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1.
Mem Inst Oswaldo Cruz ; 115: e200142, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33053076

RESUMO

BACKGROUND: Calpains are present in almost all organisms and comprise a family of calcium-dependent cysteine peptidases implicated in crucial cellular functions. Trypanosoma cruzi, the causative agent of Chagas disease, presents an expansion on this gene family with unexplored biological properties. OBJECTIVES: Here, we searched for calpains in the T. cruzi genome, evaluated the mRNA levels, calpain activity and the protein expression and determined the cellular localisation in all three parasite life cycle forms. METHODS/FINDINGS: Sixty-three calpain sequences were identified in T. cruzi CL Brener genome, with fourteen domain arrangements. The comparison of calpain mRNA abundance by quantitative polymerase chain reaction (qPCR) revealed seven up-regulated sequences in amastigotes and/or bloodstream trypomastigotes and five in epimastigotes. Western Blotting analysis revealed seven different molecules in the three parasite forms, and one amastigote-specific, while no proteolytic activity could be detected. Flow cytometry assays revealed a higher amount of intracellular calpains in amastigotes and/or trypomastigotes in comparison to epimastigotes. Finally, ultrastructural analysis revealed the presence of calpains in the cytoplasm, vesicular and plasma membranes of the three parasite forms, and in the paraflagellar rod in trypomastigotes. CONCLUSION: Calpains are differentially expressed and localised in the T. cruzi life cycle forms. This study adds data on the calpain occurrence and expression pattern in T. cruzi.


Assuntos
Calpaína/genética , Trypanosoma cruzi/genética , Animais , Western Blotting , Calpaína/metabolismo , Doença de Chagas , Estágios do Ciclo de Vida , RNA Mensageiro/genética
2.
Life Sci ; 259: 118391, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891610

RESUMO

AIMS: Dyslipidemia-associated diabetic retinopathy is featured by macular edema and retinal angiogenesis. This study investigated the in vitro lipotoxicity of free fatty acids and their modulatory roles in regulation of autophagy and angiogenic factor production in cultured human retinal pigment epithelium (RPE) ARPE-19 cells. MAIN METHODS: ARPE-19 cells were exposed to monounsaturated oleic acid (OA), saturated palmitic acid (PA), or both. Cell viability, cell cycle distribution, migration, and autophagy of the treated cells were monitored. Angiogenic factor production was determined by RT-qPCR and ELISA. KEY FINDINGS: OA, but not PA, at doses higher than 500 µM significantly induced cytostasis and lipotoxicity in ARPE-19 cells. OA exposure not only markedly enhanced autophagy flux, but also enhanced cell migration, while PA suppressed motility of RPE cells. Meanwhile, OA stimulated de novo synthesis of angiogenic factors including VEGF and bFGF in ARPE-19 cells. Mechanistically, OA treatment stimulated not only AMPK/mTOR/p70S6K signaling, but also induced hyperphosphorylation of MAPK pathway mediators, including ERK, JNK and p38 MAPK, as well as NF-κB activation. Kinase inhibition assays showed that blockade of PI3K/Akt, MAPK and NF-κB pathways prevented the OA-upregulated VEGF transcription and its peptide release. Comparatively, only NF-κB inhibition significantly suppressed bFGF peptide release from ARPE-19 cells. SIGNIFICANCE: Out findings support the OA-exhibited cytostasis, autophagy modulation and angiogenic factor production in RPE cells. This study sheds light on the interrelationship between metabolic disorder and retinopathy and provides molecular strategies for preventing and treating choroidal neovascularization in diabetic retinopathy.


Assuntos
Indutores da Angiogênese/metabolismo , Autofagia , Ácido Oleico/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Sistema de Sinalização das MAP Quinases , Ácido Palmítico/metabolismo , Epitélio Pigmentado da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
3.
Life Sci ; 259: 118375, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891612

RESUMO

OBJECTIVE: Short-chain fatty acids were reported to be the precursors of milk fat and can stimulate the de novo synthesis of fatty acids in bovine mammary epithelial cells (bMECs). However, the mechanism has not been elucidated. The purpose of this study was to investigate the effects of sodium butyrate (NaB) on milk fat synthesis in bMECs and explore its potential mechanism. METHODS: Bovine mammary epithelial cells (bMECs) were isolated for subsequent experimental uses. BODIPY staining and triglyceride kit were used to detect the milk fat synthesis in bMECs. Western blotting and RT-PCR assays were performed to detect the expression of related genes in bMECs. Immunoprecipitation was used to detect the acetylation of SREBP1 in bMECs. RESULTS: The results showed that NaB significantly promoted milk fat synthesis, promoted the activity of mechanistic target of rapamycin (mTOR) and S6 kinase (S6K), inhibited the activity of AMP-activated protein kinase (AMPK), and promoted the gene expression of G protein-coupled receptor 41 (GPR41). Knockdown of GPR41 and sterol regulatory element binding protein 1 (SREBP1) and overexpression of sirtuin1 (SIRT1), mTOR inhibitor (rapamycin), and AMPK activator (AICIR) eliminated these effects. These results indicated that NaB increased the nuclear translocation of SREBP1 via the GPR41/AMPK/mTOR/S6K signalling pathway, promoted the acetylation of mature SREBP1a via GPR41/AMPK/SIRT1, and then promoted milk fat synthesis. CONCLUSION: Taken together, these results demonstrated that NaB increased nuclear translocation and acetylation of SREBP1 to promote milk fat synthesis by activating GPR41 and its downstream signalling pathways.


Assuntos
Ácido Butírico/farmacologia , Glicolipídeos/biossíntese , Glicoproteínas/biossíntese , Glândulas Mamárias Animais/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Carbazóis , Bovinos , Células Cultivadas , Feminino , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Naftalenos , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
4.
Life Sci ; 259: 118374, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891613

RESUMO

OBJECTIVE: Dipeptidyl peptidase IV (DPP4) has been indicated as a possible prognostic biomarker in papillary thyroid cancer (PTC). However, the mechanism of DPP4 during metastasis of PTC remains unclear. In this study, we investigated whether lysine acetyltransferase 5 (KAT5) and FBJ murine osteosarcoma viral oncogene homolog B (FosB) synergistically regulate high DPP4 expression in PTC. METHODS: PTC tissues and matched paracancerous tissues were harvested, followed by the establishment of IHH-4 and TPC-1 cells with downregulation of DPP4. The relevance of DPP4 on the metastasis of PTC cells was assessed. Subsequently, the effect of KAT5 on the transcription of DPP4 was verified. The binding relationship between FosB and DPP4 was predicted by a bioinformatics website. Functional rescue experiments were performed to evaluate cell activities after overexpression of KAT5 or FosB in cells with DPP4 knockdown. RESULTS: DPP4 was overexpressed in PTC tissues and cell lines, which was correlated with higher risks for metastases and poorer survival. DPP4 downregulation curtailed cell growth and metastasis. Moreover, KAT5 acetylated DPP4 promoter histone, which promoted transcription activation of DPP4. Subsequently, FosB recruited KAT5 at the DPP4 promoter, thereby enhancing DPP4 transcriptional activation. Further overexpression of KAT5 or FosB in cells with low expression of DPP4 promoted cell activity. Finally, DPP4 expedited p62 nuclear translocation to elevate Keap1/Nrf2 expression, thus facilitating the growth and metastasis of PTC cells. CONCLUSION: FosB enhanced the growth and metastasis of PTC cells by recruiting histone acetyltransferases KAT5 to increase DPP4 transcription and activate the p62/Keap1/Nrf2 signaling.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Lisina Acetiltransferase 5/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo
5.
Life Sci ; 259: 118383, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896555

RESUMO

AIMS: Previous studies have shown that the widespread use of estrogen preparations can cause adverse outcomes such as thrombosis and cardiovascular disease. Autophagy is a biochemical process necessary to maintain cell homeostasis. The present study investigated whether E-2 mediates autophagy-induced endothelial cell dysfunction. The role of aspirin in this process was then studied. MAIN METHODS: Western blot, fluorescence microscopy, electron transmission microscopy, plasma construction and transfection, vasoreactivity study in wire myograph are all used in this study. KEY FINDINGS: We found that E-2 activated the PI3K/mTOR signaling pathway and inhibited the formation of the Atg14L-Beclin1-Vps34-Vps15 complex, thereby inhibiting autophagy. Aspirin promoted Beclin1 phosphorylation in autophagy initiation complexes and enhanced autophagy. Furthermore, E-2 treatment of HAECs resulted in endothelial dysfunction by inhibiting autophagy and leading to accumulation of α-smooth muscle actin (α-SMA). E-2 inhibited the activation of eNOS and reduced the expression of eNOS protein. In the mouse aortic vascular function test, E-2 disrupted endothelium-dependent vasodilation. An α-SMA-shRNA lentivirus eliminated the disruption to endothelium-dependent vasodilation by E-2. Aspirin inhibited α-SMA accumulation by enhancing autophagy, reversed endothelial functional impairment caused by E-2, and promoted endothelium-dependent vasodilation. SIGNIFICANCE: This study provides new evidence that E-2 inhibits autophagy and induces abnormal accumulation of α-SMA, resulting in endothelial cell dysfunction and affecting vasodilation. Aspirin can effectively restore the endothelial cell function disrupted E-2.


Assuntos
Actinas/metabolismo , Aspirina/farmacologia , Autofagia/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Estradiol/metabolismo , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Animais , Western Blotting , Células Cultivadas , Endotélio Vascular/ultraestrutura , Feminino , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Fosforilação/efeitos dos fármacos
6.
Life Sci ; 259: 118390, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896556

RESUMO

AIMS: This study aimed to evaluate the function and pathway of ATP-binding cassette transporter member A1 (ABCA1)-induced anti-inflammatory response in cells at the feto-maternal interface. MAIN METHODS: The primary amniotic mesenchymal cells (AMCs), chorion cells and decidual cells were isolated from placental membranes of women with uncomplicated pregnancies at full-term (not in labor) using enzymatic digestion. Flow cytometry was used to measure the purity of isolated cells. Immunofluorescence assay was performed to detect the location of ABCA1 and toll-like receptor 4 (TLR4). Reverse transcription PCR and western blotting analyses were used to examine ABCA1, TLR4 and inflammatory factor expression in primary cells. ELISA was used to detect cytokine secretions from the primary cells. KEY FINDINGS: ABCA1 and TLR4 were mainly located in the cell nucleus and cytoplasm of feto-maternal interface cells. ABCA1 expression remained the highest in chorion cells, medium in decidual cells, and weakest in AMCs. Upregulated expression of ABCA1 decreased expression of TLR4 and the levels of pro-inflammatory factors, but increased cytoprotective factors in all cell types. In contrast, downregulated expression of ABCA1 increased the expression of TLR4 and pro-inflammatory factors, but decreased the levels of cytoprotective factors. Downregulated ABCA1 expression followed by decreased TLR4 expression using a small interference RNA (siRNA) induced reduction of interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α) in all cell types. SIGNIFICANCE: ABCA1 at feto-maternal interface acts as an anti-inflammatory role by reducing the expression of TLR4 in uncomplicated pregnancies. ABCA1 might be a potential therapeutic target for preventing gestational diseases.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Troca Materno-Fetal , Receptor 4 Toll-Like/metabolismo , Western Blotting , Células Cultivadas , Córion/metabolismo , Decídua/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Confocal , Placenta/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Life Sci ; 259: 118397, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896557

RESUMO

There is increasing evidence that Bazedoxifene, as an FDA-approved selective estrogen inhibitor, approved by FDA, not only inhibits estrogen receptors, but also has other pharmacological effects. The purpose of this study was to investigate the effects of Bazedoxifene on the functional changes of vascular smooth muscle cells (VSMCs) after PDGF-BB stimulation. VSMCs were divided into control group, PDGF-BB treatment group, and PDGF-BB treatment group with different concentrations of Bazedoxifene. CCK-8 and EdU staining were used to determine the VSMCs viability and proliferation. Western blot was used to detect the expressions of vimentin, SMA, ERK, p-ERK, STAT3, p-STAT3, AKT, p-AKT, and LC3 I/II. Wound healing method was used to detect the migration of VSMCs. PDGF-BB treatment significantly enhanced the viability and proliferation of VSMCs as indicated by CCK-8 and EdU assays (P < 0.01), while Bazedoxifene pretreatment could reduce the increased viability and proliferation of VSMCs caused by PDGF-BB (P < 0.05). Wound healing test also showed Bazedoxifene significantly attenuated the migration in the PDGF-BB stimulated VSMCs (P < 0.01). PDGF-BB also induced the phenotypic switch and decreased the autophagy level in VSMCs, manifested as a reduction in vimentin, SMA, and LC3 II (P < 0.01). These effects of PDGF-BB were partially reversed by Bazedoxifene (P < 0.05). Bazedoxifene may inhibit the proliferation and migration of VSMCs through up-regulate the autophagy level after PDGF-BB stimulation.


Assuntos
Autofagia/efeitos dos fármacos , Becaplermina/farmacologia , Indóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Becaplermina/antagonistas & inibidores , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Músculo Liso Vascular/citologia , Fenótipo
8.
Life Sci ; 259: 118380, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32898524

RESUMO

AIMS: Benign prostatic hyperplasia (BPH) is a progressive disease, which severely affects men's health. Here, we sought to analyze the functions and mechanism of action of the tripartite motif protein 52 (TRIM52), a novel prostate basal cell biomarker in BPH. MATERIALS AND METHODS: Immunohistochemistry assay was performed in sectioned human BPH tissues, BPH-1 cells, and prostate RWPE-1 cells, to detect the expressions of TRIM52 and NF-κB. Western blotting and qRT-PCR analyses were conducted to measure the relative protein and mRNA expression levels, respectively. Further, lentiviral transfection was performed in BPH-1 and RWPE-1 cells to study the overexpression and siRNA knockdown of TRIM52. Dual-luciferase reporter assay was applied to evaluate the relationship between NF-κB and TRIM52. Furthermore, CCK-8 assay and flow cytometry were employed to analyze cell proliferation and apoptosis. KEY FINDINGS: TRIM52 and NF-κB levels were elevated in BPH tissues, and TRIM52 expression positively correlated with NF-κB expression. TRIM52 silencing suppressed the growth of BPH-1 cells and decreased the promoter activity of NF-κB. Moreover, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed TRIM52-induced proliferation of RWPE-1 cells and inhibited NF-κB promoter activity in oeTRIM52 transfected RWPE-1 cells. Silencing TRIM52 also inhibited TRAF2 ubiquitination in BPH-1 cells. Further, NF-κB promoter activity in siNC transfected cells was enhanced by the recombinant protein TNF-α and inhibited by siTRIM52. SIGNIFICANCE: TRIM52 accelerated the growth of BPH-1 cells by upregulating NF-κB, and TRIM52 could promote TRAF2 ubiquitination. These findings might contribute to the understanding of the biological functions and action mechanisms of TRIM52 in BPH.


Assuntos
NF-kappa B/metabolismo , Hiperplasia Prostática/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Western Blotting , Progressão da Doença , Citometria de Fluxo , Humanos , Masculino , Hiperplasia Prostática/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação
9.
Life Sci ; 259: 118382, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32898532

RESUMO

AIM: Vancomycin (VCM) is a glycopeptide antibiotic widely used to treat serious infections caused by methicillin-resistant Staphylococcus aureus and has been associated with some severe side effects such as hepatotoxicity and nephrotoxicity. However, the underlying mechanism of VCM-induced hepatotoxicity is not yet fully understood. Therefore, the current study was designed to evaluate the protective effects of zingerone (Zin) against VCM-induced hepatotoxicity in rats. MATERIALS AND METHODS: VCM was intraperitoneally administered at a dose of 200 mg/kg body weight (b.w.) for 7 days alone and in combination with the orally administered Zin (25 and 50 mg/kg b.w). KEY FINDINGS: Zin treatment significantly improved VCM-induced hepatic lipid peroxidation, glutathione depletion, reduced antioxidant enzyme (superoxide dismutase, catalase and glutathione peroxidase) activities and liver function markers (aspartate aminotransferase, alkaline phosphatase and alanine aminotransferase). Histopathological integrity and immunohistochemical expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the VCM-induced liver tissue were ameliorated after Zin administration. In addition, Zin reversed the changes in levels and/or activities of inflammatory and apoptotic parameters such as nuclear factor kappa B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), p53, cysteine aspartate specific protease-3 (caspase-3), cysteine aspartate specific protease-8 (caspase-8), cytochrome c, Bcl-2 associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) in the VCM-induced hepatotoxicity. SIGNIFICANCE: Collectively, these results reveal probable ameliorative role of Zin against VCM-induced hepatotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Guaiacol/análogos & derivados , Inflamação/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Vancomicina/toxicidade , Animais , Western Blotting , Ciclo-Oxigenase 2/metabolismo , Guaiacol/uso terapêutico , Interleucina-1beta/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
10.
Clin Sci (Lond) ; 134(17): 2235-2241, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32869854

RESUMO

Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin-Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Serpinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Western Blotting , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Eletroforese em Gel de Poliacrilamida , Humanos , Pandemias , Pneumonia Viral/virologia , Serpinas/uso terapêutico , Proteínas não Estruturais Virais/química
11.
Clin Sci (Lond) ; 134(17): 2235-2241, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: covidwho-738221

RESUMO

Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin-Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Serpinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Western Blotting , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Eletroforese em Gel de Poliacrilamida , Humanos , Pandemias , Pneumonia Viral/virologia , Serpinas/uso terapêutico , Proteínas não Estruturais Virais/química
12.
Mikrobiyol Bul ; 54(3): 510-522, 2020 Jul.
Artigo em Turco | MEDLINE | ID: mdl-32755525

RESUMO

Although cystic echinococcosis (CE) is quite prevalent in Turkey, it is extremely neglected due to being usually asymptomatic for years and frequently not to be reported although it is obligatory. Most of the data on the prevalence of CE in humans in Turkey are based on hospital records, reported cases and the studies based on serological methods and they do not reflect the truth. The fact that detecting no cysts in most of the seropositive cases limits the value of researches based on serological tests. The most valuable epidemiologic data on CE are obtained by mass screening surveys with the use of portable ultrasonography (US) and it took the place of serological tests, especially in the last 20 years. Two of 190 cases older than 20 years were found to be positive for CE in a village of Konya city at the first study that US was performed in Turkey. At the first research performed on preliminary school children in Manisa Province; of the 630 students examined by US, serology and chest X-ray, 2 (0.3%) were diagnosed as CE by US. Only US was used at the second study, and hydatid cysts were observed in 3 (0.5%) of the 575 students in two villages; these data suggested that the use of US alone was more easy, fast and beneficial in the field studies. In the third research, 6093 students from 37 different schools of Manisa Province were selected as a representative sample, and 9 (0.2%) children (two previously operated) were found to be positive for CE by US. The only response to the invitation to use this model in different regions of Turkey was from Elazig Province and of the 2500 students selected, six cases (one previously operated) were detected, and the prevalence was found to be 0.2% in Elazig Province. During the same years, of the 102 cases sharing the same living space with 40 patients operated due to CE, 13 (12.7%) were radiologically diagnosed as CE in Van, while CE was diagnosed in 1 (0.5%) of the 209 cases in an area dealing with animal husbandry in Aydin. At the fourth research in Manisa, 4275 students from university were examined by US, while 2034 of these were also serologically tested by ELISA and Western Blot (WB). The efficacy of WB as a screening test in CE was investigated for the first time in the world; six new and three operated cases were detected, and the prevalence was 0.2%. During the research in the rural areas of Bulgaria, Romania and Turkey, of the 8618 cases living in six cities (Ankara, Aksaray, Balikesir, Bitlis, Edirne, Sanliurfa) of Turkey, 53 (0.6%) abdominal CE cases were detected by US and one of every 163 cases in Turkey was found to be infected with CE. This ratio shows that CE is one of the most important public health problems in Turkey. Control of CE is possible with "One Health" concept. An effective control program and changes in valid laws are needed in Turkey. In this review, the value of different diagnostic procedures have also been discussed.


Assuntos
Equinococose , Animais , Western Blotting , Criança , Equinococose/diagnóstico por imagem , Equinococose/epidemiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Prevalência , Turquia/epidemiologia , Ultrassonografia
13.
Nat Commun ; 11(1): 4111, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807776

RESUMO

Mutational inactivation of VHL is the earliest genetic event in the majority of clear cell renal cell carcinomas (ccRCC), leading to accumulation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCC and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Herein we show using an autochthonous ccRCC model that Hif1a is essential for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are required for the clear cell phenotype. Transcriptomic and proteomic analyses reveal that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. HIF-2α-deficient tumours are characterised by increased antigen presentation, interferon signalling and CD8+ T cell infiltration and activation. Single copy loss of HIF1A or high levels of HIF2A mRNA expression correlate with altered immune microenvironments in human ccRCC. These studies reveal an oncogenic role of HIF-1α in ccRCC initiation and suggest that alterations in the balance of HIF-1α and HIF-2α activities can affect different aspects of ccRCC biology and disease aggressiveness.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Células 3T3 , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Linfócitos T CD8-Positivos/metabolismo , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imuno-Histoquímica , Inflamação/genética , Inflamação/metabolismo , Neoplasias Renais/genética , Espectrometria de Massas , Camundongos , Proteômica/métodos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologia
14.
Nat Commun ; 11(1): 4117, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807785

RESUMO

Strategies for eradicating cancer stem cells (CSCs) are urgently required because CSCs are resistant to anticancer drugs and cause treatment failure, relapse and metastasis. Here, we show that photoactive functional nanocarbon complexes exhibit unique characteristics, such as homogeneous particle morphology, high water dispersibility, powerful photothermal conversion, rapid photoresponsivity and excellent photothermal stability. In addition, the present biologically permeable second near-infrared (NIR-II) light-induced nanocomplexes photo-thermally trigger calcium influx into target cells overexpressing the transient receptor potential vanilloid family type 2 (TRPV2). This combination of nanomaterial design and genetic engineering effectively eliminates cancer cells and suppresses stemness of cancer cells in vitro and in vivo. Finally, in molecular analyses of mechanisms, we show that inhibition of cancer stemness involves calcium-mediated dysregulation of the Wnt/ß-catenin signalling pathway. The present technological concept may lead to innovative therapies to address the global issue of refractory cancers.


Assuntos
Raios Infravermelhos , Nanotecnologia/métodos , Células-Tronco Neoplásicas/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Western Blotting , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/metabolismo , Via de Sinalização Wnt
15.
Nat Commun ; 11(1): 4115, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807795

RESUMO

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.


Assuntos
Anticorpos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células A549 , Anticorpos/genética , Western Blotting , Calorimetria , Cristalografia por Raios X , Citometria de Fluxo , Polarização de Fluorescência , Imunofluorescência , Humanos , Espectrometria de Massas , Ligação Proteica , Domínios Proteicos/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Biologia Sintética
16.
Medicine (Baltimore) ; 99(31): e20076, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32756072

RESUMO

C-terminal binding protein-2 (CtBP2) a transcriptional corepressor, has been reported to involve in tumorigenesis and progression and predict a poor prognosis in several human cancers. However, few studies on CtBP2 in lung cancer tissues have been performed. In the present study, we first explored the CtBP2 gene expression profile from the the cancer genome atlas (TCGA) datasets, then western blot analysis and immunohistochemistry were performed to investigate and verified whether lung adenocarcinoma (LUAD) tissues exhibit deregulated CtBP2 expression. We evaluated the correlations between CtBP2 expression and the clinicopathological characteristics, and Kaplan-Meier survival analyses were performed to estimate the effect of CtBP2 expression on prognosis of LUAD patients. The results revealed that CtBP2 expression was significantly upregulated in LUAD tissues compared with normal lung tissues. Furthermore, increasing CtBP2 expression in LUAD was significantly associated with tumor differentiation (P = .028), tumor node metastasis (TNM) stage (P = .042). CtBP2 expression was significantly correlated with LUAD patients' survival (P = .028). In conclusion, the present study revealed that CtBP2 protein is a novel prognostic marker for LUAD. A further large-scale study is needed to confirm the present results.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Oxirredutases do Álcool/análise , Proteínas Correpressoras/análise , Neoplasias Pulmonares/diagnóstico , Adenocarcinoma de Pulmão/química , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/análise , Western Blotting , Feminino , Humanos , Pulmão/química , Neoplasias Pulmonares/química , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sobrevida
17.
Nat Commun ; 11(1): 4060, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792512

RESUMO

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid-binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain-hindbrain lineages and self-renewal-differentiation choices in NPCs.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Drosophila , Epigênese Genética/genética , Citometria de Fluxo , Imunofluorescência , Histona-Lisina N-Metiltransferase/genética , Imunoprecipitação , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética
18.
Life Sci ; 259: 118290, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32822713

RESUMO

AIMS: Atrial fibrillation (AF) is a common arrhythmia which is associated with higher risk of stroke, heart failure and all-cause mortality. Abnormal Ca2+ handling in diabetes mellitus (DM) can cause delayed depolarization involved with increased NCX activity. Complicated mechanisms are involved in atrial remodeling, of which CaMKII may be a key node signal. Therefore, we intend to explore whether CaMKII activation induces atrial electrical remodeling by regulating NCX expression in this study. MAIN METHODS: Adult male SD rats were used to establish a diabetic rat model, divided into three groups: the control group, DM group and allopurinol group. Hemodynamic and ECG indicators were recorded, after which electrophysiological studies were conducted. The protein expression of CaMKII, p-CaMKII, XO, MnSOD and NCX was measured by Western blot and immunohistochemistry. H&E and Masson staining were applied for observing myocardial fibrosis. HL-1 cells were cultured for the measurement of ROS generation. KEY FINDINGS: The arrangement of atrial myocytes was disordered and the collagen volume fraction of the atrium tissue was elevated in the DM group compared with the control group, and improved by allopurinol. Higher incidence of inducible AF, reduced conduction velocity and higher conduction inhomogeneity were observed in diabetic rats. These electrophysiological abnormalities were accompanied by higher oxidative stress and protein expression of p-CaMKII and NCX. Allopurinol prevented the development of these abnormal changes. SIGNIFICANCE: Allopurinol can improve atrial electrical remodeling by inhibiting CaMKII activity and protein expression of NCX. These data indicate xanthine oxidase inhibition can reduce oxidative stress and ameliorate atrial electrical remodeling.


Assuntos
Alopurinol/farmacologia , Remodelamento Atrial/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Xantina Oxidase/antagonistas & inibidores , Animais , Western Blotting , Ecocardiografia , Hemodinâmica/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real
19.
Life Sci ; 259: 118356, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32861798

RESUMO

Curculigoside (CUR) is natural ingredient from Curculigo orchioides Gaertn with multiple biological activities. However, whether CUR protects from ulcerative colitis (UC) and underlying mechanisms are unclear. Herein, mice challenged with dextran sulfate sodium (DSS) were established and administrated with CUR for 7 days. Then histological pathologies and ferroptosis regulators were determined in vivo. The ferroptotic IEC-6 cells were prepared to investigate the underlying mechanism of CUR. Results showed that CUR inhibited the disease activity index, histological damage and cell death in mice with colitis. We also found that ferroptosis was induced in mice with colitis, as evidenced by iron overload, GSH depletion, ROS and MDA production, accompanied by decreased expression of SOD and GPX4. CUR treatment significantly reversed these alterations of ferroptotic features in DSS-induced mice. Furthermore, similar effects of CUR on ferroptosis were observed in IEC-6 cells under the combined treatment of H2O2 and iron chloride hexahydrate. Interestingly, we found that CUR could increase the selenium sensitivity and promote GPX4 transcription level in IEC-6 cells. Knockdown of GPX4 significantly blocked the protective effects of CUR on cell death, GSH and MDA contents as well as LDH activity in ferroptotic IEC-6 cells. Taken together, these findings suggest that CUR protects against ferroptosis in UC by the induction of GPX4, which presents a potential agent for UC treatment.


Assuntos
Benzoatos/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Ferroptose/efeitos dos fármacos , Glucosídeos/uso terapêutico , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Animais , Western Blotting , Linhagem Celular , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/química , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Ferro/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Reação em Cadeia da Polimerase em Tempo Real
20.
Exp Parasitol ; 217: 107962, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763249

RESUMO

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.


Assuntos
FMN Redutase/metabolismo , Ferro/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Membrana Celular/enzimologia , Doença de Chagas/parasitologia , Colorimetria , FMN Redutase/análise , FMN Redutase/química , Imunofluorescência , Humanos , Filogenia , Distribuição de Poisson , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Trypanosoma cruzi/classificação , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Regulação para Cima
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