Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated ß-myosin activity.

It has been reported that muscle functional unloading is accompanied by an increase in motoneuronal excitability despite the elimination of afferent input. Thus, we hypothesized that pharmacological potentiation of spontaneous contractile soleus muscle activity during hindlimb unloading could activate anabolic signaling pathways and prevent the loss of muscle mass and strength. To investigate these aspects and underlying molecular mechanisms, we used ß-myosin allosteric effector Omecamtiv Mekarbil (OM). We found that OM partially prevented the loss of isometric strength and intrinsic stiffness of the soleus muscle after two weeks of disuse. Notably, OM was able to attenuate the unloading-induced decrease in the rate of muscle protein synthesis (MPS). At the same time, the use of drug neither prevented the reduction in the markers of translational capacity (18S and 28S rRNA) nor activation of the ubiquitin-proteosomal system, which is evidenced by a decrease in the cross-sectional area of fast and slow muscle fibers. These results suggest that chemically-induced increase in low-intensity spontaneous contractions of the soleus muscle during functional unloading creates prerequisites for protein synthesis. At the same time, it should be assumed that the use of OM is advisable with pharmacological drugs that inhibit the expression of ubiquitin ligases.

[Endogenous content of the nitric oxide in the cell layers of the eye retina].

Nitric oxide is a universal molecule that regulates many different functions in an organism. In the eye retina nitric oxide plays both a regulatory role by modulation of the synaptic transmission between photoreceptors and bipolar cells and a toxic role in apoptosis induction in the outer nuclear layer and in the layer of ganglion cells. In this paper there has been made the first attempt to estimate the endogenous NO concentration in retina layers in vivo. The concentration of the nitric oxide was determined by two indepen- dent techniques: ESR spectrometry using spin trap for in vivo determination and NO-sensitive microelectrode for in situ determination in the survival isolated frog retina. The distinct NO con- centration was detected only in the ganglion cells layer (~0.25 µM) and in the inner segments layer of the photoreceptors (~0.6 µM). The activity and the kinetic characteristic of the NO-synthase localized in the same layers were also determined. Key words: retina cells layers, nitric oxide, ESR, NO-sensitive microelectrodes.

[Tear fluid proteome variability in healthy donors].

The variability of control samples is the main problem in clinical proteomics and reliable sources for estimation of variability of normal content of different proteins are not numerous. Most of the investigations of normal human tear proteome include pool of samples as reference materials. But it is impossible to use such experimental approach to establish the range of variability of different tear proteome components. This study is the first attempt to estimate the variability of proteins content in healthy donors tear using high-performance liquid chromatography and tandem mass spectrometry. The protein profiles from 11 individual healthy donors were analyzed. Essential variability of the minor proteins was revealed while the presence of 6 major proteins in all 11 samples was invariable. We found the Lacritin glycosylation in all samples of tear fluid received from healthy donors. It seems that this modification is typical for healthy donors tear. The analysis of the pool samples was also carried out to estimate the availability for this approach for the search of ophthalmologic diseases biomarkers.

[Photoinduced production of reactive oxygen species by retinal derivatives and conjugates].

The formation of reactive oxygen species (ROS) during all trans-retinal illumination was studied by ESR. The quantity of ROS was estimated by the ESR signal of a spin adduct of DEPMPO (5-dietoxyphosphoryl-5 methyl-1-pirroline-N-oxide). The ESR signal was found to be a superposition of the adducts: 80%--superoxide anion radical, 15%--hydroxyl radical and 5%--an unknown radical. It was shown that the addition of an equimolar albumin concentration resulted in the 5-fold decrease of the relative quantum yield for ROS generation, and a 4-fold albumin excess led to decreased ROS production by 30-fold. It is supposed that ATR and A2E in the photoreceptor membrane are the sources of photodamage induced by ROS. ATR in the cell is known to be transported by interphotoreceptor retinal binding proteins that belong to the albumin family. The ATR binding with proteins is supposed to prevent the ROS production in the photoreceptor cell and thus produces the light damage protection action.

[Modification of the nitric oxide concentration regulates the development of the apoptosis in the eye retina].

Using model elaborated it was shown that the retinal ischemia initiated the development of the apoptosis in the inner layers of the retina. Administration of NOS inhibitor prevented the development of the apoptosis in the retina. To ascertain if nitric oxide could induce the retinal apoptosis by itself the nontoxic donor of nitric oxide (dinitrosyl iron complex) was injected intravitreally. Administration of DNIC in low concentrations induced the development of the apoptosis in the same retinal layers as in ischemia. The injection of dinitrosyl iron complex at the higher concentration resulted in the decrease of the apoptosis level. Administration of dinitrosyl iron complex with excess of glutathione didn't lead to the development of the retinal apoptosis. The obtained data demonstrates the neurotoxic properties of the excess of nitric oxide in the retina.

Role of NO-synthase in regulation of protein metabolism of stretched rat m. soleus muscle during functional unloading.

Gravitational unloading causes atrophy of muscle fibers and can lead to destruction of cytoskeletal and contractile proteins. Along with the atrophic changes, unloaded muscle frequently demonstrates significant shifts in the ratio of muscle fibers expressing fast and slow myosin heavy chain isoforms. Stretching of the m. soleus during hindlimb suspension prevents its atrophy. We supposed that neuronal NO-synthase (NOS) (which is attached to membrane dystrophin-sarcoglycan complex) can contribute to maintenance of protein metabolism in the muscle and prevent its atrophy when m. soleus is stretched. To test this hypothesis, we used Wistar rats (56 animals) in experiments with hindlimb suspension during 14 days. The group of hindlimb suspended rats with stretched m. soleus was injected with L-NAME to block NOS activity. We found that m. soleus mass and its protein content in hindlimb-suspended rats with stretched m. soleus were preserved due to prevention of protein degradation. NOS is involved in maintenance of expression of some muscle proteins. Proliferation of satellite cells in stretched m. soleus may be due to nNOS activity, but maintenance of muscle mass upon stretching is regulated not by NOS alone.

Protective effect of L-Arginine administration on proteins of unloaded m. soleus.

Cytoskeletal and contractile proteins degenerate during functional unloading of muscle. The ratio of myosin heavy chain (MHC) expression changes simultaneously. We have supposed that NO can be a signal molecule related to the regulation of protein metabolism upon muscle unloading. To test this hypothesis, Wistar rats underwent functional unloading for 14 days without and with peroral administration of L-arginine (500 mg/kg) as NO precursor. Significant decreases in m. soleus mass, NO, nNOS, dystrophin, Hsp90, p-S6K, and type I MHC mRNA contents were found in the group of animals with unloading without preparation compared to those in control and in the group with unloading and administration of L-arginine; at the same time, increased contents of atrogin-1/MAFbx and MuRF-1 (p < 0.05) were found. No difference in the IGF-1 mRNA content between all three groups was found. Atrophy was significantly less pronounced in the group with unloading and L-arginine administration compared to that without the amino acid, and no destruction of cytoskeletal proteins was observed. We conclude that administration of L-arginine upon functional unloading decreases the extent of m. soleus atrophy, prevents the decrease in it of type I MHC mRNA, and blocks destructive changes in some cytoskeletal proteins. Such effect can be due to the absence of increase in this group of the content of some ubiquitin ligases and decreased intensity of the p70S6 kinase synthesis marker.

[Nitrites can be reduced in retinal vessels during hypoxia and protect the retina against ischemia and apoptosis].

The possibility and the mechanism of the reduction of nitrites in retinal vessels under the acute hypoxia in vivo have been investigated. An experimental model of the rat retina ischemia was elaborated using the laser coagulation of retinal vessels. It was demonstrated that the vessel thrombosis does not occur if the nitrite concentration in the vessels is increased. It was proposed that, under acute hypoxia, nitrites are reduced to NO, which results in a drastic vasodilatation. Considering that the effect takes less than a minute, this reduction cannot be due to hypoxic acidosis but is more likely associated with NO reduction by heme proteins. It was found that the increased concentration of nitrites protects the retina from the development of ischemia progress and that the preliminary administration of nitrites prevents apoptosis in the retina and a decrease in its photoelectric activity.

[Molecular mechanisms of retinal ischemia].

The review discusses the molecular mechanisms of retinal damage, which are associated with retinal ischemia. Ischemia is one of the key factors determining the pathophysiology of many retinal diseases, such as diabetic retinopathy, glaucoma, anterior ischemic optic neuropathy, age-related macular degeneration, retinopathy of prematurity. Hypoxia and ischemia impair retinal neuronal energy metabolism, by launching a cascade of trigger reactions resulting in cell death. Oxidative stress, excitotoxicity, cell acidosis, inflammation, and others mechanisms acting in tandem are of considerable importance in ischemia. Neuronal apoptosis and neovascularization are the most important sequels of ischemia. Among all retinal neurons, ganglion cells are most susceptible to ischemia, which determines their early structural and functional changes in many ischemia-associated retinal diseases. The molecular mechanisms underlying the pathophysiology of ischemia-associated retinal diseases should be understood to substantiate and develop new therapy modalities.

[Protective effect of nitric oxide on cytoskeletal proteins in skeletal muscles under eccentric exercise].

The protective effect of nitric oxide (NO) on the cytoskeletal muscle proteins desmin and dystrophin has been studied under eccentric exercise. Experiments were performed on 28 male Wistar rats, which were divided into four groups: cage control (C, n = 7); group of eccentric exercise (running down a motor-driven treadmill, inclination 16 degrees) (20 m/min, 40 min running) (R, n = 7); eccentric exercise + L-arginine group (RA, n = 7) (with a daily supplementation of 500 mg/kg wt L-arginine for 3 days before the running); and eccentric exercise + L-NAME group (RN, n = 7) (with a daily supplementation of 90 mg/kg wt L-NAME (Nomega-nitro-L-arginine methyl ester, nNOS blocker) for 3 days before the running). It was found that increasing the NO concentration (in RA group) prevents the disruption of the dystrophin layer and decreases the loss of desmin in m. soleus under eccentric contraction, whereas in the R and RN groups the level of damage to dystrophin and desmin was significantly higher compared to the control rats. The inhibition of nNOS (by L-NAME) increases the nNOS mRNA level in the m. soleus, whereas increasing the NO concentration in m. soleus (L-arginine administration) does not affect the level of nNOS mRNA during the eccentric running. It was concluded that NO has a protective action on the cytoskeletal proteins desmin and dystrophin in skeletal muscle under eccentric contraction-induced damage.

Structural and catalytic polymorphism of human enzymes: Novel potential platforms for biomedical diagnostics.

This review focuses on some intermediate results on the path from the gene and enzyme structure to physiological responses and personalised medicine. Bioinformatics of genetic and protein-structural polymorphisms, theoretical methods of predicting the influence of single amino acid substitutions on the structure and catalytic activity of enzymes are considered. For a large group of enzymes, interrelations between genetic modifications, structural changes of the proteins and the detected physiological and clinical manifestations are discussed. In this respect, highly productive techniques to determine the catalytic activity of an enzyme as well as non-invasive proteomic approaches are of particular interest. A non-invasive proteomic analysis using mass-spectrometric protein identification of human exhaled breath condensate and tear fluids has been chosen.

Experimental model of acute ischemia of the retina in rats.

We studied the development of retinal ischemia in rat eye after laser coagulation of blood vessels. Typical signs of ischemia manifested in the retina after 24 h: development of stable retinal edema, decrease in the b/a index (ratio of the electroretinogram b and a-wave amplitudes) to 1-2 units, pronounced disorders in the retinal microcirculation system, leading to ischemia of the inner layers of the retina. The proposed model is convenient for studies of the development of acute retinal ischemia, is easily realized, and reproduces some acute ischemic diseases of the retina.

[Antibodies to retinal S antigen in the tear and serum as an indicator of the nature of a course of endogenous uveitis in children].

Antibodies (IgM and IgG) to retinal S antigen were studied by enzyme immunoassay in the tear (n = 135) and blood serum (n = 221) samples taken from 45 children with chronic endogenous uveitis during a follow-up (for 6 months to 5 years). The studies have confirmed the early evidence that detection of serum autoantibodies may be used for the diagnosis of autoimmune and infectious-and-autoimmune uveitis, the verification and prediction of renal alterations, risks for lesions in the second eye in unilateral uveitis and prerecurrence in bilateral uveitis. Criteria for detecting antibodies to lacrimal S antigen were developed. Its has been ascertained that it is expedient to use this procedure as a method for controlling the efficiency of treatment in uveitis involving the posterior parts of the eye, confirming, and predicting the risk for involvement of the retina into an inflammatory process, detecting trends for development of chronic forms of both bilateral and unilateral uveitis. Simultaneous accumulation of IgM and/or IgG antibodies in the lacrimal fluid and serum, as well as imbalance, i.e. a prolonged enhancement of only local or only systemic response to S antigen (of both classes), are prognostically unfavorable.

[The protein arrestin as a regulator of the phototransduction process and as a factor of pathogenesis of the eye diseases]. / Belok arestin kak reguliator protsessa fototransduktsii i kak patogeneticheskii faktor glaznykh zabolevanii.

In the first part of the paper, the results of the investigation of the rhodopsin arrestin interaction are presented. The results were mainly obtained with the technique of the selective labelling of the rhodopsin and arrestin SH-groups and the rhodopsin limited proteolysis. These results are discussed in the frame of the latest data on the three-dimensional structure of arrestin. In the second part of the paper, results of the antigenic properties of arrestin (S-antigen) and its role in the pathogenesis of the retina diseases are summarized. The data on the role of the autoimmune processes in the pathogenesis of diabetic retinopathy are presented. We have also described the results of the use of the elaborated technique of the immune diagnostics in the prognosis of the diabetic retinopathy and retinopathy of the premature babies.

[Humoral immune response to the retinal S-antigen in premature infants and its role in the development and course of retinopathy of prematurity]. / Gumoral'nyi immunnyi otvet na S-antigen setchatki u nedonoshennykh detei i ego rol' v razvitii i techenii retinopatii nedonoshennykh.

The humoral immune response to the retinal S-antigen was studied in premature babies with active and cicatricle stages of retinopathy of the premature (RP) and in the controls (102 serum test). The presence of antibodies to the S-antigen of classes IgG and IgM was established by the enzyme multiplied analysis and by an authors' developed test-system. A definite specific humoral response to the S-antigen correlated with the nature of the course and prognosis of the disease was found in 71% of cases. The first peak of IgM-antibodies was found in babies of the risk-group (preretinopathy), it correlated with a poor prognosis (progression of RP). The second peak was highly pronounced in babies with the terminal cicatricle stages of RP. The wave-like dynamics of IgG-antibodies suggests that specific circulating immune complexes shape up. The role of immune responses, induced by the retinal S-antigen, in the RP pathogenesis is discussed.

[Manifestations of immunopathology and validation of immunotherapy of diabetic retinopathy]. / Proiavleniia immunopatologii i obosnovanie immunoterapii diabeticheskoi retinopatii.

Three-year clinical and immunological studies of 378 patients with diabetic retinopathy revealed involvement of the T- and B-immunity systems and the specific features of the local autoimmune response to the retinal S-antigen, which manifest during the early preclinical stage of diabetic retinopathy. The progress of retinopathy is associated with augmenting insufficiency of T-suppressors, inhibition of lymphocyte blastogenesis with phytohemagglutinin, and an increase in the levels of B-lymphocytes, IgM, and circulating immune complexes in the blood. Two abnormalities of autoimmune response of the eye to S-antigen were observed in patients with the proliferative forms: hypersensitivity and insufficiency of the immune response. A moderate level of S-antigen in tears is a protective factor, stabilizing retinopathy. The authors validate the significance of immunological test for predicting the course of retinopathy and emphasize the differentiated approach to local and total-system's correction of immune disorders in diabetics.

pH changes in frog rods upon manipulation of putative pH-regulating transport mechanisms.

Rod intracellular pH (pHi) in the intact frog retina was measured fluorometrically with the dye 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein under treatments chosen to affect putative pH-regulating transport mechanisms in the plasma membrane. The purpose was to relate possible pHi changes to previously reported effects on photoresponses. In nominally bicarbonate-free Ringer, application of amiloride (1 mM) or substitution of 95 mM external Na+ by K+ or choline triggered monotonic but reversible acidifications, consistent with inhibition of Na+/H+ exchange. Bicarbonate-dependent mechanisms were characterized as follows: (1) Replacing half of a 12 mM phosphate buffer by bicarbonate caused a sustained rise of pHi. (2) Subsequent application of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2',2'-disulphonic acid (DIDS, 0.2 mM) set off a slow acidification. (3) Substitution of external Cl- by gluconate (95 mM) caused a rapid pHi rise both in normal Na+ and low-Na+ perfusion. (4) This effect was inhibited by DIDS. The results support a consistent explanation of parallel electrophysiological experiments on the assumption that intracellular acidifications reduce and alkalinizations (in a certain range) augment photoresponses. It is concluded that both Na+/H+ exchange and bicarbonate transport control rod pHi, modulating the light-sensitive current. Part of the bicarbonate transport is by Na(+)-independent HCO3-/Cl- exchange, but a further Na(+)-coupled bicarbonate import mechanism is implicated.

[The clinico-immunological validation of a differentiated approach to the use of corticosteroids in treating S-dependent uveoretinitis]. / Kliniko-immunologicheskoe obosnovanie differentsirovannogo podkhoda k primeneniiu kortikosteroidov pri lechenii S-zavisimykh uveoretinitov.

A total of 630 patients with uveitis of different etiology aged 5 to 70 were examined. Signs of autoimmunization with retinal S-antigens (mol. weight 48 kD) associated with retinal involvement were detected in more than half of them. A moderate level of S-antigen in the tears was found to promote a favorable outcome of uveoretinitis. Deficit or a drastic increase of S-antigen titers in the tears, accumulation of S-antibodies and circulating S-antigen-antibody complexes in the serum, and blood leukocyte migration inhibition in response to S-antigen were observed in chronic and complicated cases. As many as 75% focal chorioretinitis and panuveitis cases, 50% of posterior and 37% of peripheral uveitis were referred to S-related forms. The authors validate a differentiated approach to the treatment of S-uveoretinitis. If S-antibodies are accumulated only in the tears, local corticosteroids are effective; if both local and total-systems reactions are boosted, the maximal effect may be achieved by local and systemic corticosteroids and cytostatics. In case of a deficiency of S-antibodies in the tears immunodepressive therapy was of no avail, and good results could be attained only by immunomodulating therapy: corticosteroids+hemoperfusion in adults and corticosteroids+thymus preparations and/or interferonogens in children.