[The Effects of the Hydrogen Sulfide Donor GYY4137 on the Proteasome Pool of Colorectal Cancer Cells].

Cancer cells are characterized by an increased level of metabolism and are highly dependent on the correct functioning of the processes that ensure homeostasis. Reactive sulfur species (RSS) are important molecular modulators of metabolic processes in both healthy and tumor cells. The effect of RSS and, in particular, H2S, on key cellular systems, including the ubiquitin-proteasome system (UPS), which provides the destruction of most intracellular proteins, has been shown. The main components of the UPS are proteasomes, multisubunit protein complexes, within which proteolysis occurs. At the same time, data on the effect of H2S directly on the pool of proteasomes in tumor cells are insufficient. Here, we studied the effect of incubation of SW620B8-mCherry colorectal adenocarcinoma cells expressing a fluorescently labeled proteasome subunit with 50, 100, and 200 µM of the hydrogen sulfide donor GYY4137. The effect of the substance on the proteasome pool was assessed 6, 24, 48, and 72 h after administration. It was shown that the chymotrypsin-like and caspase-like proteasome activity decreases in cells incubated with 200 µM of the GYY4137 for 24 h. This coincided with an increase in the expression of proteasome subunit genes. In lysates of cells incubated with 200 µM GYY4137 for 48 h an increase in the content of the constitutive ß5 subunit was observed and the activity of proteasomes leveled off. Following prolonged incubation with GYY4137 (72h), an increase in the expression levels of some proteasome genes was also observed, although this did not have a significant effect on the activity and subunit composition of proteasomes. Thus, the obtained data indicate the modulation of proteasome activity by the hydrogen sulfide donor and the effect of GYY4137 on transcription and translation of proteasome genes.

[Changes in the Activities and Contents of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice].

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer's disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune ß-subunits (by 2.8 folds, ß1i; 2.2 folds, ß2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.

[Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging].

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the ß5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the ß5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit ß5; increased expression of genes encoding immune proteasome subunits ß5i and ß1i. In tissue homogenates of aged mice, an increase in the content of immune subunits ß1i and ß2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the ß5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the ß5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

[Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts].

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically ß1, ß2, and ß5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits ß1, ß2, and ß5 can be substituted by the immune ßli, ß2i, and ß5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

[The contribution of M. N. Akhutin in field surgery (to the 75th anniversary of Victory in the Great Patriotic war)].

The article is devoted to M. N. Akhutin (1899-1848), one of the founders of the Russian military field surgery, lieutenant general of the medical service, professor, corresponding member of the USSR Academy of medical Sciences, chief of the Military medical Academy (1940-1941). M. N. Akhutin, disciple of V. A. Oppel, was active participant in the battles at the lake Hasan and the Khalkhin-Gol river (1838-1939). During the Great Patriotic War, he was the chief surgeon of the Bryansk, 2nd Baltic and 1st Ukrainian fronts. Since 1945 he was deputy chief surgeon of the Soviet Army. He is author of works on military field surgery. He was the first director of the A. V. Vishnevsky institute of surgery and the head of the department of faculty surgery of the I. M. Sechenov 1st Moscow medical Institute.

H2S counteracts proinflammatory effects of LPS through modulation of multiple pathways in human cells.

BACKGROUND: Hydrogen sulfide donors reduce inflammatory signaling in vitro and in vivo. The biological effect mediated by H2S donors depends on the kinetics of the gas release from the donor molecule. However, the molecular mechanisms of H2S-induced immunomodulation were poorly addressed. Here, we studied the effect of two different hydrogen sulfide (H2S)-producing agents on the generation of the LPS-induced inflammatory mediators. Importantly, we investigated the transcriptomic changes that take place in human cells after the LPS challenge, combined with the pretreatment with a slow-releasing H2S donor-GYY4137. METHODS: We investigated the effects of GYY4137 and sodium hydrosulfide on the release of proinflammatory molecules such as ROS, NO and TNF-α from LPS-treated human SH-SY5Y neuroblastoma and the THP-1 promonocytic cell lines. Transcriptomic and RT-qPCR studies using THP-1 cells were performed to monitor the effects of the GYY4137 on multiple signaling pathways, including various immune-related and proinflammatory genes after combined action of LPS and GYY4137. RESULTS: The GYY4137 and sodium hydrosulfide differed in the ability to reduce the production of the LPS-evoked proinflammatory mediators. The pre-treatment with GYY4137 resulted in a drastic down-regulation of many TNF-α effectors that are induced by LPS treatment in THP-1 cells. Furthermore, GYY4137 pretreatment of LPS-exposed cells ameliorates the LPS-mediated induction of multiple pro-inflammatory genes and decreases expression of immunoproteasome genes. Besides, in these experiments we detected the up-regulation of several important pathways that are inhibited by LPS. CONCLUSION: Based on the obtained results we believe that our transcriptomic analysis significantly contributes to the understanding of the molecular mechanisms of anti-inflammatory and cytoprotective activity of hydrogen sulfide donors, and highlights their potential against LPS challenges and other forms of inflammation.

Thermodynamic properties and lattice dynamics investigation of LuB2C: experiment and ab initio calculations.

A sample of lutetium carboboride LuB2C was synthesized from a mixture of lutetium hydride, boron and carbon by annealing in argon. The temperature dependence of the heat capacity Cp(T) (2-300 K) and lattice parameters a(T), b(T), and c(T) (5-300 K) of the carboboride was experimentally determined. The experimental values of the heat capacity were fitted with the approximation Cp(T) = aT + ΣCD + CE + CTLS(T). Here the first term is the electronic contribution, the second is the sum of the Debye components, the third is the Einstein contribution, and the fourth is the contribution to the heat capacity due to the vibrations of the two-level systems which are formed in the Lu-subsystem due to the asymmetry of the B-C atomic arrangement around the Lu3+-ions and, as a consequence, the possible transition of the lutetium atoms between spatially close, but energetically non-equivalent positions. A strong anisotropy of the thermal expansion of the carboboride was revealed. Along the c axis the coefficient of thermal expansion monotonically increases; in the basal plane, the expansion is practically not observed. The temperature dependence of the unit cell volume Vu(T) has been analyzed in the Debye-Einstein approximation taking into account the electronic contribution and effect of two-level systems. The values of the Gruneisen parameters corresponding to different modes of the phonon spectrum of the carboboride have been determined. The frequencies of the lattice vibrations, determined in a Raman scattering experiment, are in satisfactory agreement with the parameters obtained from Cp(T) using the Debye-Einstein approximation. Using ab initio band theory methods and an exchange-correlation functional in the PBE form in the VASP package, it was established that the total energies of these two crystal structures differ by no more than 0.01 eV f.u.-1. Calculations of the thermodynamic properties of LuB2C yielded similar results for orthorhombic and tetragonal phases of the carboboride.

[Dynamics of the Functional Activity and Expression of Proteasome Subunits during Cellular Adaptation to Heat Shock].

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.

Psychometric properties of the Russian version of the Pediatric Daytime Sleepiness Scale (PDSS).

Insufficient sleep could severely impair both cognitive and learning skills. More prominent changes are found in children and adolescents. Tools used to estimate sleepiness in the adult population are commonly inappropriate for children. The objective of our study was to provide a reliable instrument to measure excessive sleepiness for upcoming studies in Russian-speaking children, applying the Russian version of Pediatric Daytime Sleepiness Scale (PDSS). The following tasks were resolved in our study: translation, validation, and analysis of psychometric properties of the Russian adaptation of the PDSS by standard tests. After the semantic validation of the instrument through a multi-stage translation process we checked its psychometric validation. A total of 552 students, consisting of N = 285 for the exploratory factor analysis (EFA), N = 267 for the confirmatory factor analysis (CFA) and N = 204 for test-retest analysis of public elementary schools located in Northern Russia completed the PDSS and Munich Chronotype Questionnaire to estimate sleep parameters in the classroom during the lessons. Response rate was 90%; excluded cases contained no data. Further, 204 of our participants completed the PDSS in a 3 months interval to check the test-retest reliability. Internal consistency was measured by Cronbach's alpha coefficients and CFA was used to test factorial validity of the tool. Concurrent validity and test-retest reliability were assessed via intra-class coefficient. Internal consistency of the PDSS scale was high (Cronbach's α = 0.8). The construct validity of the PDSS was supported by CFA (factor loadings were from 0.438 to 0.727) and the test-retest reliability demonstrated by the intra-class coefficient was 0.70. The total PDSS score was independent of sex. The mean total value of PDSS was 11.95 ± 6.24. Higher scores on PDSS were negatively correlated with sleep duration. Thus, the construct validity of the instrument remains valid and could be used for Russian-speaking youth samples in the evaluation of daytime sleepiness. It could be useful in future applications by sleep scientists and health practitioners.

[Circadian rhythms of antioxidant enzyme's activity in young and adult rats under light deprivation conditions.]

We studied the age-related features of circadian rhythms of superoxide dismutase (SOD) and catalase activity in the liver of rats under light deprivation. In standard light conditions (LD), significant daily fluctuations in SOD activity with a maximum at 07:00 am was detected only in young animals (1,5 months) but catalase activity was observed in young (1,5 months) and in adults (7,5 months) with peak at 04:00 am. The daily dynamics of total and specific activity of SOD and catalase in the liver in young and adult rats differed depending on the period of ontogeny in which the impact of light deprivation began. When the females and offspring were moved to darkness after birth (group LD/DD), the circadian rhythms of SOD and catalase activitys were observed in young and were absent in adult rats. However, circadian rhythms of the antioxidant enzymes (AOE) activities were inherent only adult rats when light deprivation impacted on pregnant females (group DD/DD). Changes in circadian rhythms under light deprivation were characterized either by a phase shift of the enzymes activity (in the LD/DD group) or by a violation of their develpoment in ontogeny (in the DD/DD group). During aging significant decreasing of catalase activity was compensated by an increase in the amplitude of circadian rhythms of activity of this enzyme in animals of all groups. A distinctive feature of daily fluctuations in AOЕ activity in young rats in LD and LD/DD groups can be considered the presence of an ultradian rhythm in the general circadian cycle, which had a second peak with a smaller amplitude and shorter period.

Video-tactile pneumatic sensor for soft tissue elastic modulus estimation.

BACKGROUND: A new sensor for estimating elasticity of soft tissues such as a liver was developed for minimally invasive surgery application. METHODS: By measuring deformation and adjusting internal pressure of the pneumatic sensor head, the sensor can be used to do palpation (indentation) of tissues with wide range of stiffness. A video camera installed within the sensor shell is used to register the radius of the contact area. Based on finite element model simulations and the measured data, elastic modulus of the indented soft tissue can be calculated. RESULTS AND CONCLUSIONS: Three phantom materials, namely plastic, silicone and gelatin, with varied stiffness were tested. The experimental results demonstrated that the new sensor can obtain highly reliable data with error less than 5%. The new sensor might be served as an instrument in laparoscopic surgery for diagnosis of pathological tissues or internal organs.

[Heat-shock protein HSP70 decreases activity of proteasomes in human neuroblastoma cells treated by amyloid-beta 1-42 with isomerized Asp7].

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer's disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-ß (Aß42) and its more toxic form with isomerized Asp7 (isoAß42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAß42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAß42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aß peptides, proteasomes, and HSP70.

[Role of military medicine in delivering of health care to ministry of internal affairs staff].

Role of military medicine in delivering of health care to ministry of internal affairs staff. The article describes the creation of the Medical Service Ministry of Internal Affairs of the Russian system. Throughout its history, medical-mechanical maintenance of employees of law enforcement bodies, such as in pre-revolutionary period, and in the early years of the Soviet regime was closely linked with military medicine of the Russian Empire and the Red Army. Many police officers, and later the police received medical care in military hospitals. Start the NKVD- MVD departmental health laid in 1921 with the creation of oadmissionso to provide medical care to police officers. As part of the system of state-sensible care, the medical service of the Russian Interior Ministry system and now introduces non-assessable contribution to its development.

Effects of Geroprotectors on Age-Related Changes in Proteolytic Digestive Enzyme Activities at Different Lighting Conditions.

We studied the effect of melatonin and epithalon on age-related changes in proteolytic digestive enzyme activity in the pancreas and gastric mucosa of rats kept under different lighting conditions. In rats kept under standard illumination, pepsin activity and the total proteolytic activity in the stomach and pancreas increased by the age of 12 months, but then decreased. Constant and natural lighting disturbed the age dynamics of proteolytic digestive enzyme activity. Administration of melatonin and epithalon to animals exposed to constant lighting restored age dynamics of pepsin activity and little affected total proteolytic activity.

The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal.

[For fortieth anniversary of organization of national narcological service].

The 1975 is a year of organization in the USSR of independent service with the competence to regulate problems of alcoholism, drug addiction and toxicomania. The narcological dispensary, public narcological posts and night-time preventoriums were organized. The persons with chronic alcoholism were directed to forced treatment for preventive purpose.

[DNA vaccine encoding alpha-fetoprotein with fused ornithine decarboxylase degradation signal significantly suppresses hepatocellular carcinoma growth in mice].

Hepatocellular carcinoma (HCC) is among the most frequent malignancies in humans. HCC therapy is not efficient and the usual outcome is poor. In this regard, novel approaches to treat and prevent HCC are urgently needed. The Alpha-fetoprotein (AFP) is a serological marker of HCC. Recently it has been shown, that the DNA vaccines expressing AFP are capable in generating immune response against AFP. However, both, the immunization procedures and DNA vaccines used before were complex and not always very effective. We have shown that DNA vaccine encoding HIV-1 reverse transcriptase (RT) with fused ornithine decarboxylase (ODC) degradation signal induced a strong Th-1 immune response against RT in mice. Using this approach we designed a set of novel DNA vaccines bearing AFP and ODC degradation signal. Results obtained on transfected cells demonstrated efficient expression and fast proteasomal degradation of the recombinant AFP. The anti-tumor immune response stimulation was shown in immunized animals and most importantly a notable retardation of tumor growth was observed as a result of protective vaccination.

[From the history of health resort treatment in the Armed Forces of the Russian Federation].

The health resort treatment takes an important place in the system of treatment and prophylaxis activities aimed to conservation and restoration of the health of military personnel. It is ninety years since the foundation of the first military health resorts. During this time a unique system of resort treatment in the armed forces of the Russian Federation has been formed and is neatly functioning. It is nowadays one of the component parts of medical support of military personnel, retirees and members of their families.

[Aviation medicine: yesterday, today and tomorrow].

The article describes the history and current state of medical support of the Air Force. On December 1, 2009 Air Force Medical Service was renamed into the service of aviation medicine of Command Air Forces, and many of the functions of medical support of the Air Force transferred to the newly formed Air Force Center of Aviation Medicine. April 29, 2011 office of Aviation Medicine of the High Command of the Air Force has stepped 95-year milestone. The changes affected all structures entrusted with the issues of medical support of the Air Force. Today, the medical service of the Air Force faces challenges--ensuring safety, the study of flight conditions and their impact on health, job performance and psychological characteristics of flight personnel.

[Critical amino acids of ornitin decarboxylase degron: the presence and C-terminal arrangement is insufficient for alfa-fetoprotein degradation].

Mouse ornithine decarboxylase (ODC) degrades in proteasome in an ubiquitin-independent manner with an averagehalf-life of 2 h. The 37 amino acid long C-terminal fragment known as a degradation signal (degron) is responsible for the effective degradation of ODC. Recently, amino acids being critical for degradation in the ODC-degron have been mapped. Mutations of Cys441 and Ala442 led to protein stabilization, while a substitution of other amino acids composing ODC-degron had almost no effect on the protein turnover; whereas insertions or deletions in region between Ala442 and ODC C-terminus diminished greatly rate of protein degradation, e.g. positioning of the key amino acids from the C-terminus was shown to be crucial. Using these data we introduced both key amino acids into the alfa-fetoprotein with truncated exportation signal (deltaAFP), at the same distance from the C-terminus as they being in the ODC (deltaAFPCAG and deltaAFPLCAG). Removal of N-terminal exportation signal prevented secretion of modified proteins. Using in silico approach we demonstrated no significant difference in hydrophobicity or secondary structure between C-terminus of deltaAFP and mutated proteins. The degradation kinetics of deltaAFP, deltaAFPCAG, deltaAFPLCAG in cyloheximide-chase and proteasome inhibition assay (using MG132) was identical. Obtained results suggest that introduced substitutions are insufficient for effective recognition of mutated deltaAFP by26S proteasome. We assume thatadditional amino aci ds composing ODC-degron or their combine action could also affect degradation. Besides that, one cannot exclude that conformation of the mutated deltaAFP limits its C-terminus accessibility to proteasome.