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1.
J Strength Cond Res ; 33(9): 2344-2351, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31343553

RESUMO

Pickering, C, Suraci, B, Semenova, EA, Boulygina, EA, Kostryukova, ES, Kulemin, NA, Borisov, OV, Khabibova, SA, Larin, AK, Pavlenko, AV, Lyubaeva, EV, Popov, DV, Lysenko, EA, Vepkhvadze, TF, Lednev, EM, Leonska-Duniec, A, Pajak, B, Chycki, J, Moska, W, Lulinska-Kuklik, E, Dornowski, M, Maszczyk, A, Bradley, B, Kana-ah, A, Cieszczyk, P, Generozov, EV, and Ahmetov, II. A genome-wide association study of sprint performance in elite youth football players. J Strength Cond Res 33(9): 2344-2351, 2019-Sprint speed is an important component of football performance, with teams often placing a high value on sprint and acceleration ability. The aim of this study was to undertake the first genome-wide association study to identify genetic variants associated with sprint test performance in elite youth football players and to further validate the obtained results in additional studies. Using micro-array data (600 K-1.14 M single nucleotide polymorphisms [SNPs]) of 1,206 subjects, we identified 12 SNPs with suggestive significance after passing replication criteria. The polymorphism rs55743914 located in the PTPRK gene was found as the most significant for 5-m sprint test (p = 7.7 × 10). Seven of the discovered SNPs were also associated with sprint test performance in a cohort of 126 Polish women, and 4 were associated with power athlete status in a cohort of 399 elite Russian athletes. Six SNPs were associated with muscle fiber type in a cohort of 96 Russian subjects. We also examined genotype distributions and possible associations for 16 SNPs previously linked with sprint performance. Four SNPs (AGT rs699, HSD17B14 rs7247312, IGF2 rs680, and IL6 rs1800795) were associated with sprint test performance in this cohort. In addition, the G alleles of 2 SNPs in ADRB2 (rs1042713 & rs1042714) were significantly over-represented in these players compared with British and European controls. These results suggest that there is a genetic influence on sprint test performance in footballers, and identifies some of the genetic variants that help explain this influence.


Assuntos
Desempenho Atlético/fisiologia , Grupo com Ancestrais do Continente Europeu/genética , Corrida/fisiologia , Futebol/fisiologia , 17-Hidroxiesteroide Desidrogenases/genética , Aceleração , Adolescente , Alelos , Angiotensinogênio/genética , Criança , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Fator de Crescimento Insulin-Like II/genética , Interleucina-6/genética , Masculino , Polônia , Polimorfismo de Nucleotídeo Único , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Receptores Adrenérgicos beta 2/genética , Federação Russa , Reino Unido , Adulto Jovem
2.
J Clin Med ; 8(5)2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100888

RESUMO

The present study is centered on molecular mechanisms of the cytoprotective effect of geranylgeraniol (GGOH) in skeletal muscle harmed by statin-associated myopathy (SAM). GGOH via autophagy induction was purportedly assumed to prevent skeletal muscle viability impaired by statins, atorvastatin (ATR) or simvastatin (SIM). The C2C12 cell line was used as the 'in vitro' model of muscle cells at different stages of muscle formation, and the effect of ATR or SIM on the cell viability, protein expression and mitochondrial respiration were tested. Autophagy seems to be important for the differentiation of muscle cells; however, it did not participate in the observed GGOH cytoprotective effects. We showed that ATR- and SIM-dependent loss in cell viability was reversed by GGOH co-treatment, although GGOH did not reverse the ATR-induced drop in the cytochrome c oxidase protein expression level. It has been unambiguously revealed that the mitochondria of C2C12 cells are not sensitive to SIM, although ATR effectively inhibits mitochondrial respiration. GGOH restored proper mitochondria functioning. Apoptosis might, to some extent, explain the lower viability of statin-treated myotubes as the pan-caspase inhibitor, N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK), partly reversed ATR- or SIM-induced cytotoxic effects; however, it does not do so in conjunction with caspase-3. It appears that the calpain inhibitor, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLM), restored the viability that was reduced by ATR and SIM (p < 0.001). GGOH prevents SAM, in part, as a consequence of a caspase-3 independent pathway, probably by calpain system inactivation.

3.
Int J Mol Sci ; 20(6)2019 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-30909654

RESUMO

In this study we attempted to verify the hypothesis that the mevalonate pathway affects amyloid beta precursor protein (AßPP) processing and regulates clusterin protein levels. AßPP expression was monitored by green fluorescence (FL) and Western blot (WB). WB showed soluble amyloid protein precursor alpha (sAßPPα) presence in AßPP-wt cells and Aß expression in AßPP-sw cells. Nerve growth factor (NGF)-differentiated rat neuronal pheochromocytoma PC-12 cells were untreated/treated with statins alone or together with non-sterol isoprenoids. Co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol demonstrated statin-dependent neurotoxicity resulted from the attenuated activity of mevalonate pathway rather than lower cholesterol level. Atorvastatin (50 µM) or simvastatin (50 µM) as well as cholesterol chelator methyl-ß-cyclodextrin (0.2 mM) diminished cell viability (p < 0.05) and clusterin levels. Interestingly, co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol stimulated (p < 0.05) clusterin expression. Effects of non-sterol isoprenoids, but not water soluble cholesterol (Chol-PEG), were the most significant in mock-transfected cells. Geranylgeraniol (GGOH) overcame atorvastatin (ATR)-dependent cytotoxicity. This effect does not seem to be dependent on clusterin, as its level became lower after GGOH. The novelty of these findings is that they show that the mevalonate (MEV) pathway rather than cholesterol itself plays an important role in clusterin expression levels. In mock-transfected, rather than in AßPP-overexpressing cells, GGOH/farnesol (FOH) exerted a protective effect. Thus, protein prenylation with GGOH/FOH might play substantial role in neuronal cell survival.


Assuntos
Precursor de Proteína beta-Amiloide/genética , Colesterol/farmacologia , Clusterina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mutação , Terpenos/farmacologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colesterol/química , Clusterina/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Ácido Mevalônico/metabolismo , Células PC12 , Ratos
4.
Int J Mol Sci ; 19(10)2018 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-30241315

RESUMO

This review addresses the issue of the numerous roles played by Rap1 GTPase (guanosine triphosphatase) in different cell types, in terms of both physiology and pathology. It is one among a myriad of small G proteins with endogenous GTP-hydrolyzing activity that is considerably stimulated by posttranslational modifications (geranylgeranylation) or guanine nucleotide exchange factors (GEFs), and inhibited by GTPase-activating proteins (GAPs). Rap1 is a ubiquitous protein that plays an essential role in the control of metabolic processes, such as signal transduction from plasma membrane receptors, cytoskeleton rearrangements necessary for cell division, intracellular and substratum adhesion, as well as cell motility, which is needed for extravasation or fusion. We present several examples of how Rap1 affects cells and organs, pointing to possible molecular manipulations that could have application in the therapy of several diseases.


Assuntos
Proteínas rap1 de Ligação ao GTP/fisiologia , Imunidade Adaptativa , Diferenciação Celular , Transformação Celular Neoplásica , Modelos Moleculares , Prenilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/metabolismo
5.
Oxid Med Cell Longev ; 2018: 6463807, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29951166

RESUMO

The present study investigated the cytotoxic effects of statins (atorvastatin (ATR) and simvastatin (SIM), resp.) and methyl-beta-cyclodextrin (MßCD), at their respective IC50 concentrations, on muscle regeneration in the in vitro model of murine C2C12 myoblasts. Cotreatment with mevalonate (MEV), farnesol (FOH), geranylgeraniol (GGOH), or water-soluble cholesterol (Chol-PEG) was employed to determine whether the statin-dependent myotoxicity resulted from the lower cholesterol levels or the attenuated synthesis of intermediates of mevalonate pathway. Our findings demonstrated that while GGOH fully reverted the statin-mediated cell viability in proliferating myoblasts, Chol-PEG exclusively rescued MßCD-induced toxicity in myocytes. Statins caused loss of prenylated RAP1, whereas the GGOH-dependent positive effect was accompanied by loss of nonprenylated RAP1. Geranylgeranyltransferases are essential for muscle cell survival as inhibition with GGTI-286 could not be reversed by GGOH cotreatment. The increase in cell viability correlated with elevated AKT 1(S463) and GSK-3ß(S9) phosphorylations. Slight increase in the levels of autophagy markers (Beclin 1, MAP LC-3IIb) was found in response to GGOH cotreatment. Autophagy rose time-dependently during myogenesis and was inhibited by statins and MßCD. Statins and MßCD also suppressed myogenesis and neither nonsterol isoprenoids nor Chol-PEG could reverse this effect. These results point to GGOH as the principal target of statin-dependent myotoxicity, whereas plasma membrane cholesterol deposit is ultimately essential to restore viability of MßCD-treated myocytes. Overall, this study unveils for the first time a link found between the GGOH- and Chol-PEG-dependent reversal of statin- or MßCD-mediated myotoxicity and cytoprotective autophagy, respectively.


Assuntos
Autofagia/efeitos dos fármacos , Diterpenos/uso terapêutico , GTP Fosfo-Hidrolases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Mialgia/induzido quimicamente , Mialgia/tratamento farmacológico , Animais , Diterpenos/farmacologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Camundongos , Mialgia/patologia
6.
FEBS J ; 284(9): 1370-1387, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28342290

RESUMO

Verapamil, an L-type calcium channel blocker, has been used successfully to treat cardiovascular diseases. Interestingly, we have recently shown that treatment of cancer cells with verapamil causes an effect on autophagy. As autophagy is known to modulate chemotherapy responses, this prompted us to explore the impact of verapamil on autophagy and cell viability in greater detail. We report here that verapamil causes an induction of autophagic flux in a number or tumor cells and immortalized normal cells. Moreover, we found that inhibition of autophagy in COLO 205 cells, via treatment with the chloroquine (CQ) or by CRISPR/Cas9-mediated disruption of the autophagy genes Atg7 and Atg5, causes an upregulation of apoptotic markers in response to verapamil. In search of a mechanism for this effect and because autophagy can often mitigate metabolic stress, we examined the impact of verapamil on cellular metabolism. This revealed that in normal prostate cells, verapamil diminishes glucose and glycolytic intermediate levels leading to adenosine 5'-triphosphate (ATP) depletion. In contrast, in COLO 205 cells it enhances aerobic glycolysis and maintains ATP. Importantly, we found that the autophagic response in these cells is related to the activity of l-lactate dehydrogenase A (LDHA, EC 1.1.1.27), as inhibition of LDHA reduces both basal and verapamil-induced autophagy and consequently decreases cell viability. In summary, these findings not only identify a novel mechanism of cytoprotective autophagy induction but they also highlight the potential of using verapamil together with inhibitors of autophagy for the treatment of malignant disease. ENZYMES: l-lactate dehydrogenase (LDHA, EC 1.1.1.27).


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Citoproteção/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Verapamil/farmacologia , Antimaláricos/farmacologia , Antineoplásicos/efeitos adversos , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Biomarcadores/metabolismo , Sistemas CRISPR-Cas , Bloqueadores dos Canais de Cálcio/efeitos adversos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cloroquina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Microscopia Eletrônica de Transmissão , Neoplasias/metabolismo , Neoplasias/ultraestrutura , Verapamil/efeitos adversos
7.
PLoS One ; 11(8): e0161693, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27551730

RESUMO

Both myoblasts and mesenchymal stem cells (MSC) take part in the muscle tissue regeneration and have been used as experimental cellular therapy in muscular disorders treatment. It is possible that co-transplantation approach could improve the efficacy of this treatment. However, the relations between those two cell types are not clearly defined. The aim of this study was to determine the reciprocal interactions between myoblasts and MSC in vitro in terms of the features important for the muscle regeneration process. Primary caprine muscle-derived cells (MDC) and bone marrow-derived MSC were analysed in autologous settings. We found that MSC contribute to myotubes formation by fusion with MDC when co-cultured directly, but do not acquire myogenic phenotype if exposed to MDC-derived soluble factors only. Experiments with exposure to hydrogen peroxide showed that MSC are significantly more resistant to oxidative stress than MDC, but a direct co-culture with MSC does not diminish the cytotoxic effect of H2O2 on MDC. Cell migration assay demonstrated that MSC possess significantly greater migration ability than MDC which is further enhanced by MDC-derived soluble factors, whereas the opposite effect was not found. MSC-derived soluble factors significantly enhanced the proliferation of MDC, whereas MDC inhibited the division rate of MSC. To conclude, presented results suggest that myogenic precursors and MSC support each other during muscle regeneration and therefore myoblasts-MSC co-transplantation could be an attractive approach in the treatment of muscular disorders.


Assuntos
Comunicação Celular , Células-Tronco Mesenquimais/metabolismo , Mioblastos/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Fusão Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Cabras , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Células-Tronco Mesenquimais/citologia , Desenvolvimento Muscular , Mioblastos/citologia
8.
Pharmacol Rep ; 68(4): 707-14, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27126697

RESUMO

BACKGROUND: The timing and consequences of alternations in substrate utilization in heart failure (HF) and their relationship with structural changes remain unclear. This study aimed to analyze metabolic changes associated with transition to overt heart failure in transgenic mouse model of HF resulting from cardiac-specific overexpression of constitutively active Gαq*. METHODS: Structural changes quantified by morphometry, relative cardiac mRNA and protein expression of PPARα, FAT/CD36, CPT-1, GLUT-4 and glycolytic efficiency following administration of 1-(13)C glucose were investigated in 4-14-month-old Tgαq*44 mice (TG), compared with age-matched FVB wild type mice (WT). RESULTS: Initial hypertrophy in TG (4-10-month of age) was featured by an accelerated glycolytic pathway that was not accompanied by structural changes in cardiomyocytes. In 10-month-old TG, cardiomyocyte elongation and hypertrophic remodeling and increased glycolytic flux was accompanied by relatively low expression of FAT/CD36, CPT-1 and PPARα. During the transition phase (12-month-old TG), a pronounced increase in PPARα with an increase in relative fatty acid (FA) flux was associated with anomalies of cardiomyocytes with accumulation of lipid droplets and glycogen as well as cell death. At the stage of overt heart failure (14-month-old TG), an accelerated glycolytic pathway with a decline in FA oxidation was accompanied by further structural changes. CONCLUSION: Tgαq*44 mice display three distinct phases of metabolic/structural changes during hypertrophy and progression to HF, with relatively short period of increase in FA metabolism, highlighting a narrow metabolic changes associated with transition to overt heart failure in Tgaq*44 mice that have therapeutic significance.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Fatores Etários , Animais , Antígenos CD36/biossíntese , Carnitina O-Palmitoiltransferase/biossíntese , Morte Celular , Ácidos Graxos/biossíntese , Transportador de Glucose Tipo 4/biossíntese , Insuficiência Cardíaca/patologia , Hipertrofia/metabolismo , Hipertrofia/patologia , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , PPAR alfa/biossíntese
9.
Oxid Med Cell Longev ; 2016: 1805304, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26881014

RESUMO

This review is focused on the possible causes of mitochondrial dysfunction in AD, underlying molecular mechanisms of this malfunction, possible causes and known consequences of APP, Aß, and hyperphosphorylated tau presence in mitochondria, and the contribution of altered lipid metabolism (nonsterol isoprenoids) to pathological processes leading to increased formation and accumulation of the aforementioned hallmarks of AD. Abnormal protein folding and unfolded protein response seem to be the outcomes of impaired glycosylation due to metabolic disturbances in geranylgeraniol intermediary metabolism. The origin and consecutive fate of APP, Aß, and tau are emphasized on intracellular trafficking apparently influenced by inaccurate posttranslational modifications. We hypothesize that incorrect intracellular processing of APP determines protein translocation to mitochondria in AD. Similarly, without obvious reasons, the passage of Aß and tau to mitochondria is observed. APP targeted to mitochondria blocks the activity of protein translocase complex resulting in poor import of proteins central to oxidative phosphorylation. Besides, APP, Aß, and neurofibrillary tangles of tau directly or indirectly impair mitochondrial biochemistry and bioenergetics, with concomitant generation of oxidative/nitrosative stress. Limited protective mechanisms are inadequate to prevent the free radical-mediated lesions. Finally, neuronal loss is observed in AD-affected brains typically by pathologic apoptosis.


Assuntos
Doença de Alzheimer/metabolismo , Mitocôndrias/patologia , Estresse Oxidativo , Resposta a Proteínas não Dobradas , Idoso , Precursor de Proteína beta-Amiloide/química , Animais , Apoptose , Encéfalo/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Glicosilação , Homeostase , Humanos , Neurônios/metabolismo , Fosforilação Oxidativa , Oxigênio/metabolismo , Fosforilação , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas tau
10.
PLoS One ; 11(1): e0146726, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26785133

RESUMO

Myogenesis and muscle hypertrophy account for muscle growth and adaptation to work overload, respectively. In adults, insulin and insulin-like growth factor 1 stimulate muscle growth, although their links with cellular energy homeostasis are not fully explained. Insulin plays critical role in the control of mitochondrial activity in skeletal muscle cells, and mitochondria are essential for insulin action. The aim of this study was to elucidate molecular mechanism(s) involved in mitochondrial control of insulin-dependent myogenesis. The effects of several metabolic inhibitors (LY294002, PD98059, SB216763, LiCl, rotenone, oligomycin) on the differentiation of C2C12 myoblasts in culture were examined in the short-term (hours) and long-term (days) experiments. Muscle cell viability and mitogenicity were monitored and confronted with the activities of selected genes and proteins expression. These indices focus on the roles of insulin, glycogen synthase kinase 3 beta (GSK-3ß) and forkhead box protein O1 (FOXO1) on myogenesis using a combination of treatments and inhibitors. Long-term insulin (10 nM) treatment in "normoglycemic" conditions led to increased myogenin expression and accelerated myogenesis in C2C12 cells. Insulin-dependent myogenesis was accompanied by the rise of mtTFA, MtSSB, Mfn2, and mitochondrially encoded Cox-1 gene expressions and elevated levels of proteins which control functions of mitochondria (kinase--PKB/AKT, mitofusin 2 protein--Mfn-2). Insulin, via the phosphatidylinositol 3-kinase (PI3-K)/AKT-dependent pathway reduced transcription factor FOXO1 activity and altered GSK-3ß phosphorylation status. Once FOXO1 and GSK-3ß activities were inhibited the rise in Cox-1 gene action and nuclear encoded cytochrome c oxidase subunit IV (COX IV) expressions were observed, even though some mRNA and protein results varied. In contrast to SB216763, LiCl markedly elevated Mfn2 and COX IV protein expression levels when given together with insulin. Thus, inhibition of GSK-3ß activity by insulin alone or together with LiCl raised the expression of genes and some proteins central to the metabolic activity of mitochondria resulting in higher ATP synthesis and accelerated myogenesis. The results of this study indicate that there are at least two main targets in insulin-mediated myogenesis: notably FOXO1 and GSK-3ß both playing apparent negative role in muscle fiber formation.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Insulina/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular , Animais , Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Camundongos , Mitocôndrias/metabolismo , Células Musculares/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sistemas do Segundo Mensageiro
11.
FEMS Yeast Res ; 15(6)2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26091838

RESUMO

Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level.


Assuntos
Glutationa/metabolismo , Homeostase , Mitocôndrias/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Cádmio/toxicidade , Metabolismo Energético , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Oxirredução , Fitoquelatinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética
12.
Biomed Res Int ; 2015: 535908, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25961024

RESUMO

Monitoring and control of infections are key parts of surveillance systems and epidemiological risk prevention. In the case of influenza A viruses (IAVs), which show high variability, a wide range of hosts, and a potential of reassortment between different strains, it is essential to study not only people, but also animals living in the immediate surroundings. If understated, the animals might become a source of newly formed infectious strains with a pandemic potential. Special attention should be focused on pigs, because of the receptors specific for virus strains originating from different species, localized in their respiratory tract. Pigs are prone to mixed infections and may constitute a reservoir of potentially dangerous IAV strains resulting from genetic reassortment. It has been reported that a quadruple reassortant, A(H1N1)pdm09, can be easily transmitted from humans to pigs and serve as a donor of genetic segments for new strains capable of infecting humans. Therefore, it is highly desirable to develop a simple, cost-effective, and rapid method for evaluation of IAV genetic variability. We describe a method based on multitemperature single-strand conformational polymorphism (MSSCP), using a fragment of the hemagglutinin (HA) gene, for detection of coinfections and differentiation of genetic variants of the virus, difficult to identify by conventional diagnostic.


Assuntos
Coinfecção/genética , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Infecções por Orthomyxoviridae/genética , Animais , Coinfecção/transmissão , Coinfecção/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/transmissão , Influenza Humana/virologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Polônia , Polimorfismo Conformacional de Fita Simples , Sus scrofa , Suínos
14.
Nanoscale Res Lett ; 10: 98, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25852394

RESUMO

Recently, it has been shown that silver nanoparticles (AgNPs) provide a unique approach to the treatment of tumors, especially those of neuroepithelial origin. Thus, the aim of this study was to evaluate the impact of AgNPs on proliferation and activation of the intrinsic apoptotic pathway of glioblastoma multiforme (GBM) cells cultured in an in ovo model. Human GBM cells, line U-87, were placed on chicken embryo chorioallantoic membrane. After 8 days, the tumors were divided into three groups: control (non-treated), treated with colloidal AgNPs (40 µg/ml), and placebo (tumors supplemented with vehicle only). At the end of the experiment, all tumors were isolated. Assessment of cell proliferation and cell apoptosis was estimated by histological, immunohistochemical, and Western blot analyses. The results show that AgNPs can influence GBM growth. AgNPs inhibit proliferation of GBM cells and seem to have proapoptotic properties. Although there were statistically significant differences between control and AgNP groups in the AI and the levels of active caspase 9 and active caspase 3, the level of these proteins in GBM cells treated with AgNPs seems to be on the border between the spontaneous apoptosis and the induced. Our results indicate that the antiproliferative properties of silver nanoparticles overwhelm proapoptotic ones. Further research focused on the cytotoxic effect of AgNPs on tumor and normal cells should be conducted.

15.
Biomed Res Int ; 2015: 352794, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25821797

RESUMO

Autophagy is a basic catabolic process, serving as an internal engine during responses to various cellular stresses. As regards cancer, autophagy may play a tumor suppressive role by preserving cellular integrity during tumor development and by possible contribution to cell death. However, autophagy may also exert oncogenic effects by promoting tumor cell survival and preventing cell death, for example, upon anticancer treatment. The major factors influencing autophagy are Ca(2+) homeostasis perturbation and starvation. Several Ca(2+) channels like voltage-gated T- and L-type channels, IP3 receptors, or CRAC are involved in autophagy regulation. Glucose transporters, mainly from GLUT family, which are often upregulated in cancer, are also prominent targets for autophagy induction. Signals from both Ca(2+) perturbations and glucose transport blockage might be integrated at UPR and ER stress activation. Molecular pathways such as IRE 1-JNK-Bcl-2, PERK-eIF2α-ATF4, or ATF6-XBP 1-ATG are related to autophagy induced through ER stress. Moreover ER molecular chaperones such as GRP78/BiP and transcription factors like CHOP participate in regulation of ER stress-mediated autophagy. Autophagy modulation might be promising in anticancer therapies; however, it is a context-dependent matter whether inhibition or activation of autophagy leads to tumor cell death.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Homeostase , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Autofagia , Humanos , Estresse Oxidativo
16.
Biomed Res Int ; 2015: 746092, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25821818

RESUMO

Pheochromocytoma PC-12 cells are immune to physiological stimuli directed to evoke programmed cell death. Besides, metabolic inhibitors are incapable of sensitizing PC-12 cells to extrinsic or intrinsic apoptosis unless they are used in toxic concentrations. Surprisingly, these cells become receptive to cell deletion after human APP-sw gene expression. We observed reduced cell viability in GFP vector + APP-sw-nucleofected cells (drop by 36%) but not in GFP vector - or GFP vector + APP-wt-nucleofected cells. Lower viability was accompanied by higher expression of Aß 1-16 and elevated secretion of Aß 1-40 (in average 53.58 pg/mL). At the ultrastructural level autophagy-like process was demonstrated to occur in APP-sw-nucleofected cells with numerous autophagosomes and multivesicular bodies but without autolysosomes. Human APP-sw gene is harmful to PC-12 cells and cells are additionally driven to incomplete autophagy-like process. When stimulated by TRAIL or nystatin, CLU protein expression accompanies early phase of autophagy.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Autofagia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Transfecção/métodos , Precursor de Proteína beta-Amiloide/genética , Animais , Núcleo Celular/genética , Sobrevivência Celular , Terapia Genética/métodos , Neoplasias Experimentais/genética , Células PC12 , Ratos , Resultado do Tratamento , Regulação para Cima
17.
Biomed Res Int ; 2014: 462609, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25401102

RESUMO

Brugada Syndrome (BS) is an inherited channelopathy associated with a high incidence of sudden cardiac death. The paper presents the discovery of new genetic variants of SCN5A gene which might be associated with the development of a concealed form of Brugada Syndrome. The study involved a group of 59 patients (37 men) with suspected concealed form of Brugada Syndrome. Pharmacological provocation with intravenous ajmaline administration was performed. Six patients with positive test results were subjected to molecular analysis of SCN5A gene with MSSCP method. Additionally, MSSCP genotyping was performed for samples obtained from the family members with Brugada Syndrome, despite the fact that they had negative ajmaline challenge test results. Genetic examinations of the SCN5A gene at 6 positive patients showed 6 known polymorphisms, 8 new single nucleotide point (SNP) variants located at exons, and 12 new single nucleotide point variants located at introns. Among new SNPs localized in SCN5A gene exons three SNPs affected the protein sequence.


Assuntos
Síndrome de Brugada/genética , Testes Genéticos , Variação Genética , Adolescente , Adulto , Síndrome de Brugada/patologia , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5 , Polônia , Polimorfismo de Nucleotídeo Único
18.
Acta Biochim Pol ; 61(3): 479-83, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25180222

RESUMO

Influenza viruses are the worldwide major causative agents of human and animal acute respiratory infections. Some of the influenza subtypes have caused epidemics and pandemics among humans. The varieties of methods are available for the rapid isolation and identification of influenza viruses in clinical and environmental samples. Since nucleic acids amplification techniques such as RT-PCR have been adapted, fast and sensitive influenza type and subtype determination is possible. However, in some ambiguous cases other, more detailed assay might be desired. The genetic material of influenza virus is highly unstable and constantly mutates. It is known that single nucleotide polymorphisms (SNPs) results in resistance to commercially available anti-viral drugs. The genetic drift of the virus could also result in weakening of immune response to infection. Finally, in a substantial number of patients co-infection with various virus strains or types has been confirmed. Although the detection of co-infection or presence of minor genetic variants within flu-infected patients is not a routine procedure, a rapid and wide spectrum diagnostics of influenza virus infections could reveal an accurate picture of the disease and more importantly, is crucial for choosing the appropriate therapeutics and virus monitoring. Herein we present the evidences that native gel electrophoresis and MSSCP--a method based on multitemperature single strand conformation polymorphism could furnish a useful technique for minor variants, which escape discovery by conventional diagnostic assays.


Assuntos
Eletroforese , Técnicas de Genotipagem , Influenzavirus A/genética , Influenzavirus A/isolamento & purificação , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Influenza Humana/diagnóstico , Influenza Humana/virologia , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Polimorfismo Conformacional de Fita Simples , RNA Viral/química
19.
Med Oncol ; 31(10): 176, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25173530

RESUMO

Discoidin death receptor 2 (DDR2) receptor belongs to a DDR family that shows a tyrosine kinase activity. The somatic mutations in DDR2 gene, reported in non-small cell lung cancer (NSCLC), are involved in up-regulation of cells' migration, proliferation and survival. A S768R substitution in DDR2 gene was commonly reported in squamous cell lung carcinoma. Clinical data of patients carrying the DDR2 gene mutation suggest that its presence can be independent of gender and age. The effectiveness of an oral dual-specific (Src and Abl) multikinase inhibitors-dasatinib-was observed in different cell lines and in some NSCLC patients with identified DDR2 mutation. In the present study, we have used three molecular methods (ASP-real-time PCR, ASP-DNA-FLA PCR and direct sequencing) to detect the DDR2 gene mutation in 143 patients with NSCLC metastases to the central nervous system (CNS). The prevalence of the DDR2 gene mutation was correlated with the occurrence of mutations in the EGFR, KRAS, HER2 and BRAF genes. We identified three patients (2.1% of studied group) with DDR2 mutation. The mutation was observed in two patients with low differentiated squamous cell lung cancer and in one patient with adeno-squamous cell carcinoma (ADSCC). In ADSCC patients, DDR2 mutation coexisted with G12C substitution in KRAS gene. According to the current knowledge, examination of the presence of the DDR2 gene mutation in metastatic lesion is the first such report worldwide. The information, that these driver mutations are present in CNS metastases of NSCLC, could broaden therapeutic choices in such group of patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias do Sistema Nervoso Central/genética , Éxons/genética , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos/genética , Análise de Sequência de DNA/métodos , Idoso , Carcinoma Pulmonar de Células não Pequenas/secundário , Neoplasias do Sistema Nervoso Central/secundário , Receptores com Domínio Discoidina , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Estudos Retrospectivos , Sensibilidade e Especificidade
20.
PLoS One ; 9(4): e94259, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24722055

RESUMO

α-Synuclein (ASN) plays an important role in pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3ß (GSK-3ß) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3ß inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3ß via increasing of protein level and activation of this enzyme. GSK-3ß activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3ß activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3ß in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3ß is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3ß-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3ß inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Microtúbulos/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Animais , Apoptose , Citoesqueleto/metabolismo , Glicogênio Sintase Quinase 3 beta , Indóis/química , Maleimidas/química , Células PC12 , Fosforilação , Ratos , Fatores de Tempo , Tubulina (Proteína)/metabolismo
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