochemical destruction, but not of conformational changes of protein, because there is no red shift in the fluorescence maximum. The differential scanning calorimetry (DSC) profiles of the samples of UV-irradiated and wild type ß(L)-crystallin were registered. The area under curves, which is proportional to the amount of the native protein, decreased exponentially with increasing the irradiation dose. The shape of the DSC profiles for the samples of UV-irradiated ß(L)-crystallin was identical to that for wild type ß(L)-crystallin. The DSC data allowed estimating the portion of UV-denatured ß(L)-crystallin, which is not registered by DSC, and the portion of the combined fraction consisting of native and UV-damaged molecules retaining the native structure. A conclusion has been made that UV-induced denaturation of ß(L)-crystallin follows the one-hit model. The study of the kinetics of thermal aggregation of UV-irradiated ß(L)-crystallin at 37 °Ð¡ using dynamic light scattering showed that the initial stage of aggregation was that of formation of the start aggregates with the hydrodynamic radius of 20 nm. Further sticking of the start aggregates proceeded in the regime of reaction-limited cluster-cluster aggregation. Splitting of the aggregate population into two components occurred above a definite point in time.

Thermodynamic study of protein phases formation and clustering in model water-protein-salt solutions.

Thermodynamic analysis of the water-protein-salt system, based on the description of the spinodal curve, has been carried out in various coordinate systems: (water chemical potential, protein concentration m(2)); (protein "solubility" log S, salt concentration m(3)); (effective temperature, critical composition of the system m(2)/m(3)). Such presentations explain the existence of diagrams with normal and retrograde protein solubility as a result of straightforward effect of ions present in solution as well as some features of the widely used phase diagram in coordinates (temperature, protein concentration). Analytic expressions for coefficients K and b of the salting out equation log S=-K.m(3)+b as functions of protein charge and protein adsorbed ions have been obtained and identified with the spinodal characteristic points reflecting quasi-equilibrium between protein-lean phase and dense protein-rich phase. Liquid-liquid, liquid-solid phase transitions, dynamic protein clusters and second virial coefficient that characterize interaction between solution components have been thus interrelated. The results of our thermodynamic analysis have been compared with the data reported for lysozyme .