Autoregulation and Autoinhibition of the Main NO Synthase Isoforms (Brief Review).

Nitric oxide (II) (NO) is the most important mediator of a wide range of physiological and pathophysiological processes. It is synthesized by NO synthases (NOSs), which have three main isoforms differing from each other in terms of activation and inhibition features, levels of NO production, subcellular localization, etc. At the same time, all isoforms are structurally very similar, and these differences are determined by NOS autoregulatory elements. The article presents an analysis of the autoregulatory and autoinhibitory mechanisms of the NOS reductase domain that determine differences in the productivity of isoforms, as well as their dependence on the concentration of Ca2+ ions. The main regulatory elements in NOS that modulate the electron transfer from flavin to heme include calmodulin (CaM), an autoinhibitory insert (AI), and the C-terminal tail (C-tail). Hydrophobic interactions of CaM with the surface of the NOS oxidase domain are assumed to facilitate electron transfer from flavin mononucleotide (FMN). CaM binding causes a change in the inter-domain distances, a shift of AI and the C-tail, and, as a result, a decrease in their inhibitory effect. CaM also shifts the conformational equilibrium of the reductase domain towards more open conformations, reduces the lifetime of conformations, their stereometric distribution, and accelerates the flow of electrons through the reductase domain. The AI element, apparently, induces a conformational change that hinders electron transfer within the reductase domain, similar to the hinge domain in cytochrome P450. Together with CaM, the C-tail regulates the electron flow between flavins, the distance and relative orientation of isoalloxane rings, and also modulates the electron flow from FMN to the terminal acceptor. Together with the C-tail, AI also predetermines the dependence of neuronal and endothelial forms of NOS on the concentration of Ca2+ ions, and the C-tail length affects differences in the productivity of NO synthesis. The inhibitory effect of the C-tail is likely to be reduced by CaM binding due to the C-tail shift due to the electrostatic repulsive forces of the negatively charged phosphate and aspartate residues. The autoregulatory elements of NOS require further study, since the mechanisms of their interaction are complex and multidirectional, and hence provide a wide range of characteristics of the observed isoforms.

Protective role of exogenous recombinant peroxiredoxin 6 under ischemia-reperfusion injury of kidney.

Peroxiredoxin 6 (Prx6) is an important antioxidant enzyme with various functions in the cell. Prx6 reduces a wide range of peroxide substrates, playing a leading role in maintaining the redox homeostasis of mammalian cells. In addition to the peroxidase activity, a phospholipase A2-like activity was demonstrated for Prx6, which plays an important role in the metabolism of membrane phospholipids. Besides that, due to its peroxidase and phospholipase activities, Prx6 participates in intracellular and intercellular signal transduction, thus triggering regenerative processes in the cell, suppressing apoptosis caused by various factors, including ischemia-reperfusion injuries. A nephroprotective effect of exogenous recombinant Prx6 administered before ischemia-reperfusion injury was demonstrated on an animal model. Exogenous Prx6 effectively alleviates the severeness of renal ischemia-reperfusion injuries and facilitates normalization of their structural and functional conditions. Infusion of exogenous Prx6 increases the survival rate of experimental animals by almost 3 times. Application of exogenous Prx6 can be an effective approach in the prevention and treatment of renal ischemia-reperfusion kidney lesions and in preserving isolated kidneys during transplantation.

[Effect of conditioned medium from mesenchymal stem cells on regeneration of endothelium at HCl-induced damage trachea in rats].

The purpose. Respiratory epithelium regeneration is studied in rats with tracheal damage induced by inhaling hydrochloric acid vapor. Method. Regeneration process after the chemical burn was activated by intratracheal administration of preparations obtained from the same-species mesenchymal stem cells (MSC). Results. Tracheal epithelium is shown to recover almost completely on day 3-7 after applying MSC compositions (MSCs). Closed structures containing ciliated cells similar to ciliated cells of the respiratory epithelium lining the trachea are formed in the submucosal epithelium during regeneration. These structures migrate towards epithelium and get incorporated into the damaged epithelium. This phenomenon is apparently indicative of the special mechanism of respiratory epithelium regeneration after HCl-induced injury. Conclusion. It is demonstrated in this study that cell-free MSCs instilled intratracheally promote the recovery of normal submucosal epithelium by either preventing or reducing necrosis and inflammation. Such topical MSCs administration significantly accelerates migration of ciliated cell towards the surface and de novo formation of the ciliary epithelium.

Nephroprotective Effect Exogenous Antioxidant Enzymes during Ischemia/Reperfusion-Induced Damage of Renal Tissue.

Nephroprotective effect of exogenous chimeric antioxidant enzyme with combined superoxide dismutase and peroxide activities (PSH protein) was studied on the model of ischemia/reperfusion damage of the renal tissue. It was shown that post-ischemic (25- and 45-min ischemia) intravenous administration of PSH protein significantly normalized the levels of creatinine and urea. Histological studies showed that as distinct from ischemic kidney, the structure of renal corpuscles and tubules remained unchanged, the number of atrophied glomeruli and glomeruli with exudates and protein inclusions decreased in the capsular teeth after postischemic intravenous administration of PSH protein. Immunohistochemical investigations showed that post-ischemic intravenous injection of PSH protein significantly reduced the intensity of apoptosis in ischemic renal tissues.

Protective Effect of Peroxiredoxin 6 in Ischemia/Reperfusion-Induced Damage of Small Intestine.

BACKGROUND: Strong oxidative stress starting in the epithelium upon restoration of blood cell circulation is a major cause of necrosis of the intestinal epithelium in ischemia/reperfusion-induced damage. AIM: The purpose of this study was to investigate the tissue-protective effect of exogenous peroxiredoxin 6 (Prx6) in ischemia/reperfusion-induced damage of small intestine. METHODS: The research was carried out using a model of acute superior mesenteric artery occlusion in Wistar male rats. Exogenous Prx6 was administrated intravenously 15 min prior to small intestine ischemia. The distribution of endogenous Prx6 in the small intestine was determined by immunohistochemical analysis. The expression level of antioxidant enzymes was evaluated by RT-PCR in real time. RESULTS: Exogenous Prx6 injected to animals intravenously was detected in blood vessel lumens, and its diffuse distribution was subsequently confirmed in the intestinal epithelium. Expression analysis of genes coding for major antioxidant enzymes demonstrated a significant activation of SOD 1, SOD 3, Prx6, GPx2, GPx7 expression during I/R-induced damage of the small intestine. Injection of exogenous Prx6 prior to induced ischemia resulted in minimization of oxidative injury by reducing necrosis and apoptosis, by normalization of gene activity of antioxidant enzyme. It eventually led to a reduction of epithelium destruction in the small intestine. By contrast, administration of a purified mutant form of Prx6 (Prx6C47S) without peroxidase activity had no protective effect. CONCLUSION: The application of exogenous Prx6 enables normalization of the antioxidant status of the small intestine and reduction of cell destruction upon I/R-induced organ damage.

Effect of Conditioned Medium and Bone Marrow Stem Cell Lysate on the Course of Acetaminophen-Induced Liver Failure.

A composition containing culture medium conditioned by mesenchymal stem cells and mesenchymal stem cell lysate improves biochemical parameters, reduces inflammation, and stimulates regenerative processes in the liver.

[The ototoxic action of cisplatin on the middle ear].

The objective of the present study was to elucidate the mechanisms underlying the ototoxic action of cisplatin after its single administration using an experimental model (white mice). Short latency acoustically evoked potentials (SAEP) and distortion-product otoacoustic emissions (DPOAE) were recorded for the purpose. The results of the study confirm the possibility of using the proposed model for the estimation of the cisplatin-induced loss of hearing. It was shown that a single cisplatin dose causes hearing impairment in the mice within 7 days after its administration; with the maximum effect becoming apparent on day 30.

[Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)].

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 hours 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 adsorpted 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 hours.

[Use of perfluorocarbon emulsions for administration of photosensitizing preparations into bone marrow stem cells].

It has been shown that, upon incubation of mouse bone marrow stem cells (BMSC) in vitro with the nanoparticles of perfluorocarbon (PFC) emulsion stabilized by proxanol 268, these nanoparticles penetrate into cells and stay there for a long time (up to 20 days of observation). It has been found that, under in vitro conditions, mouse BMSC loaded with the nanoparticles of both the original emulsion and the emulsion preliminarily incubated with radachlorine do not differ from control stem cells in the rate of division, stretching on a plastic support, and the formation of a monolayer. It has been shown that the exposure to laser radiation of BMSC incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine under in vitro conditions leads to the death of these cells due to the destruction of the cell membrane. The treatment with laser radiation of BMSC incubated with the nanoparticles of the starting PFC emulsion (without preliminarily incubation with radachlorine) causes no death of these cells. It has been shown in in vivo experiments that, when transplanted to the organism of a recipient mouse, BMSC of a donor mouse incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine retain their functional activity, in particular the ability to migrate in the animal body. In this case, radachlorine contained in these stem cells retains its major function, to induce the death of stem cells by the action of laser radiation due to the destruction of the cell membrane. The observation period after the transplantation was 5-7 days.

[Improvement of myocardial remodelling and functioning in patients undergoing surgical treatment of chronic cardiac insufficiency by two-staged elevation of the immunoregulatory reserve of autological bone marrow cells and their intramyocardial application].

The aim of the investigation was to study a possibility to improve left ventricular (LV) volume and function remodelling in patients with chronic cardiac insufficiency (CCI) by means oftwo-stage bone marrow cell (BMC) activation: first in vivo and then ex vivo within the process of their cultivation. Two groups of CCI patients were recruited. Group 1 (controls) consisted of 50 patients undergoing conventional aorto-coronary bypass surgery (ACBS). Group 2 consisted of 57 patients injected with 200 million autological mononuclear BMC intramyocardially during ACBS. In Group 2 patients, the severity of immune dysregulation was assessed initially using blood leukocyte stimulation index (SI) values. Sixteen patients with SI >1 and moderate immunographic alterations were considered to have a favorable prognosis for BMC treatment; 41 subjects with SI <1 and pronounced immunographic abnormalities were regarded as having an unfavorable prognosis for BMC application. Twenty-two patients with SI <1 were administered a preliminary immunocorrective course (in vivo BMC activation). Mononuclear BMC obtained from 38 patients with SI >1 and 19 patients with SI <1 were cultured for 5 to 7 days (ex vivo BMC activation). Positive changes in BMC phenotypical pattern were observed only in patients with SI >1: CD3, CD4, CD8, CD25, and some other measurements increased. Significant positive effects on LV function parameters and Duke Activity Status Index (DASI) values were revealed in patients with SI >1 six months after BMC administration. In vivo immunocorrection in combination with subsequent ex vivo BMC activation (n=38) promoted significant improvements in LV volume characteristics 6 months after ACBS vs. the controls (ACBS without BMC, n=50). In conclusion, to make the administration of autological BMC in CCI patients effective, two-staged BMC activation should be performed: in vivo activation with immunocorrectors should be followed by ex vivo activation of cultured cells.