GAPDH binders as potential drugs for the therapy of polyglutamine diseases: Design of a new screening assay

AbstractProteins with long polyglutamine repeats form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti-aggregation compounds with a focus on the aggregation-promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and −(−)deprenyl, and the benzothiazole derivative PGL-135 which exhibits anti-aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia

Pyrrolylquinoxaline-2-one derivative as a potent therapeutic factor for brain trauma rehabilitation

Traumatic brain injury (TBI) often causes massive brain cell death accompanied by the accumulation of toxic factors in interstitial and cerebrospinal fluids. The persistence of the damaged brain area is not transient and may occur within days and weeks. Chaperone Hsp70 is known for its cytoprotective and antiapoptotic activity, and thus, a therapeutic approach based on chemically induced Hsp70 expression may become a promising approach to lower post-traumatic complications. To simulate the processes of secondary damage, we used an animal model of TBI and a cell model based on the cultivation of target cells in the presence of cerebrospinal fluid (CSF) from injured rats. Here we present a novel low molecular weight substance, PQ-29, which induces the synthesis of Hsp70 and empowers the resistance of rat C6 glioma cells to the cytotoxic effect of rat cerebrospinal fluid taken from rats subjected to TBI. In an animal model of TBI, PQ-29 elevated the Hsp70 level in brain cells and significantly slowed the process of the apoptosis in acceptor cells in response to cerebrospinal fluid action. The compound was also shown to rescue the motor function of traumatized rats, thus proving its potential application in rehabilitation therapy after TBI. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.Ministry of Education and Science of the Russian Federation, Minobrnauka: 0124-2019-002Russian Foundation for Basic Research, RFBR: 20-33-70102Russian Science Foundation, RSF: 18-74-10087Funding: This research was funded by Russian Science Foundation, research project #18-74-10087 (V.F.L., E.A.D., M.A.M., E.R.M.), Russian Foundation for Basic Research, research project #20-33-70102 (I.A.U., O.N.C., V.N.C, M.?.T., I.V.G.), and by The Ministry of Education and Science of Russian Federation № 0124-2019-002 (R.V.S., N.D.A., B.A.M.)

Dataset of NMR-Spectra Pyrrolyl- and Indolylazines and Evidence of Their Ability to Induce Heat Shock Genes Expression in Human Neurons

These data are related to our previous paper “Synthesis and approbation of new neuroprotective chemicals of pyrrolyl- and indolylazine classes in a cell model of Alzheimer's disease” (Dutysheva et al., 2021), in which we demonstrate neuroprotective abilities of pyrrolyl- and indolylazines in a cell model of Alzheimer's disease. Using a novel procedure of photocatalysis we have synthesized a group of new compounds. The current article presents nuclear magnetic resonance spectra including heteronuclear single quantum coherence spectra of chemicals synthesized by us. The effect of new compounds have on heat shock proteins genes expression in reprogrammed human neurons are presented. We also presented data that verify neuronal phenotype of reprogrammed cells. © 2021Funding: This work was supported by the Russian Foundation for Basic Research [Grant No. 20–33–70102], and by the Russian Science Foundation [grant number 18–74–10087]

Investigation of Packaging Films with Electret Effect

The possibility of using electret polymeric materials for milk packaging has been investigated. It is noted that the electric field of the packaging material can inhibit the growth of bacteria harmful to dairy production

Тепловой стресс стимулирует секрецию клетками колоректальной карциномы специфической популяции нановезикул с повышенным содержанием БТШ70 и измененным составом микроРНК

Background. Heat stress (HS) induces the cellular secretion of heat shock proteins (HSP ) and extracellular nanovesicles (ENVs). The biological link between these phenomena is poorly understood. In the case of colorectal cancer (CRC) cells, the secretion of HSP s and ENV may be involved in the clinical response to intraperitoneal therapy of peritoneal carcinomatosis.Material and Methods. Established colon cancer cell lines COLO 320, HCT 116, HT29 and DLD 1 were used. ENVs were isolated from culture media by differential ultra-centrifugation and analyzed by dynamic light scattering, nanoparticle tracking analysis, atomic force microscopy and flow cytometry. Super-paramagnetic particles (SPMP ) covered by antibodies to the membrane form of Hsp70 were used for isolation and quantification of Hsp70(+) ENVs. Vesicular microRNA was assayed by RT-qPC R.Results. HS induces the secretion of ENVs by CRC cells, the resistance to HS correlates with the activity of HS-induced ENVs secretion. HS induces the secretion of a specific population of ENVs enriched by membrane form Hsp70 (mHsp70). The microRNA content of mHsp70(+) ENVs has qualitative and quantitative features. The concentration of miR-126-3p, -181-5p, -155-5p, -223 is increased in mHSP 70(+) ENVs secreted by three CRC cell lines.Conclusion. HS induces the secretion of mHSP 70(+) ENVs by CRC cells. This phenomenon may be involved in a clinical response to intraperitoneal chemo-hyperthermic perfusion therapy of peritoneal carcinomatosis.Введение. Температурный стресс стимулирует секрецию клетками белков теплового шока (БТШ) и внеклеточных нановезикул (ВНВ). Биологическая связь между этими явлениями изучена слабо. В случае клеток колоректального рака (КРР) секреция БТШ и ВНВ может участвовать в формировании клинического ответа на внутрибрюшные методы терапии перитонеального карциноматоза.Цель исследования – оценка эффекта теплового шока (ТШ) на способность клеток КРР секретировать ВНВ in vitro, выделение, количественный и качественный анализ популяции ВНВ, мембрана которых содержит БТШ70 (мБТШ70(+) ВНВ), анализ эффекта ТШ на активность секреции мБТШ70(+) ВНВ, оценка состава микроРНК в популяции ТШ-индуцированных ВНВ.Материал и методы. В работе использованы стабильные линии клеток КРР: COLO 320, HCT 116, HT29, DLD 1. Внеклеточные везикулы выделены методом дифференцированного ультрацентрифугирования, для их анализа использованы методы лазерной корреляционной спектроскопии, анализ траекторий наночастиц, атомная силовая микроскопия и проточная цитометрия. Для выделения и количественной оценки БТШ70(+) ВНВ были использованы суперпарамагнитные частицы (СПМЧ), «декорированные» антителами к мембранной форме БТШ70. Анализ микроРНК в составе ВНВ был проведен методом обратной транскрипции и последующей ПЦР.Результаты. Тепловой шок индуцировал секрецию клетками КРР мБТШ70(+)ВНВ, наблюдалась корреляция между резистентностью клеток к ТШ и активностью ТШ-индуцированной секреции ВНВ. Состав микроРНК БТШ70(+)ВНВ имел качественные и количественные особенности. Концентрация miR-126-3p, – 181-5p, -155-5p, -223 была повышена в БТШ70(+)ВНВ, секретируемых тремя линиями клеток КРР.Заключение. Тепловой шок стимулирует секрецию мБТШ70(+)ВНВ клетками КРР. Этот феномен может участвовать в формировании клинического ответа на интраперитонеальную химио-гипертермическую перфузию, проводимую в рамках терапии перитонеального карциноматоза

Simulation of Stress Response in Animals by the Delivery of Heat Shock Protein 70 kDa

Proceedings of the 9th International Multidisciplinary Conference «Stress and Behavior» Saint-Petersburg, Russia, 16–19 May 2005.Heat shock proteins (Hsps) are thought to exert two major functions: proper folding of cellular protein (chaperonic activity) and cytoprotection. Unless known as almost exclusively intracellular protein Hsp70 was shown to persist in extracellular locales and biological fluids of an organism, for example, in blood (Febbraio M.A. et al., 2002) and in saliva (Fabian T.K. et al., 2003). It was shown that the appearance of Hsp70 in circulation in most cases is due to its export from living cells, rather than to the release from dying cells (Hunter-Lavin C. et al., 2004). Extracellular Hsp70 was found also to enter macrophages or endothelial cells and into other cells, for example brain cells. The quantity of Hsp70 in blood increases after an action of different stressors such as heating, exercises. The main possible site of Hsp70 internalization and externalization is cellular membrane lipid rafts, which are sphingolipid-cholesterol-rich structures (Broquet A.H. et al., 2003). Besides the latter Hsp70 can associate with some cellular membrane receptors such as toll-like receptors, CD14 (Asea A et al., 2000), CD40 (Becker T. et al., 2002) and CD91 (Noessner E. et al., 2002). Basing on the data obtained in vitro models we suggest that the main function of exogenous internalized Hsp70 is to protect cells from damaging factors (Tytell M. et al, 1986, Guzhova I.V. et al., 2001). It is also worth-mentioning that Hsp70 after its entry to a cell can modulate the activity of certain cellular proteins (Guzhova I.V. et al., 1997), receptors, and to influence the expression of some genes whose products take part in stress reaction and determine inflammatory status of an organism. The goal of this investigation was to simulate stress response of the whole organism by the delivery of Hsp70 preparation in a brain and to explore how this delivery can influence on key functions of control or subjected to stressful conditions animals.Methods. The preparation of Hsp70 was obtained from the bovine muscle and was consisted of two isoforms: Hsp70 and Hsc70. Hsp70 preparation was injected into normal and stressed males of unbred and Wistar rats in cisterna magna (up to 10 ug), central nucleus of amygdala (up to 1 ug), and in both nostrils (10 ug). To reveal the possible sites of Hsp70 localization the protein was labeled with NHS-ester-Alexa Fluor (Molecular Probes, USA). Control and stressed rats were behaviourally tested. Biological samples were analyzed using biochemical and morphological methods.Results and discussion. First experiments revealed that Hsp70 preparation exerted dose-dependent suppressive effect on exploratory and motor activity of rats, which resembles some stress situation when depression of behavior activity takes place (Andreeva L.I. et al., 2004). Simultaneously, emotionality of animals was found to elevate in rats accepted Hsp70. After the protein (0.2 and 0.5 ug) delivery into cisterna magna the behavior activity in the open field test was not altered even there was a tendency to some activation. When doses of the protein were elevated up to 1–10 ug, dose-dependent inhibitory effect was observed. Hsp70 in a maximal doses caused increased emotionality of rats. High doses of Hsp70 increased in some extent the content of corticosteron in serum 4 h after injections (514 ± 125 nM saline;656 ± 104 and 614 ± 93 nM, 3 and 10 ug of Hsp70 respectively). Repeated intranasal HSP70 administration (once a day, 3 days) also inhibited motor and exploratory activity compared with controls. Corticosteron content in sera of 43 % rats received Hsp70 was significantly lower than in control group (saline) indicating that anti-stress effect of Hsp70 was profound in a half of animals. This effect was also found under double Hsp70 intranasal administration following both psycho-emotional and physical stressors representing «terrorist-hostage» model and characterized by hyper-excitability of animals. The behavior testing showed that the Hsp70 administration led to restriction of excitation after the end of stress treatment. Biochemical analysis demonstrated the decreasing of calcium concentration, urea, creatinine, some intracellular enzymes in serum after applying of exogenous Hsp70. The tissue content of inducible isoform of Hsp70 (stress marker) in brain and in liver of stressed rats was also lower as compared with control stressed animals (saline). The results of experiments with injection of Hsp70 into central nucleus of amigdala («center of emotions») showed that Hsp70 (0.5 and 1 ug) was able to activate self-stimulation in rats. On the contrary, when the protein was applied to rats which were subjected to the social isolation and alcogolization the protein was found to suppress self-stimulation. Preliminary data on localization of exogenously delivered Hsp70 showed that fluorescent label localized to sites proximal to ependimal cells, vessel endothelial cells and to Purkinje cells of cerebellum.Conclusion. Simulation of stress response in a whole organism by Hsp70 administration (a transition from norm to stress) revealed a dose-dependent effect of the protein from slight activation to growing depression of motor and exploratory behavior. Increased emotionality after Hsp70 administration may be explained by interactions of Hsp70 with limbic structures. Hsp70 plays an important role in development of stress reaction. Alterations of motor activity maybe associated with internalization of Hsp70 by motor neurons (Purkinje cells). The study was supported by the Russian Foundation for Basic Research (project no. 03–03-33024).ReferencesAndreeva L.I. et al. Dokl Acad Nauk. 2004; 394 (6): 835–839. Asea A. et al. Cell Stress Chaperones. 2000; 5 (5): 425–431. Becker T. et al. J Cell Biol. 2002; 158 (7): 1277–1285. Broquet A.H. et al. J Biol Chem. 2003; 278 (24): 21601–21606. Fabian T.F. et al. Med Sci Monit. 2003; 9(1): BR62–65. Febbraio M.A. et al. J Physiol 2002; 544(3): 957–961. Guzhova I.V. et al. Cell Stress & Chap. 1997; 2: 132–139.Guzhova I.V. et al. Brain Res. 2001; 914: 66–73. Hunter-Lavin C. et al. Biochem Biophys Res Commun. 2004; 324: 511–517. Noessner E. et al. J Immunol. 2002; 169: 5424–5432. Tytell M. et al. Brain Res. 1986; 363: 161–164

The mTOR Pathway in Pluripotent Stem Cells: Lessons for Understanding Cancer Cell Dormancy

Currently, the success of targeted anticancer therapies largely depends on the correct understanding of the dormant state of cancer cells, since it is increasingly regarded to fuel tumor recurrence. The concept of cancer cell dormancy is often considered as an adaptive response of cancer cells to stress, and, therefore, is limited. It is possible that the cancer dormant state is not a privilege of cancer cells but the same reproductive survival strategy as diapause used by embryonic stem cells (ESCs). Recent advances reveal that high autophagy and mTOR pathway reduction are key mechanisms contributing to dormancy and diapause. ESCs, sharing their main features with cancer stem cells, have a delicate balance between the mTOR pathway and autophagy activity permissive for diapause induction. In this review, we discuss the functioning of the mTOR signaling and autophagy in ESCs in detail that allows us to deepen our understanding of the biology of cancer cell dormancy

Simulation of Stress Response in Animals by the Delivery of Heat Shock Protein 70 kDa

Proceedings of the 9th International Multidisciplinary Conference «Stress and Behavior» Saint-Petersburg, Russia, 16–19 May 2005.Heat shock proteins (Hsps) are thought to exert two major functions: proper folding of cellular protein (chaperonic activity) and cytoprotection. Unless known as almost exclusively intracellular protein Hsp70 was shown to persist in extracellular locales and biological fluids of an organism, for example, in blood (Febbraio M.A. et al., 2002) and in saliva (Fabian T.K. et al., 2003). It was shown that the appearance of Hsp70 in circulation in most cases is due to its export from living cells, rather than to the release from dying cells (Hunter-Lavin C. et al., 2004). Extracellular Hsp70 was found also to enter macrophages or endothelial cells and into other cells, for example brain cells. The quantity of Hsp70 in blood increases after an action of different stressors such as heating, exercises. The main possible site of Hsp70 internalization and externalization is cellular membrane lipid rafts, which are sphingolipid-cholesterol-rich structures (Broquet A.H. et al., 2003). Besides the latter Hsp70 can associate with some cellular membrane receptors such as toll-like receptors, CD14 (Asea A et al., 2000), CD40 (Becker T. et al., 2002) and CD91 (Noessner E. et al., 2002). Basing on the data obtained in vitro models we suggest that the main function of exogenous internalized Hsp70 is to protect cells from damaging factors (Tytell M. et al, 1986, Guzhova I.V. et al., 2001). It is also worth-mentioning that Hsp70 after its entry to a cell can modulate the activity of certain cellular proteins (Guzhova I.V. et al., 1997), receptors, and to influence the expression of some genes whose products take part in stress reaction and determine inflammatory status of an organism. The goal of this investigation was to simulate stress response of the whole organism by the delivery of Hsp70 preparation in a brain and to explore how this delivery can influence on key functions of control or subjected to stressful conditions animals.Methods. The preparation of Hsp70 was obtained from the bovine muscle and was consisted of two isoforms: Hsp70 and Hsc70. Hsp70 preparation was injected into normal and stressed males of unbred and Wistar rats in cisterna magna (up to 10 ug), central nucleus of amygdala (up to 1 ug), and in both nostrils (10 ug). To reveal the possible sites of Hsp70 localization the protein was labeled with NHS-ester-Alexa Fluor (Molecular Probes, USA). Control and stressed rats were behaviourally tested. Biological samples were analyzed using biochemical and morphological methods.Results and discussion. First experiments revealed that Hsp70 preparation exerted dose-dependent suppressive effect on exploratory and motor activity of rats, which resembles some stress situation when depression of behavior activity takes place (Andreeva L.I. et al., 2004). Simultaneously, emotionality of animals was found to elevate in rats accepted Hsp70. After the protein (0.2 and 0.5 ug) delivery into cisterna magna the behavior activity in the open field test was not altered even there was a tendency to some activation. When doses of the protein were elevated up to 1–10 ug, dose-dependent inhibitory effect was observed. Hsp70 in a maximal doses caused increased emotionality of rats. High doses of Hsp70 increased in some extent the content of corticosteron in serum 4 h after injections (514 ± 125 nM saline;656 ± 104 and 614 ± 93 nM, 3 and 10 ug of Hsp70 respectively). Repeated intranasal HSP70 administration (once a day, 3 days) also inhibited motor and exploratory activity compared with controls. Corticosteron content in sera of 43 % rats received Hsp70 was significantly lower than in control group (saline) indicating that anti-stress effect of Hsp70 was profound in a half of animals. This effect was also found under double Hsp70 intranasal administration following both psycho-emotional and physical stressors representing «terrorist-hostage» model and characterized by hyper-excitability of animals. The behavior testing showed that the Hsp70 administration led to restriction of excitation after the end of stress treatment. Biochemical analysis demonstrated the decreasing of calcium concentration, urea, creatinine, some intracellular enzymes in serum after applying of exogenous Hsp70. The tissue content of inducible isoform of Hsp70 (stress marker) in brain and in liver of stressed rats was also lower as compared with control stressed animals (saline). The results of experiments with injection of Hsp70 into central nucleus of amigdala («center of emotions») showed that Hsp70 (0.5 and 1 ug) was able to activate self-stimulation in rats. On the contrary, when the protein was applied to rats which were subjected to the social isolation and alcogolization the protein was found to suppress self-stimulation. Preliminary data on localization of exogenously delivered Hsp70 showed that fluorescent label localized to sites proximal to ependimal cells, vessel endothelial cells and to Purkinje cells of cerebellum.Conclusion. Simulation of stress response in a whole organism by Hsp70 administration (a transition from norm to stress) revealed a dose-dependent effect of the protein from slight activation to growing depression of motor and exploratory behavior. Increased emotionality after Hsp70 administration may be explained by interactions of Hsp70 with limbic structures. Hsp70 plays an important role in development of stress reaction. Alterations of motor activity maybe associated with internalization of Hsp70 by motor neurons (Purkinje cells). The study was supported by the Russian Foundation for Basic Research (project no. 03–03-33024).ReferencesAndreeva L.I. et al. Dokl Acad Nauk. 2004; 394 (6): 835–839. Asea A. et al. Cell Stress Chaperones. 2000; 5 (5): 425–431. Becker T. et al. J Cell Biol. 2002; 158 (7): 1277–1285. Broquet A.H. et al. J Biol Chem. 2003; 278 (24): 21601–21606. Fabian T.F. et al. Med Sci Monit. 2003; 9(1): BR62–65. Febbraio M.A. et al. J Physiol 2002; 544(3): 957–961. Guzhova I.V. et al. Cell Stress & Chap. 1997; 2: 132–139.Guzhova I.V. et al. Brain Res. 2001; 914: 66–73. Hunter-Lavin C. et al. Biochem Biophys Res Commun. 2004; 324: 511–517. Noessner E. et al. J Immunol. 2002; 169: 5424–5432. Tytell M. et al. Brain Res. 1986; 363: 161–164