Restoration of Parkinson's Disease-Like Deficits by Activating Autophagy through mTOR-Dependent and mTOR-Independent Mechanisms in Pharmacological and Transgenic Models of Parkinson's Disease in Mice.

We studied the possibilities of inhibition of neurodegeneration in MPTP-induced model of Parkinson's disease (PD) in C57Bl/6J mice and transgenic model of early PD stage (5-monthold B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J mice) by autophagy activation through mTOR-dependent and mTOR-independent pathways with rapamycin and trehalose, respectively. Therapy with autophagy inducers in a "postponed" mode (7 days after MPTP intoxication) restored the expression of the dopaminergic neuron marker tyrosine hydroxylase and markedly improved cognitive function in the conditioned passive avoidance response (CPAR; fear memory). The transgenic model also showed an increase in the expression of tyrosine hydroxylase in the nigrostriatal system of the brain. An enhanced therapeutic effect of the combined treatment with the drugs was revealed on the expression of tyrosine hydroxylase, but not in the CPAR test. Thus, activation of both pathways of autophagy regulation in PD models with weakened neuroinflammation can restore the dopaminergic function of neurons and cognitive activity in mice.

Hypolipidemic Effect of Mannan in Mice with Acute Lipemia Induced by Poloxamer 407.

We studied biological effects of mannan, a polysaccharide immunomodulator from C. albicans, that interacts with mannose receptor in vivo. It is shown that preliminary administration of mannan (5 times in a dose of 50 mg/kg or 2 times in a dose of 100 mg/kg) to mice with acute lipemia induced by lipase inhibitor poloxamer 407 (300 mg/kg) reduces the serum concentrations of atherogenic LDL, cholesterol, and triglycerides. Administration of mannan to intact mice and animals with acute lipemia reduces triglyceride concentration and causes labilization of lysosomal membranes in the liver. Serum activity of chitotriosidase, a marker of macrophage activation, was elevated in mice with acute lipemia treated with mannan. Thus, mannan from C. albicans is a promising hypolipidemic polysaccharide compound, similar by its activity to ß-glycan, a component of LPS.

Depression of Macrophages Modifies Serum Lipid Profile in Hyperlipidemia.

Suppression of functional activity of macrophages by gadolinium chloride, suppressing the macrophage population and the endocytosis velocity, was studied in vivo. Injection of GdCl3 led to an increase in serum cholesterol concentration. Preliminary injection of GdCl3 to mice with lipidemia 24 h before poloxamer 407 reduced the concentrations of triglycerides and LDL during marked depression of macrophages (in 24 h). Macrophage repopulation (days 5, 7) was associated with development of a trend to an increase of triglyceride and LDL levels. lectron microscopic study of Kupffer cells after injection of poloxamer 407 and its combination with gadoliniun chloride detected the intralysosomal accumulation syndrome in these cells (formation of auto- and heterophagolysosomes). Activity of cathepsin B, characteristic of macrophages, reduced 24 h after injections of GdCl3 and poloxamer 407 alone and restored in response to their combination.

[A Therapeutic Target for Inhibition of Neurodegeneration: Autophagy].

The role of autophagy in cell survival and suppression of neurodegeneration was considered. We discussed its involvement in Alzheimer's, Parkinson's, and Huntington's diseases connected with accumulation of amy- loid-ß, α-synuclein, and huntingtin, respectively. Autophagy is reduced in these diseases and in aging as well to various extent. Elimination of accumulated toxic proteins and structures is performed by autophagy mech- anisms (chaperon-mediated autophagy, macroautophagy, selected autophagy) in an interaction with ubiqui- tin-proteasome system. In many cases activation of mTOR-dependent autophagy and mTOR-independent regulatory pathways lead to the therapeutic effect of inhibition of neurodegeneration in cell cultures and an- imal models. Some autophagy enhancers such as resveratrol, metformin, rilmenidine, lithium, and curcumin are tested now in clinical trials.

[EXTRACELLULAR VESICLES: INTERCELLULAR INFORMATION FLOW AND MEDICAL APPLICATIONS].

The major features of extracellular vesicles secreted by mammalian cells are considered. Cell activation caused by formation of pathology stimulates the secretion acutely. The vesicles (exosomes, microvesicles) are enriched with annexin V, tetraspanin, miRNA. Exosomes are enriched especially by integrins, heat shock proteins. Microvesicles contain elevated amounts of tissue factors, phosphatidylserine, mRNA. The vesicles carry information about the pathological process, and microvesicles contain more proteins characteristic of inflammation and death than exosomes. They are important mediators of inflammation and infection in the body, have different effects on the immune system and the processes of carcinogenesis and neurodegeneration. However, antigenic profiles of extracellular vesicles differ not profoundly in various pathologies and so far they help diagnostics limitedly. The vesicles carry signals of genetic reprogramming of the cells and epigenetic stimulation, connected with both protein factors and mRNA and miRNA. Profiles of miRNA vesicles produced by the various pathological sources are studied actively and are useful as indicators of source and stage of cancer. Some ways of therapeutic use of the vesicles are also considered.

[Reparative autophagy and autophagy death of cells. Functional and regulatory aspects].

Molecular regulation of reparative/homeostatic autophagy induced by failure of vital resources and by cellular stress is considered. Extensive autophagy regulatory apparatus responds to starvation, insufficiency of growth factors and energy supply, accumulation of unfolded proteins (ER stress), reactive oxygen species, microbial invasion. Central sensor of the regulation is kinase mTOR. Part of the mTOR pool presented in lysosomes responds to the local level of amino acids and is able to induce autophagy under low rates of intralysosomal proteolysis. Autophagy is a self-regulated cell process, the peak of autophagy is followed by its regulatory weakening by amino acids formed in autophagolysosomes. Protective effect of autophagy is associated mainly with removal of permeabilized mitochondria generating ROS, and elimination of abnormally folded proteins. Autophagy has optimum of its activity: its deficiency leads to accelerated cellular aging, and the excessive autophagy brings to the deficiency of cellular survival resources and cell death. Autophagy cell death seems like a hyper-stimulated self-eating of the cell, but more probably that excessive autophagy disturbs cellular energy supply and switches on some specific cell death signalization (possibly associated with kinases c-Jun, DRP-1, PI3K class I etc.). Some approaches to use reparative autophagy for prevention of cell degeneration are considered.

[Lysosomal membrane permeabilization as apoptogenic factor].

Lysosomal membrane labilizing agents (incl. proapoptotic proteins of Bcl-2 family, LAPF, p53), estimation of lysosomal membrane permeabilization in living cells, the new data on differential permeabilization of lysosomal membranes, membrane stabilizing factors (incl. Hsp70), relations between lysosomal membrane damage, and initiation of apoptosis were considered. Signal effect of lysosomal membrane permeabilization is caused preferentially by release of cathepsin B and D in cytosol. Subsequent numerous pathways of apoptogenic signalization include proteolytic attack/activation on signal cytosolic proteins, mitochondria, procaspases, cell nuclei. The mainstream of the cell damage is connected with activation pf proapoptotic Bid and Bax, leading to permeabilization of the outer mitochondrial membrane, release of cytochrome c into cytosol and activation of caspase cascade. Translocation of the lysosoma enzymes in cytosol is capable to induce both the caspase-dependent and caspase-independent paths of apoptosis.

[Multifactorial evaluation of endogenous intoxication in patients with chronic viral hepatitis C].

The integral laboratory indices of endogenous intoxication (EI) (effective albumin concentration (EAC) and albumin binding reserve (ABR)) were studied in 104 patients with chronic hepatitis C (CHC) in the context of data on the current manifestations of the disease and the pattern of and association between different types of somatic pathology and toxic exposures. The patients with CHC were found to have significant changes in EI indices as compared with the control group wherein the indices were steady-state during conventional pathogenetic therapy. There was no clear correlation between the clinical symptoms of intoxication and EAC and ABR. More considerable changes were ascertained in CHC patients with degenerative diseases, biliary system diseases, and chronic constipation and in those with the signs of negative changes in the body's responsiveness.

[Effect of whole-body controlled hyperthermia on lipid peroxidation products and lysosomal enzyme levels in blood serum from Wistar rats with Walker-256 carcinosarcoma].

Whole-body controlled hyperthermia (up to 43.5 degrees C) of Wistar rats with Walker-256 carcinosarcoma was followed by symptoms of enhanced lipid peroxidation for 3-14 days and release of lysosomal enzymes into blood on day 14. Our data, in totality, point to a potential to stimulate tumor cell apoptosis.

[Serum albumin binding capacity values in the clinical evaluation of endogenous intoxication in acute viral hepatitis].

The fluorescence technique was used to determine the effective albumin concentration (EAC) and albumin binding reserve (ABR) in 130 patients with acute viral hepatitis A and B in order to establish their values in the evaluation of endogenous intoxication (EI). The most considerable EAC and ABR reduction was found in patients with previous comorbidity and with negative responsiveness changes underlying a high baseline EI level. Despite the resolution of the clinical symptoms of intoxication, the long existence of EAC and ABR changes in the course of the disease in these patients is associated with the tension of the body's key systems in impaired somatic regulation and determines the increased consumption of defense reserves and their rapid exhaustion. Thus, the informative value of the study biochemical parameters of EI increased within the multifactor system of its clinical evaluation.

Effect of benz(a)pyrene and constant light exposure on rat liver lysosomes and biliary excretion of lysosomal enzymes.

Threefold administration of benz(a)pyrene in a dose of 20 mg/kg markedly stimulated biliary excretion of lysosomal enzymes from rat liver without signs of lysosomal damage. Constant light exposure induced changes attesting to functional activation of the lysosomal apparatus in liver cells and inhibited constitutive biliary excretion of lysosomal enzymes. Combined treatment decreased, but not abolished the stimulatory effect of benz(a)pyrene on vesicular transport of lysosomal enzymes to the bile.

Effect of transitory ischemia on liver lysosomal apparatus in rats with different resistance to hypoxia.

We studied the state of lysosomal apparatus and pro- and antioxidant activity in the liver of rats with different resistance to hypoxia during postischemic recovery. Under normal conditions the lysosomal apparatus did not differ in highly and low resistant animals. During ischemia and reperfusion the damage to hepatic lysosomal membranes in rats highly resistant to hypoxia was less pronounced than in low resistant animals. These differences also concerned labilization of lysosomes during exposure to damaging factors (hypotonia and Triton X-100). The rats highly resistant to hypoxia differed from low resistant animals by higher stability of lysosomal membranes, lower prooxidant activity (malonic dialdehyde content), and higher tissue concentration of alpha-tocopherol during reperfusion.

Sucrose-stimulated release of FITC-dextran into the bile in the dynamics of its storage and elimination from the liver.

Stimulation of release of FITC-dextran endocytosed in the liver by an injection of hypertonic sucrose led to the appearance of a less pronounced and prolonged peak (with the maximum at min 15-25) at later terms after injection of the marker (30 days). Biliary release of FITC-dextran was 4-9-fold accelerated on days 1-14 in comparison with the constitutive release, and after 30 days it was accelerated 19-fold, which reflects high capacity of the long storage compartment to stimulated release of FITC-dextran into the bile.

Protective effect of enterosgel on rat liver lysosomes during cytostatic treatment.

Polychemotherapy with a complex of cytostatics (cyclophosphamide, doxorubicin, vincristine, prednisolone) induces progressive damage to hepatocyte membranes, which manifested in labilization of lysosomes and activation of lysosomal enzymes and serum transaminases. Enterosgel stabilized liver lysosomes and reduced manifestation of hepatocyte cytolysis.

[Examining fibrotic process in lung during treatment of chronic murine tuberculosis with lysosomotropic drug isonaizid]. / Ussledovanie protsessov fibrozirovaniia v legkikh pri lechenii lizosomotropnym preparatom izoniazida khronicheskogo tuberkuleza u myshei.

Light and ultrastructural morphometries were used to evaluate the effects of dextran-conjugated isoniazid (MW 30-40 kDa) on the morphology of chronic pulmonary tuberculous inflammation induced by BCG vaccine in mice. The revealed antifibrotic effect of the conjugated drug is accompanied by much less damage to the alveolar macrophageal ultrastructures (the mitochondrial apparatus in particular) than that of free isoniazid. It is concluded that the lysosomotropic properties imparted to isoniazid basically change its effect on fibrogenesis and are essential in the prevention of antiinflammatory pneumoscleroses.

[Elimination of asialoorosomucoid and its catabolism in liver of rats with toxic CCl4-induced cirrhosis]. / Vyvedenie iz krovi i katabolizm asialoorozomukoida v pecheni krys s toksicheskim CCl4-tsirrozom.

Human 125I-acyaloorosomucoid was intravenously injected to male Wistar rats. Hepatic cirrhosis was induced by oily CCl4 solution (0.04 ml toxin per 100 g body weight, twice a week for 2 months). Cirrhosis was found not to prevent blood elimination of 125I-acyaloorosomucoid (t1/2 = 2.53 versus 2.50 in the control). Ten minutes after injection, there was 75.2% of the administered radioactive dose in the liver versus 77.1% in the control. The 60-minute hepatic elimination of substrate in rats with cirrhosis was even more intensive than in the control animals, which is in agreement with the high levels of acid-soluble radioactivity (for 10 minutes). By large, CCl4-induced hepatic cirrhosis did not lead to disturbances of the hepatocytic function aimed at endocytosis and catabolism of acyaloorosomucoid.

[Adverse effects of Ferrum Lec (clinico-experimental study)]. / Ob otritsatel'nykh éffektakh preparata Ferrum Lek (kliniko- éksperimental'noe issledovanie).

Summation of negative effects of hypoxia and lysosome-tropic properties of Ferrum Lec was observed in patients with severe stage of iron-deficiency anemia (IDA), and in rabbits with experimental IDA, during treatment by intravenous injections of Ferrum Lec. These effects were expressed in exageration of the destabilizing action of hypoxia on lysosome membranes, and attended by a significant rise in the lysosomal enzyme levels of blood plasma, by the development of dystrophic processes in hepatocytes, and by a decrease in the number of sinusoidal cells. All these facts should be considered in clinical practice.

[A stable reagent for the-single stage determination of inorganic phosphate]. / Stabil'nyi reaktiv dlia odnostadiinogo opredeleniia neorganicheskogo fosfata.

A recipe of a simple reagent for phosphorus detection has been developed, consisting of ammonium molybdate (4 mM), sulfuric acid (0.2 N), and Tween-80 (0.2%). The developing phosphate staining may be registered in 15 min at a wavelength of 350 nm. The product molar extinction is equal to 1.20.10(4) M-1.cm-1, this being close to that of molybdic blue. Phosphate staining is characterized by the stability of results and insensitivity to the presence of a number of substances used in enzymology. The prepared reagent is fit for experiments within a fortnight if stored in the cold.

[The effect of single and repeated administration of chloroquine on the activity of lysosomal proteinases in rat liver cells]. / Effekt odnokratnogo i povtornogo vvedeniia khlorokhina na aktivnost' proteinaz lizosom kletok pecheni krys.

Effects of single and repeated injections of lysosomotropic agent chloroquine on lysosomal proteolytic activity and physico-chemical properties of rat liver lysosomes have been studied. Chloroquine was administered intraperitoneally to rats at a dose of 30 mg/kg of body mass. Osmotic properties, lysosomal enzymes activity and functional state of the system of mononuclear phagocytes were estimated. No alterations of colloid carbon clearance followed by a single dose of chloroquine administration were noted. Distinct alterations in osmotic properties, weak labilization of lysosomes and an increase in acid hydrolases activity were similar after single and/or repeated chloroquine administrations, whereas activation of cysteine proteinases and cathepsin D were most pronounced. Chloroquine accumulation by rat liver cells proved to be similar, but the drug excretion was longer after repeated injections. The lysosomal disorders noted were similar to those symptoms of lysosomal storage disease.

[Effective extraction of lysosomal enzymes with digitonin]. / Effektivnaia ékstraktsiia lisosomal'nykh fermentov s pomoshch'iu digitonina.

A method for rapid and effective extraction of rat liver lysosomal enzymes has been elaborated. It includes isolation of lysosomal-mitochondrial fraction by means of differential centrifugation, selective destruction of the lysosomal membrane by digitonin and centrifugal obtaining of the lysosomal matrix. Total labilization of the lysosomal membrane is achieved at 0.3 mM of the detergent. The maximal enrichment of an extract by lysosomal enzymes is observed in the range of 0.3-0.4 mM of digitonin. The level of lysosomal enzyme purification is 30.7 for cysteine cathepsins B, L, H, 24.9- for beta-galactosidase, 14.1- for acid phosphatase. The method gives high yield of lysosomal enzymes (40-80%).