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1.
Nat Neurosci ; 24(10): 1488-1500, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34426698

RESUMO

Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.


Assuntos
Encéfalo/fisiopatologia , Epilepsia/fisiopatologia , Neurônios , Adulto , Benzotiazóis/farmacologia , Encéfalo/crescimento & desenvolvimento , Sinalização do Cálcio , Pré-Escolar , Epilepsia/diagnóstico por imagem , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Proteína 2 de Ligação a Metil-CpG/genética , Rede Nervosa/fisiopatologia , Neurogênese/genética , Neuroimagem , Síndrome de Rett/diagnóstico por imagem , Síndrome de Rett/fisiopatologia , Análise de Célula Única , Sinapses , Tolueno/análogos & derivados , Tolueno/farmacologia , Transcriptoma
2.
Bioorg Chem ; 114: 105153, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34328851

RESUMO

A series of novel substituted phenyl 1, 3-thiazolidin-4-one sulfonyl derivatives 5 (a-t) were synthesized and screened for their in-vitro anti-microbial and anti-viral activity. The result of the anti-microbial assay demonstrated compounds 5d, 5f, 5g, 5h, 5i, 5j showed prominent inhibitory activity against all the tested Gram-positive and Gram-negative bacterial strains, while compounds 5g, 5j, 5o, 5p, 5q showed significant activity against the entire set of fungal strains as compared to standard drug Ampicillin and Clotrimazole, respectively. The antimicrobial study revealed that compounds having electron-withdrawing groups showed significant antimicrobial potency. The most active antibacterial compound 5j showed potent inhibition of S. aureus DNA Gyrase enzyme as a possible mechanism of action for antimicrobial activity. Moreover, the antiviral testing of selected compounds showed considerable activity against Herpes simplex virus-1(KOS), Herpes simplex virus-2 (G), Herpes simplex virus-1(TK- KOS ACVr), Vaccinia virus, Human Coronavirus (229E), Reovirus-1, Sindbis virus, Coxsackie virus B4, Yellow Fever virus and Influenza A, B virus. Compounds 5h exhibited low anti-viral activity against HIV-1(strain IIIB) and HIV-2 (strain ROD). The study clearly outlined that synthesized compounds endowed with good antimicrobial property together with considerable antiviral activity.


Assuntos
Fenóis/síntese química , Sulfonamidas/síntese química , Tolueno/análogos & derivados , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Humanos , Fenóis/química , Fenóis/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Tolueno/síntese química , Tolueno/química , Tolueno/farmacologia , Células Vero , Vírus/classificação , Vírus/efeitos dos fármacos
3.
J Biochem Mol Toxicol ; 35(8): e22822, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091999

RESUMO

The neurotoxicity of local anesthetics (LAs) has attracted more and more attention, However, they lack preventive and therapeutic measures. Many studies have shown that apoptosis plays an important role in the process of LA-induced neurotoxicity. As an important signaling molecule to activate apoptosis, p53 has been proved to be involved in the neurotoxicity induced by LAs, but the mechanism is unclear. In this study, we explored the effect of pifithrin-α (PFT-α), a p53 inhibitor, on apoptosis by ropivacaine (Rop) in vivo and in vitro. Cell viability and apoptosis detected by CCK-8 and a JC-1 apoptosis detection kit, the changes of spinal cord structure observed after hematoxylin and eosin staining, apoptosis of the spinal cord measured by terminal deoxynucleotidyl transferase dUTP nick end labeling staining, behavioral assessment of the nerve Injury evaluated by the detection of sciatic nerve conduction velocity (SNCV) andmechanical withdrawal threshold (MWT), the expression of p53 and many apoptosis-related genes included Bax, Bcl-2, and caspase-3 detected by quantitative real-time polymerase chain reaction, Western blot analysis, immunofluorescence, and immunohistochemistry. Results showed that PC12 cell viability decreased because of Rop, but the pretreatment of PFT-α could protect it. And PFT-α reduced the injuries in the spinal cord by Rop included vacuoles or edema. The results of immunofluorescence and immunohistochemistry testing showed that PFT-α inhibited the p53 protein upregulated by Rop. Apoptosis rate and many proapoptotic genes include p53, Bax, caspase-3 messenger RNA, and proteins were increased by Rop, but PFT-α could decrease it. In conclusion, PFT-α inhibited cell apoptosis and spinal cord injuries induced by Rop.


Assuntos
Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Mitocôndrias/metabolismo , Ropivacaina/efeitos adversos , Traumatismos da Medula Espinal/metabolismo , Tolueno/análogos & derivados , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Células PC12 , Ratos , Ratos Sprague-Dawley , Ropivacaina/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Tolueno/farmacologia , Proteína Supressora de Tumor p53/metabolismo
4.
J Ind Microbiol Biotechnol ; 48(5-6)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34137896

RESUMO

Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.


Assuntos
Hexosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Pichia/metabolismo , Alginatos/química , Carboidratos/análise , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Imobilizadas , Fermentação , Hexosiltransferases/genética , Humanos , Microbiologia Industrial , Inulina/metabolismo , Melaço , Pichia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales , Sacarose , Tolueno/farmacologia , Trissacarídeos/biossíntese
5.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33827945

RESUMO

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-µ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Memória Imunológica , Vírus da Influenza A Subtipo H1N1/imunologia , Células Matadoras Naturais/imunologia , Infecções por Orthomyxoviridae/imunologia , Transferência Adotiva , Animais , Antígenos Ly/análise , Antígenos Ly/metabolismo , Benzotiazóis/farmacologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cocultura , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Subfamília C de Receptores Semelhantes a Lectina de Células NK/análise , Receptor 1 Desencadeador da Citotoxicidade Natural/análise , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Baço/citologia , Baço/imunologia , Tolueno/análogos & derivados , Tolueno/farmacologia
6.
Theranostics ; 11(10): 4710-4727, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754023

RESUMO

Background: Telomere shortening and dysfunction may cause metabolic disorders, tissue damage and age-dependent pathologies. However, little is known about the association of telomere-associated protein Rap1 with mitochondrial energy metabolism and cardiac aging. Methods: Echocardiography was performed to detect cardiac structure and function in Rap1+/+ and Rap1-/- mice at different ages (3 months, 12 months and 20 months). Telomere length, DNA damage, cardiac senescence and cardiomyocyte size were analyzed using the real-time PCR, Western blotting, senescence associated ß-galactosidase assay and wheat germ agglutinin staining, respectively. Western blotting was also used to determine the level of cardiac fatty acid metabolism related key enzymes in mouse and human myocardium. Chromatin immunoprecipitation assay was used to verify the direct link between p53 and PPARα. The p53 inhibitor, Pifithrin-α and PPARα activator WY14643 were utilized to identify the effects of Rap1/p53/PPARα signaling pathway. Results: Telomere was shortened concomitant with extensive DNA damage in aged Rap1-/- mouse hearts, evidenced by reduced T/S ratios and increased nuclear γH2AX. Meanwhile, the aging-associated phenotypes were pronounced as reflected by altered mitochondrial ultrastructure, enhanced senescence, cardiac hypertrophy and dysfunction. Mechanistically, acetylated p53 and nuclear p53 was enhanced in the Rap1-/- mouse hearts, concomitant with reduced PPARα. Importantly, p53 directly binds to the promoter of PPARα in mouse hearts and suppresses the transcription of PPARα. In addition, aged Rap1-/- mice exhibited reduced cardiac fatty acid metabolism. Pifithrin-α alleviated cardiac aging and enhanced fatty acid metabolism in the aged Rap1-/- mice. Activating PPARα with WY14643 in primarily cultured Rap1-/- cardiomyocytes restored maximal oxygen consumption rates. Reduced Rap1 expression and impaired p53/PPARα signaling also presented in aged human myocardium. Conclusion: In summary, Rap1 may link telomere biology to fatty acid metabolism and aging-related cardiac pathologies via modulating the p53/PPARα signaling pathway, which could represent a therapeutic target in preventing/attenuating cardiac aging.


Assuntos
Envelhecimento/genética , Cardiomegalia/genética , Senescência Celular/genética , Miócitos Cardíacos/metabolismo , PPAR alfa/genética , Proteínas de Ligação a Telômeros/genética , Proteína Supressora de Tumor p53/genética , Animais , Benzotiazóis/farmacologia , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/fisiopatologia , Dano ao DNA , Ecocardiografia , Ácidos Graxos/metabolismo , Cardiopatias/diagnóstico por imagem , Cardiopatias/genética , Cardiopatias/fisiopatologia , Histonas/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Teste de Campo Aberto , PPAR alfa/metabolismo , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Telômero/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo
7.
Bioorg Med Chem Lett ; 30(24): 127653, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33129992

RESUMO

We synthesized a series of amides with a benzo[d][1,3]dithiol core. The chemical library of compounds was tested for their cytotoxicity and inhibiting activity against influenza virus A/California/07/09 (H1N1)pdm09 in MDCK cells. For each compound, values of CC50, IC50 and selectivity index (SI) were determined. Compounds of this structure type were for the first time found to exhibit anti-influenza activity. The structure of an amide substituent in the tested compounds was demonstrated to have a significant effect on their activity against the H1N1 influenza virus and cytotoxicity. Compound 4d has a high selectivity index of about 30. 4d was shown to be most potent at early stages of viral cycle. In direct fusogenic assay it demonstrated dose-dependent activity against fusogenic activity of hemagglutinin of influenza virus. Based on molecular docking and regression analysis data, viral hemagglutinin was suggested as possible target for these new antiviral agents.


Assuntos
Antivirais/química , Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Tolueno/análogos & derivados , Animais , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/tratamento farmacológico , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Infecções por Orthomyxoviridae/tratamento farmacológico , Tolueno/química , Tolueno/farmacologia
8.
PLoS Genet ; 16(8): e1008953, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776944

RESUMO

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.


Assuntos
Proteínas de Ligação a DNA/genética , Surdez/genética , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Benzotiazóis/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteínas de Ligação a DNA/deficiência , Surdez/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas Internas/patologia , Humanos , Camundongos , Mutação , RNA Guia/genética , Transdução de Sinais/efeitos dos fármacos , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Sequenciamento Completo do Exoma , Peixe-Zebra/genética
9.
Life Sci ; 259: 118254, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32800833

RESUMO

The biological and renal effects made by simultaneous and non-simultaneous exposure to toluene and noise were investigated. Twenty-four New Zealand white rabbits were exposed to 100 dB of white noise and 1000 ppm of toluene vapor for two weeks. The examined biochemical factors were urea, uric acid, creatinine, glucose, triglyceride, cholesterol, and albumin serum levels, measured on different days after the end of the exposure. Moreover, glutathione peroxidase activity (GPX), malondialdehyde dismutase activity (MDA), and superoxide dismutase (SOD) parameters were measured in the kidney tissue. The hematoxylin and eosin staining method was used for histopathological experiments. Overall, the noise increased albumin, uric acid, creatinine, and glucose levels, but it decreased urea, cholesterol, and triglyceride levels. Toluene decreased albumin, uric acid, and urea levels, while it increased creatinine, triglyceride, cholesterol, and glucose levels. Simultaneous exposure to noise and toluene decreased albumin, uric acid, cholesterol, and urea levels, whereas it increased creatinine, glucose, and triglyceride levels. GPX, MDA, and SOD levels increased by simultaneous and non-simultaneous exposure to noise and toluene. Furthermore, massive tubular degeneration, tubular cell vacuolization, glomerular disorganization, congestion, glomerular cell shrinkage, and unclear brush border were detected in the kidney tissue.


Assuntos
Rim/efeitos dos fármacos , Ruído/efeitos adversos , Tolueno/toxicidade , Animais , Antioxidantes/farmacologia , Catalase/metabolismo , Colesterol/metabolismo , Creatinina/metabolismo , Feminino , Glucose/metabolismo , Glutationa Peroxidase/metabolismo , Rim/metabolismo , Nefropatias/patologia , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Coelhos , Superóxido Dismutase/metabolismo , Tolueno/metabolismo , Tolueno/farmacologia , Ureia/metabolismo , Ácido Úrico/metabolismo
10.
Ecotoxicol Environ Saf ; 206: 111123, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32861005

RESUMO

Hormesis of soil enzyme that involved in heavy metal has been attracting much more attention for risk assessment of heavy metal toxicity, but insufficient studies were conducted to define the hormetic responses induced by toluene or other organic pollutions. The objectives of this study were to investigate the hormetic responses of soil enzyme induced by toluene and explore the potential enzyme kinetic mechanism. Soil alkaline phosphatase (ALP) activity was regarded as the endpoint to explore the hormetic responses under different doses of toluene (0.0, 0.1, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0, 50.0 and 100.0 µL g-1). Subsequently, we conducted the experiments of enzymatic reaction kinetics and pure enzyme to further verify the potential mechanisms of soil ALP's hormesis. Results showed that ALP activities at 0.1-1.0 µL g-1 toluene were significantly increased in contrast to the control (0 µL g-1 toluene) (P < 0.05) at the exposure time of 30, 36, 48 and 54 h, with the maximum stimulation magnitudes of 24-43%. ALP activities were almost not affected by toluene (2-100 µL L-1) in the whole experimental period (6-54 h). Meanwhile, the values of catalytic efficiency (the radio Vmax/Km, Vmax: maximum reaction velocity and Km: Michaelis constant) and Vmax significantly increased compared with the control, but the value of Km decreased from 2.5 to 1.6. Overall, low dose toluene can induce hormesis of soil ALP. The potential reason is that low-dose toluene could enhance the combination of soil ALP and substrates. We believe that this study will provide a new viewpoint for ecological risk assessment of toluene contaminated soils.


Assuntos
Fosfatase Alcalina/metabolismo , Hormese/efeitos dos fármacos , Poluentes do Solo/farmacologia , Solo/química , Tolueno/farmacologia , Relação Dose-Resposta a Droga , Cinética , Medição de Risco , Poluentes do Solo/análise , Tolueno/análise
11.
PLoS Negl Trop Dis ; 14(5): e0008339, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32437349

RESUMO

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.


Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Tolueno/análogos & derivados , Trypanosoma brucei brucei/efeitos dos fármacos , Antiprotozoários/isolamento & purificação , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Ensaios de Triagem em Larga Escala , NADH NADPH Oxirredutases/química , Ligação Proteica , Conformação Proteica , Tolueno/isolamento & purificação , Tolueno/farmacologia , Trypanosoma brucei brucei/enzimologia
12.
Anim Reprod Sci ; 215: 106330, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32216931

RESUMO

The objective of this study was to investigate effects of selenium (Se) on proliferation and apoptosis of sheep spermatogonial stem cells (SSC) in vitro. The SSC were assigned to five treatment groups (0, 2.0, 4.0, 8.0 and 16.0 µmol/L Se). After treatment with Se for 96 h, cell proliferation and apoptosis were evaluated. The relative abundance of P53 mRNA transcript and protein, cell cycle and apoptosis-related genes were detected using real-time PCR and Western blot quantifications, respectively. The results indicate there were the least cell proliferation rates in the Se16.0 group. Treatments with relatively greater Se concentrations (8.0 and 16.0 µmol/L) resulted in a greater percentage of apoptotic cells, which was consistent with the relative abundances of P53, P21, P27 and pro-apoptosis mRNA transcripts. There were relatively greater ROS concentrations in the control, Se8.0 and Se16.0 groups. Compared with the control group, treatment with the Se concentration of 16.0 µmol/L resulted in an increased abundance of P53, P21, P27 and BAX proteins. Treatment with Pifithrin-α suppressed the increase in abundance of P53 and P21 proteins induced by the relatively greater concentration of Se (16.0 µmol/L), however, did not result in a change in abundances of P27 and BAX proteins. These results indicate the regulatory functions of Se on proliferation and apoptosis of sheep SSC is associated with the P21-mediated P53 signalling pathway. The P27 and BAX proteins have limited functions during the apoptotic process of SSC induced by the relatively greater concentrations of Se.


Assuntos
Células-Tronco Germinativas Adultas/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Selênio/farmacologia , Ovinos , Células-Tronco Germinativas Adultas/fisiologia , Animais , Benzotiazóis/administração & dosagem , Benzotiazóis/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Redução da Medicação , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Selênio/administração & dosagem , Tolueno/administração & dosagem , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
13.
Sci Rep ; 10(1): 1049, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974452

RESUMO

Pifithrin-α (PFT-α) is a small molecule which has been widely used as a specific inhibitor of p53 transcription activity. However, its molecular mechanism of action remains unclear. PFT-α has also been described to display potent p53-independent activity in cells. In this study, we addressed the mechanism of action of PFT-α. We found that PFT-α failed to prevent the effects of Mdm2 inhibitor Nutlin-3 on cell cycle and apoptosis in several cancer cell lines. However, PFT-α rescued normal primary fibroblasts from growth inhibition by Nutlin-3. PFT-α displayed a very limited effect on p53-dependent transcription upon its activation by Nutlin-3. Moreover, PFT-α inhibitory effect on transcription was highly dependent on the nature of the p53 target gene. PFT-α attenuated post-translational modifications of p53 without affecting total p53 protein level. Finally, we found that PFT-α can decrease the level of intracellular reactive oxygen species through activation of an aryl hydrocarbon receptor (AHR)-Nrf2 axis in a p53-independent manner. In conclusion, PFT-α inhibits only some aspects of p53 function, therefore it should be used with extreme caution to study p53-dependent processes.


Assuntos
Benzotiazóis/farmacologia , Imidazóis/metabolismo , Piperazinas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Tolueno/análogos & derivados , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HCT116 , Humanos , Células MCF-7 , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Tolueno/farmacologia , Transcrição Genética/genética , Proteína Supressora de Tumor p53/metabolismo
14.
Toxicol Appl Pharmacol ; 387: 114848, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31809756

RESUMO

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a disappointing prognosis. The aim of this study was to investigate the anticancer effect of sesamin and the underlying mechanism. The MTT assay was used to detect the proliferation of NSCLC cells. The cell cycle and apoptosis were analyzed by flow cytometry. The protein levels of Akt, p-Akt (Ser473), p53, cyclin D1, CDK2, MDM2, p-MDM2 (Ser166) were detected by western blotting. The expression of p-Akt (Ser473), p53 and Ki67 in vivo was analyzed by IHC. Histopathologic analyses of major organs (heart, liver, spleen, lung and kidney) were performed by H&E staining. The results show that sesamin suppressed cell proliferation and induced apoptosis of NSCLC cells (A549 and H1792) in a dose-dependent manner. Treatment with sesamin caused cell cycle arrest at G1 phase and inhibited cyclin D1 and CDK2 expression. In addition, sesamin inhibited Akt activity and upregulated p53 expression both in vivo and in vitro. When Akt and p53 were suppressed by LY294002 and PFTα, respectively, sesamin exerted no additional effects. The in vivo results mostly matched the in vitro findings. Specifically, sesamin exerted little damage to major organs. Taken together, this study demonstrates that sesamin suppresses NSCLC cell proliferation by induction of G1 phase cell cycle arrest and apoptosis via Akt/p53 pathway. Therefore, sesamin may be a promising adjuvant treatment for NSCLC therapy.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Dioxóis/farmacologia , Lignanas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Benzotiazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Dioxóis/uso terapêutico , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Lignanas/uso terapêutico , Neoplasias Pulmonares/patologia , Camundongos , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Laryngoscope ; 130(12): 2818-2824, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31886905

RESUMO

OBJECTIVES: In this study, we aimed at investigating the expressions of miR-145 and its well-characterized direct targets on carboplatin treatment. STUDY DESIGN: Laboratory study. METHODS: The effect of carboplatin and miR-145 on the proliferative capacity of head and neck squamous cell carcinoma cells was evaluated using Cell Viability Detection Kit-8. Expressions of miR-145 and its targets were evaluated using quantitative real-time polymerase chain reaction on carboplatin treatment and p53 inhibition. Western blot was used to measure the levels of p53 and its acetylated versions in cells treated with carboplatin and/or pifithrin-α. RESULTS: We demonstrated that carboplatin induced the expression of miR-145 in a dose-dependent manner and suppressed the expressions of miR-145 direct targets. In addition, we showed that inhibition of p53 by pifithrin-α in carboplatin-treated cells reduced miR-145 expression and reversed the suppression of miR-145 direct targets. CONCLUSIONS: Considering all these findings together, one of the proposed mechanisms of carboplatin to kill cells might be the induction of miR-145 and deregulation of its targets in parallel, via p53 activation, which happens through carboplatin's DNA-damaging property. To the best of our knowledge, these findings are the first to reveal the relationship between carboplatin and miR-145 in cancer cells. LEVEL OF EVIDENCE: NA Laryngoscope, 2019.


Assuntos
Carboplatina/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Benzotiazóis/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolueno/análogos & derivados , Tolueno/farmacologia , Transfecção
16.
Circulation ; 141(4): 285-300, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31707831

RESUMO

BACKGROUND: Current differentiation protocols to produce cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations as determined by expression of cardiac troponin T. However, these cardiomyocytes remain immature, more closely resembling the fetal state, with a lower maximum contractile force, slower upstroke velocity, and immature mitochondrial function compared with adult cardiomyocytes. Immaturity of iPSC-derived cardiomyocytes may be a significant barrier to clinical translation of cardiomyocyte cell therapies for heart disease. During development, cardiomyocytes undergo a shift from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR)-signaling pathway plays a key role in nutrient sensing and growth. We hypothesized that transient inhibition of the mTOR-signaling pathway could lead cardiomyocytes to a quiescent state and enhance cardiomyocyte maturation. METHODS: Cardiomyocytes were differentiated from 3 human iPSC lines using small molecules to modulate the Wnt pathway. Torin1 (0 to 200 nmol/L) was used to inhibit the mTOR pathway at various time points. We quantified contractile, metabolic, and electrophysiological properties of matured iPSC-derived cardiomyocytes. We utilized the small molecule inhibitor, pifithrin-α, to inhibit p53 signaling, and nutlin-3a, a small molecule inhibitor of MDM2 (mouse double minute 2 homolog) to upregulate and increase activation of p53. RESULTS: Torin1 (200 nmol/L) increased the percentage of quiescent cells (G0 phase) from 24% to 48% compared with vehicle control (P<0.05). Torin1 significantly increased expression of selected sarcomere proteins (including TNNI3 [troponin I, cardiac muscle]) and ion channels (including Kir2.1) in a dose-dependent manner when Torin1 was initiated after onset of cardiomyocyte beating. Torin1-treated cells had an increased relative maximum force of contraction, increased maximum oxygen consumption rate, decreased peak rise time, and increased downstroke velocity. Torin1 treatment increased protein expression of p53, and these effects were inhibited by pifithrin-α. In contrast, nutlin-3a independently upregulated p53, led to an increase in TNNI3 expression and worked synergistically with Torin1 to further increase expression of both p53 and TNNI3. CONCLUSIONS: Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Naftiridinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Benzotiazóis/farmacologia , Linhagem Celular , Humanos , Imidazóis/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Piperazinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores
17.
Tissue Eng Part C Methods ; 25(12): 711-720, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31621504

RESUMO

The objective of this study is to design a cancer invasion model based on an interaction between cancer cells and cancer-associated fibroblasts (CAF) aggregates. The strength of this study is to incorporate gelatin hydrogel microspheres (GM) containing pifithrin-α (PFT) of a p53 inhibitor (GM-PFT) with the CAF aggregates. Incorporation of GM-PFT allowed CAF aggregates to enhance the alpha-smooth muscle actin expression level at a high concentration of PFT. When the cancer cells were cocultured with the CAF aggregates incorporating GM-PFT, the invasion rate of cancer cells was significantly high compared with CAF aggregates or CAF aggregates incorporating GM with or without the same dose of free PFT as well as two-dimension cultured CAF with or without the same dose of PFT. In addition, an inhibitor of matrix metalloproteinase decreased the cancer invasion rate for the CAF aggregates incorporating GM-PFT. It is concluded that the interaction between cancer cells and CAF aggregates incorporating GM-PFT of biological activation needs to realize the invasion of cancer cells even in vitro. Impact Statement The strength of this study is to combine with a three-dimensional cell culture system and a drug delivery system technology for a cancer invasion model. The combination enabled cancer-associated fibroblasts to enhance the biological functions. This cancer invasion model is a promising tool to mimic the tumor microenvironment for anticancer drug screening.


Assuntos
Benzotiazóis , Fibroblastos Associados a Câncer/metabolismo , Gelatina , Hidrogéis , Microesferas , Modelos Biológicos , Neoplasias/metabolismo , Tolueno/análogos & derivados , Proteína Supressora de Tumor p53/antagonistas & inibidores , Benzotiazóis/química , Benzotiazóis/farmacologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Gelatina/química , Gelatina/farmacologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Invasividade Neoplásica , Neoplasias/patologia , Tolueno/química , Tolueno/farmacologia
18.
Biosci Rep ; 39(9)2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31420372

RESUMO

Background: Cerebral ischemia-reperfusion (I/R) injury is the key to fatality in cerebrovascular accident, hence further endeavor is warranted to delineate the mechanism underlying its lethal aggravation procedure. In the present study, we aimed to elucidate the anti-autophagy and anti-apoptosis effects of ginkgetin via nuclear factor κB (NF-κB)/p53 pathway in cerebral I/R rats. Methods: Rats were administrated 2-h occlusion of right middle cerebral artery before the 24-h reperfusion followed. There were three doses of ginkgetin (25, 50, 100 mg/kg) given intraperitoneally (i.p.) after the 2-h ischemia, and Pifithrin-α (PFT-α, p53 inhibitor), SN50 (NF-κB inhibitor) and 3-methyladenine (3-MA, autophagy inhibitor) was administered 20 min before the ischemia, respectively. Results: The neurological deficits decreased significantly with the administration of ginkgetin. The concentrations of microtubule-associated protein 1 light chain 3-II and p53 were significantly decreased by PFT-α, 3-MA and ginkgetin. The concentrations of Beclin 1, damage-regulated autophagy modulator, cathepsin B and cathepsin D were significantly decreased due to the administration of PFT-α, ginkgetin and SN50. Furthermore, the concentrations of Bax and p53-upregulated modulator of apoptosis were significantly decreased with that of Bcl-2 being significantly increased by administration of SN50, PFT-α and ginkgetin. Conclusion: Ginkgetin can alleviate cerebral ischemia/reperfusion induced autophagy and apoptosis by inhibiting the NF-κB/p53 signaling pathway.


Assuntos
Biflavonoides/farmacologia , Isquemia Encefálica/tratamento farmacológico , Traumatismo por Reperfusão/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Benzotiazóis/farmacologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Humanos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores
19.
J Cell Biochem ; 120(10): 17625-17634, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31148231

RESUMO

How p53 participates in acute kidney injury (AKI) progress and what are the underlying mechanisms remain illusive. For this issue, it is important to probe into the role of p53 in cisplatin-induced AKI. We find that p53 was upregulated in cisplatin-induced AKI, yet, pifithrin-α inhibites the p53 expression to attenuated renal injury and cell apoptosis both in vivo cisplatin-induced AKI mice and in vitro HK-2 human renal tubular epithelial cells. To knock down p53 by siRNA significantly decreased the miRNA, miR-199a-3p, expression in HK-2 cells. Blockade of miR-199a-3p significantly reduced cisplatin-induced cell apoptosis and inhibited caspase-3 activity. Mechanistically, we identified that miR-199a-3p directly bound to mechanistic target of rapamycin (mTOR) 3'-untranslated region and overexpressed miR-199a-3p reduce the expression and phosphorylation of mTOR. Furthermore, we demonstrated that p53 inhibited mTOR activation through activating miR-199a-3p. In conclusion, our findings reveal that p53, upregulating the expression of miR-199a-3p affects the progress of cisplatin-induced AKI, which might provide a promising therapeutic target of AKI.


Assuntos
Injúria Renal Aguda/genética , MicroRNAs/genética , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Camundongos , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Serina-Treonina Quinases TOR/genética , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Acta Physiol (Oxf) ; 227(3): e13336, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31231946

RESUMO

AIM: mTORC1 is regarded as an important key regulator of protein synthesis and hypertrophy following mechanical stimuli in skeletal muscle. However, as excitation and tension development is tightly coupled in most experimental models, very little and largely indirect evidence exist for such a mechanosensitive pathway. Here, we sought to examine whether activation of mTORC1 signalling is dependent on tension per se in rat skeletal muscle. METHODS: To examine the mechanosensitivity of mTORC1, rat EDL muscles were exposed to either excitation-induced eccentric contractions (ECC), passive stretching (PAS) with identical peak tension (Tpeak ) and Tension-Time-Integral (TTI), or ECC with addition of inhibitors of the myosin ATPases (IMA ). To further explore the relationship between tension and mTORC1 signalling, rat EDL muscles were subjected to PAS of different magnitudes of Tpeak while standardizing TTI and vice versa. RESULTS: PAS and ECC with equal Tpeak and TTI produced similar responses in mTORC1 signalling despite different modes of tension development. When active tension during ECC was nearly abolished by addition of IMA , mTORC1 signalling was reduced to a level comparable to non-stimulated controls. In addition, when muscles were exposed to PAS of varying levels of Tpeak with standardized TTI, activation of mTORC1 signalling displayed a positive relationship with peak tension. CONCLUSIONS: The current study directly links tension per se to activation of mTORC1 signalling, which is independent of an active EC-coupling sequence. Moreover, activation of mTORC1 signalling displays a positive dose-response relationship with peak tension.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Músculo Esquelético/metabolismo , Animais , Fenômenos Biomecânicos , Retículo Endoplasmático , Regulação da Expressão Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Contração Muscular , Miosinas/antagonistas & inibidores , Miosinas/metabolismo , Ratos , Transdução de Sinais , Sulfonamidas/farmacologia , Tolueno/análogos & derivados , Tolueno/farmacologia
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