RESUMO
Ochratoxin A (OTA) is considered one of the main mycotoxins responsible for health problems and considerable economic losses in the feed industry. The aim was to study OTA's detoxifying potential of commercial protease enzymes: (i) Ananas comosus bromelain cysteine-protease, (ii) bovine trypsin serine-protease and (iii) Bacillus subtilis neutral metalloendopeptidase. In silico studies were performed with reference ligands and T-2 toxin as control, and in vitro experiments. In silico study results showed that tested toxins interacted near the catalytic triad, similar to how the reference ligands behave in all tested proteases. Likewise, based on the proximity of the amino acids in the most stable poses, the chemical reaction mechanisms for the transformation of OTA were proposed. In vitro experiments showed that while bromelain reduced OTA's concentration in 7.64% at pH 4.6; trypsin at 10.69% and the neutral metalloendopeptidase in 8.2%, 14.44%, 45.26% at pH 4.6, 5 and 7, respectively (p < 0.05). The less harmful α-ochratoxin was confirmed with trypsin and the metalloendopeptidase. This study is the first attempt to demonstrate that: (i) bromelain and trypsin can hydrolyse OTA in acidic pH conditions with low efficiency and (ii) the metalloendopeptidase was an effective OTA bio-detoxifier. This study confirmed α-ochratoxin as a final product of the enzymatic reactions in real-time practical information on OTA degradation rate, since in vitro experiments simulated the time that food spends in poultry intestines, as well as their natural pH and temperature conditions.
Assuntos
Micotoxinas , Ocratoxinas , Animais , Bovinos , Ocratoxinas/análise , Bromelaínas , Simulação de Acoplamento Molecular , Tripsina , Ração Animal/análise , MetaloendopeptidasesAssuntos
Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Antimaláricos/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Metaloendopeptidases/metabolismo , Modelos Moleculares , Estrutura Molecular , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismoRESUMO
Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.
Assuntos
Antibacterianos/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Biguanidas/farmacologia , Clorexidina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Homeostase , Humanos , Membranas/metabolismo , Metaloendopeptidases/efeitos dos fármacos , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfolipídeos/metabolismoRESUMO
AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.
Assuntos
Leishmania major/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose/imunologia , Metaloendopeptidases/imunologia , Peptídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Proteção Cruzada , Antígeno HLA-A2/imunologia , Antígeno HLA-DR1/imunologia , Imunidade Celular , Imunidade Humoral , Leishmania infantum/imunologia , Leishmaniose/prevenção & controle , Vacinas contra Leishmaniose/administração & dosagem , Vacinas contra Leishmaniose/química , Mesocricetus , Metaloendopeptidases/química , Óleo Mineral/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/químicaRESUMO
We examined the methionine aminopeptidase 2 inhibitor fumagillin in dogs consuming a high-fat and -fructose diet (HFFD). In pilot studies (3 dogs that had consumed HFFD for 3 yr), 8 wk of daily treatment with fumagillin reduced food intake 29%, weight 6%, and the glycemic excursion during an oral glucose tolerance test (OGTT) 44%. A second group of dogs consumed the HFFD for 17 wk: pretreatment (weeks 0-4), treatment with fumagillin (FUM; n = 6), or no drug (Control, n = 8) (weeks 4-12), washout period (weeks 12-16), and fumagillin or no drug for 1 wk (week 17). OGTTs were performed at 0, 4, 11, and 16 wk. A hyperinsulinemic hyperglycemic clamp was performed in week 12; 4 chow-fed dogs underwent identical clamps. Kilocalories per day intake during the treatment period was 2,067 ± 50 (Control) versus 1,824 ± 202 (FUM). Body weights (kg) increased 1.9 ± 0.3 vs. 2.7 ± 0.8 (0-4 wk) and 1.2 ± 0.2 vs. -0.02 ± 0.9 (4-12 wk) in Control versus fumagillin. The OGTT glycemic response was 30% greater in Control versus fumagillin at 11 wk. Net hepatic glucose uptake (NHGU; mg·kg-1·min-1) in the Chow, Control, and fumagillin dogs was ~1.5 ± 0.6, -0.1 ± 0.1, and 0.3 ± 0.4 (with no portal glucose infusion) and 3.1 ± 0.6, 0.5 ± 0.3, and 1.5 ± 0.5 (portal glucose infusion at 4 mg·kg-1·min-1), respectively. Fumagillin improved glucose tolerance and NHGU in HFFD dogs, suggesting methionine aminopeptidase 2 (MetAP2) inhibitors have the potential for improving glycemic control in prediabetes and diabetes.
Assuntos
Aminopeptidases/antagonistas & inibidores , Cicloexanos/farmacologia , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos Insaturados/farmacologia , Frutose/efeitos adversos , Glucose/metabolismo , Glucose/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Dieta , Cães , Ingestão de Alimentos/efeitos dos fármacos , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Resistência à Insulina , Masculino , Sesquiterpenos/farmacologiaRESUMO
PURPOSE: Coffee is widely consumed and implicated in numerous health outcomes but the mechanisms by which coffee contributes to health is unclear. The purpose of this study was to test the effect of coffee drinking on candidate proteins involved in cardiovascular, immuno-oncological and neurological pathways. METHODS: We examined fasting serum samples collected from a previously reported single blinded, three-stage clinical trial. Forty-seven habitual coffee consumers refrained from drinking coffee for 1 month, consumed 4 cups of coffee/day in the second month and 8 cups/day in the third month. Samples collected after each coffee stage were analyzed using three multiplex proximity extension assays that, after quality control, measured a total of 247 proteins implicated in cardiovascular, immuno-oncological and neurological pathways and of which 59 were previously linked to coffee exposure. Repeated measures ANOVA was used to test the relationship between coffee treatment and each protein. RESULTS: Two neurology-related proteins including carboxypeptidase M (CPM) and neutral ceramidase (N-CDase or ASAH2), significantly increased after coffee intake (P < 0.05 and Q < 0.05). An additional 46 proteins were nominally associated with coffee intake (P < 0.05 and Q > 0.05); 9, 8 and 29 of these proteins related to cardiovascular, immuno-oncological and neurological pathways, respectively, and the levels of 41 increased with coffee intake. CONCLUSIONS: CPM and N-CDase levels increased in response to coffee intake. These proteins have not previously been linked to coffee and are thus novel markers of coffee response worthy of further study. CLINICAL TRIAL REGISTRY: http://www.isrctn.com/ISRCTN12547806.
Assuntos
Ceramidases/sangue , Café/metabolismo , Metaloendopeptidases/sangue , Proteômica/métodos , Adulto , Biomarcadores/sangue , Café/enzimologia , Feminino , Finlândia , Proteínas Ligadas por GPI/sangue , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Enterotoxigenic Bacteroides fragilis (ETBF) is human intestinal commensal bacterium and a potent initiator of colitis through secretion of the metalloprotease Bacteroides fragilis toxin (BFT). BFT induces cleavage of E-cadherin in colon cells, which subsequently leads to NF-κB activation. Zerumbone is a key component of the Zingiber zerumbet (L.) Smith plant and can exhibit anti-bacterial and anti-inflammatory effects. However, whether zerumbone has anti-inflammatory effects in ETBF-induced colitis remains unknown. The aim of this study was to determine the anti-inflammatory effect of orally administered zerumbone in a murine model of ETBF infection. Wild-type C57BL/6 mice were infected with ETBF and orally administered zerumbone (30 or 60 mg/kg) once a day for 7 days. Treatment of ETBF-infected mice with zerumbone prevented weight loss and splenomegaly and reduced colonic inflammation with decreased macrophage infiltration. Zerumbone treatment significantly decreased expression of IL-17A, TNF-α, KC, and inducible nitric oxide synthase (iNOS) in colonic tissues of ETBF-infected mice. In addition, serum levels of KC and nitrite was also diminished. Zerumbone-treated ETBF-infected mice also showed decreased NF-κB signaling in the colon. HT29/C1 colonic epithelial cells treated with zerumbone suppressed BFT-induced NF-κB signaling and IL-8 secretion. However, BFT-mediated E-cadherin cleavage was unaffected. Furthermore, zerumbone did not affect ETBF colonization in mice. In conclusion, zerumbone decreased ETBF-induced colitis through inhibition of NF-κB signaling.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Bacteroides/tratamento farmacológico , Bacteroides fragilis , Colite/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Sesquiterpenos/uso terapêutico , Animais , Toxinas Bacterianas , Infecções por Bacteroides/imunologia , Bacteroides fragilis/metabolismo , Caderinas/metabolismo , Colite/imunologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/fisiopatologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células HT29 , Humanos , Interleucina-17/metabolismo , Interleucina-8/sangue , Metaloendopeptidases , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Hepatocellular carcinoma (HCC) tumors invariably develop resistance to cytotoxic and targeted agents, resulting in failed treatment and tumor recurrence. Previous in vivo short hairpin RNA (shRNA) screening evidence revealed mitochondrial-processing peptidase (PMPC) as a leading gene contributing to tumor cell resistance against sorafenib, a multikinase inhibitor used to treat advanced HCC. Here, we investigated the contributory role of the ß subunit of PMPC (PMPCB) in sorafenib resistance. Silencing PMPCB increased HCC tumor cell susceptibility to sorafenib therapy, decreased liver tumor burden, and improved survival of tumor-bearing mice receiving sorafenib. Moreover, sorafenib + PMPCB shRNA combination therapy led to attenuated liver tumor burden and improved survival outcome for tumor-bearing mice, and it reduced colony formation in murine and human HCC cell lines in vitro. Additionally, PMPCB silencing enhanced PINK1-Parkin signaling and downregulated the anti-apoptotic protein MCL-1 in sorafenib-treated HCC cells, which is indicative of a healthier pro-apoptotic phenotype. Higher pre-treatment MCL-1 expression was associated with inferior survival outcomes in sorafenib-treated HCC patients. Elevated MCL-1 expression was present in sorafenib-resistant murine HCC cells, while MCL-1 knockdown sensitized these cells to sorafenib. In conclusion, our findings advocate combination regimens employing sorafenib with PMPCB knockdown or MCL-1 knockdown to circumvent sorafenib resistance in HCC patients.
Assuntos
Carcinoma Hepatocelular/patologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/patologia , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , RNA Interferente Pequeno/administração & dosagem , Sorafenibe/administração & dosagem , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Camundongos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Peptidase de Processamento MitocondrialRESUMO
Quorum sensing (QS) is the cell density dependent communication network which coordinates the production of pathogenic determinants in majority of pathogenic bacteria. Pseudomonas aeruginosa causes hospital-acquired infections by virtue of its well-defined QS network. As the QS regulatory network in P. aeruginosa regulates the virulence determinants and antibiotic resistance, attenuating the QS system seems to be influential in developing next-generation anti-infective agents. In the current study, the QS attenuation potential of a flavonoid, mosloflavone was investigated against P. aeruginosa virulence and biofilm formation. Mosloflavone inhibited the pyocyanin production, LasB elastase and chitinase by 59.52⯱â¯2.74, 35.90⯱â¯4.34 and 61.18⯱â¯5.52% respectively. The QS regulated biofilm formation and development was also reduced when supplemented with sub-MIC of mosloflavone. The gene expression studies of mosloflavone using RT-PCR depicted its ability to down-regulate the expression levels of QS regulated virulence genes such as lasI (60.64%), lasR (91.70%), rhlI (57.30%), chiC (90.20%), rhlA (47.87%), rhlR (21.55%), lasB (37.80%), phzM (42.40%), toxA (61.00%), aprA (58.4%), exoS (78.01%), algD (46.60%) and pelA (50.45%). The down-regulation of QS virulence phenotypes by mosloflavone could be attributed to its binding affinity with the QS regulatory proteins, LasR and RhlR by competitively inhibiting the binding of natural autoinducers as evidenced from simulation studies. Mosloflavone also exhibited promising potential in controlling bacterial infection in Caenorhabditis elegans model system, in vivo. The anti-biofilm and anti-QS potential of mosloflavone in the current study illustrated the candidature of mosloflavone as a promising biocide.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Flavonoides/farmacologia , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Alginatos , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans , Quitinases/metabolismo , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metaloendopeptidases/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Piocianina/metabolismo , Transativadores/genética , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND: 2-hydroxy-3-methylanthraquinone (HMA), an anthraquinone monomer in traditional Chinese medicine Hedyotis diffusa, has been reported to inhibit the growth of several types of cancer, but its effect on lung cancer has not been adequately investigated. HYPOTHESIS/PURPOSE: This study aimed to test the hypothesis that HMA inhibit the growth, migration, and invasion of lung cancer cells in part via downregulation of interleukin (IL)-6-induced JAK2/STAT3 pathway. METHODS: Growth and apoptosis of lung cancer cells were quantitated by CCK-8 assay and Annexin V-FITC/PI flow cytometric analysis, respectively. Migration and invasion of A549 cells were determined by wound-healing assay and transwell invasion assay, respectively. The effect of HMA on cytokines expression in A549 cells was evaluated by the cytokine antibody array assay. Gene expression and protein levels of related molecular markers were quantitated by real time-PCR and Western blot analysis, respectively. RESULTS: HMA significantly inhibited IL-6-stimulated growth and colony formation of A549 cells, increased the number of apoptotic cells, and inhibited invasion associated with downregulation of expression of IL-6-induced MMP-1, MMP-2, and MMP-9 genes. IL-6 increased the levels of tyrosine phosphorylation of JAK2 and STAT3 in A549 cells, which was reversed by HMA treatment. In addition, HMA reduced the expression of a series of inflammation-related cytokines in A549 cells supernatant, including IL-6, G-CSF, IL-6R, IL-8, MCP-1, RANTES, TNF-α. CONCLUSION: These results suggest that HMA may inhibit the growth and invasion of lung cancer cells in part via downregulation of IL-6-induced JAK2/STAT3 pathway.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Janus Quinase 2/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo , Células A549 , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Medicamentos de Ervas Chinesas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Neoplasias Pulmonares/patologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The RAW264.7 cell model was employed to screen immunomodulatory selenium-containing peptides from selenium-enriched rice protein hydrolysates (SPHs). Moreover, the selenium-containing peptides of high-activity protein hydrolysates were purified by Sephadex G-25, and identified by reversed phase ultra performance liquid chromatography coupled with triple quadrupole time-of-flight mass spectrometry. The results showed that 25 peptide sequences containing selenomethionine (SeMet) information above 90% of probability confidence were found in a fraction of alcalase hydrolysates. SeMDPGQQ and TSeMMM of 100% probability confidence were speculated as two novel selenium-containing peptide sequences. The artificially synthesized peptide TSeMMM was subsequently verified by an excellent immunomodulatory activity at a concentration of 80⯵g/mL. In conclusion, the immunomodulatory activity of SPHs was correlated to SeMet sequence in the structure of selenium-containing peptides, and TSeMMM with a stronger immunomodulatory activity demonstrated potential as functional food additives for improving human health.
Assuntos
Oryza/metabolismo , Peptídeos/análise , Hidrolisados de Proteína/química , Selênio/química , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fatores Imunológicos/análise , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Oryza/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Células RAW 264.7 , Selenometionina/química , SonicaçãoRESUMO
The aim of this study was to compare the treatment effects of laser photobiomodulation (LPBM) therapy and aerobic exercise on the biomechanical properties, tissue morphology and the expression of tendon matrix molecules during early remodeling of Achilles tendon (AT) injury in diabetic rats. Animals were randomly assigned to five groups: injured non diabetic (I, n = 15), injured diabetic (ID, n = 15), injured diabetic plus LPBM (IDL, n = 16), injured diabetic plus aerobic exercise (IDE, n = 16) and injured diabetic plus aerobic exercise and LPBM (IDEAL, n = 17). Type 1 diabetes was induced via a single intravenous injection of Streptozotocin at a dose of 40 mg/kg. A partial tenotomy was performed in the right AT. LPBM was performed with an indium-gallium-aluminum-phosphide 660 nm 10 mW laser device (spot size 0.04 cm2, power density 250 mW/cm2, irradiation duration 16 s, energy 0.16 J, energy density 4 J/cm2) on alternate days for a total of 9 sessions over 3 weeks (total energy 1.44 J), using a stationary contact technique to a single point over the dorsal aspect of the AT. Moderate aerobic exercise was performed on a motorized treadmill (velocity 9 m/min for 60 minutes). At 3 weeks post-injury, biomechanical analyzes as well as assessment of fibroblast number and orientation were performed. Collagen 1 (Col1) and 3 (Col3) and matrix metalloproteinases (MMPs) -3 and 13 protein distributions were studied by immunohistochemistry; while Col1 and Col3 and MMP-2 and 9 gene expression were assessed by quantitative RT-PCR (qRT-PCR). IDEAL exhibited significant increases in several biomechanical parameters in comparison to the other groups. Moreover, IDEAL presented stronger Col1 immunoreactivity when compared to ID, and weaker Col3 immunoreactivity than IDE. Both IDL and IDEAL demonstrated weaker expression of MMP-3 in comparison to I, while IDL presented no expression of MMP-13 when compared to ID. ID, IDL and IDE showed an increased number of fibroblasts in comparison to I, while IDEAL decreased the number of these cells in comparison to ID and IDE. IDL and IDEAL groups exhibited decreased angular dispersion among the fibroblasts when compared to I. The gene expression results showed that IDE demonstrated a downregulation in Col1 mRNA expression in comparison to I and ID. IDEAL demonstrated upregulation of Col1 mRNA expression when compared to IDL or IDE alone and increased MMP-2 expression when compared to IDL and IDE. MMP-9 expression was upregulated in IDEAL when compared to I, IDL and IDE. Our results suggest a beneficial interaction of combining both treatment strategies i.e., aerobic exercise and LPBM, on the biomechanical properties, tissue morphology and the expression of matrix molecules in diabetic tendons.
Assuntos
Tendão do Calcâneo/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Traumatismos dos Tendões/terapia , Tendão do Calcâneo/metabolismo , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Fibroblastos/metabolismo , Terapia com Luz de Baixa Intensidade/métodos , Masculino , Metaloendopeptidases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Estreptozocina/farmacologia , Traumatismos dos Tendões/etiologia , Traumatismos dos Tendões/metabolismo , Traumatismos dos Tendões/fisiopatologia , Regulação para Cima/fisiologia , Cicatrização/fisiologiaRESUMO
Oxidative stress caused by free radicals has been implicated in several human disorders. Dietary antioxidants can help the body to counteract those reactive species and reduce oxidative stress. Antioxidant activity is one of the multiple health-promoting attributes assigned to bovine whey products. The present study investigated whether this activity was retained during upper gut transit using a static simulated in vitro gastrointestinal digestion (SGID) model. The capacity to scavenge free radicals and reduce ferric ion of whey protein isolate (WPI), individual whey proteins, and hydrolysates pre- and post-SGID were measured and compared using various antioxidant assays. In addition, the free AA released from individual protein fractions in physiological gut conditions were characterized. Our results indicated that the antioxidant activity of WPI after exposure to the harsh conditions of the upper gut significantly increased compared with intact WPI. From an antioxidant bioactivity viewpoint, this exposure negates the need for prior hydrolysis of WPI. The whey protein α-lactalbumin showed the highest antioxidant properties post-SGID (oxygen radical absorbance capacity = 1,825.94 ± 50.21 µmol of Trolox equivalents/g of powder) of the 4 major whey proteins tested with the release of the highest amount of the antioxidant AA tryptophan, 6.955 µmol of tryptophan/g of protein. Therefore, α-lactalbumin should be the preferred whey protein in food formulations to boost antioxidant defenses.
Assuntos
Antioxidantes/metabolismo , Trato Gastrointestinal/metabolismo , Proteínas do Soro do Leite/metabolismo , Animais , Antioxidantes/administração & dosagem , Bromelaínas/metabolismo , Bovinos , Cromanos/administração & dosagem , Cromanos/metabolismo , Digestão , Sequestradores de Radicais Livres/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Lactalbumina/metabolismo , Metaloendopeptidases/metabolismo , Proteínas do Leite/metabolismo , Estresse Oxidativo , Subtilisinas/metabolismo , Soro do Leite/química , Proteínas do Soro do Leite/administração & dosagemRESUMO
Hutchinson-Gilford progeria syndrome (HGPS) is a very rare fatal disease characterized for accelerated aging. Although the causal agent, a point mutation in LMNA gene, was identified more than a decade ago, the molecular mechanisms underlying HGPS are still not fully understood and, currently, there is no cure for the patients, which die at a mean age of thirteen. With the aim of unraveling non-previously altered molecular pathways in the premature aging process, human cell lines from HGPS patients and from healthy parental controls were studied in parallel using Next-Generation Sequencing (RNAseq) and High-Resolution Quantitative Proteomics (iTRAQ) techniques. After selection of significant proteins and transcripts and crosschecking of the results a small set of protein/transcript pairs were chosen for validation. One of those proteins, ribose-phosphate pyrophosphokinase 1 (PRPS1), is essential for nucleotide synthesis. PRPS1 loss-of-function mutants present lower levels of purine. PRPS1 protein and transcript levels are detected as significantly decreased in HGPS cell lines vs. healthy parental controls. This modulation was orthogonally confirmed by targeted techniques in cell lines and also in an animal model of Progeria, the ZMPSTE24 knock-out mouse. In addition, functional experiments through supplementation with S-adenosyl-methionine (SAMe), a metabolite that is an alternative source of purine, were done. Results indicate that SAMe has a positive effect in the proliferative capacity and reduces senescence-associated Beta-galactosidase staining of the HPGS cell lines. Altogether, our data suggests that nucleotide and, specifically, purine-metabolism, are altered in premature aging, opening a new window for the therapeutic treatment of the disease.
Assuntos
Lamina Tipo A/genética , Progéria/genética , Purinas/metabolismo , RNA Mensageiro/genética , Ribose-Fosfato Pirofosfoquinase/genética , Adulto , Animais , Linhagem Celular , Proliferação de Células , Criança , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Efeito Fundador , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lamina Tipo A/deficiência , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Progéria/tratamento farmacológico , Progéria/metabolismo , Progéria/patologia , RNA Mensageiro/metabolismo , Ribose-Fosfato Pirofosfoquinase/deficiência , S-Adenosilmetionina/farmacologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Cancer is a major cause of mortality. The present study evaluates the antitumor effects of Ferula hezarlalehzarica Y. Ajani fractions on various cancer cell lines, including the Raji Burkitt's lymphoma cells. METHODS: We evaluated the cytotoxic activity of various fractions of F. hezarlalehzarica against tumor cell lines by the MTT assay. Annexin V-PE/7-AAD and cell cycle analysis were assessed by flow cytometry. Expressions of genes associated with cell death and proliferation (Bax, Bcl-2, Fas, and c-Myc) were determined using real-time PCR. Alteration in mitochondrial membrane potential (MMP) was examined by JC-1 dye staining. RESULTS: The hexane fraction of F. hezarlalehzarica showed the highest degree of cytotoxicity against Raji cells (IC50 = 31.6 µg/ml). Flow cytometry analysis showed that 200 µg/ml of the fraction induced apoptosis in >96% of Raji cells after 24 h. In cell cycle analysis, at the same concentration, the percentage of apoptotic cells in the sub G1phase increased to 95.25 ± 1.76% at 48 h of treatment. The fraction induced cell cycle arrestat the G0/G1phase. Exposure to 100 µg/ml of the fraction after 48 h increased the percentage of G0/G1 cells (76.3 ± 6.08%) compared to the negative control (<50%). Treatment with75µg/ml of fraction reduced the expressions of Bcl-2 (0.23 ± 0.008-fold) and c-Myc (0.68 ± 0.07-fold) and increased Bax (1.75 ± 0.31-fold) and Fas (5.02 ± 0.74-fold; p < 0.01). We observed a decrease in MMP (≈0.4, p < 0.05) at ≥100 µg/ml and this effect remained almost unchanged until 48 h. CONCLUSIONS: The F. hezarlalehzarica hexane fraction induced apoptosis in Raji cells by changing the expression of apoptosis-related genes, cell cycle distribution, and MMP. These data suggested a potential effectiveness of F. hezarlalehzarica for inducing cell death in lymphoma cells. Graphical abstract á .
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Linfoma de Burkitt/genética , Ferula/química , Hexanos/farmacologia , Mitocôndrias/fisiologia , Antineoplásicos Fitogênicos/química , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Células Hep G2 , Hexanos/química , Humanos , Células K562 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metaloendopeptidases/genética , Mitocôndrias/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
Ultraviolet B radiation represents 10% of the total UV radiation that reaches the Earth's surface, being the primary responsible for the biological effects related to skin cancer and photoaging. Ilex Paraguariensis A. St. Hil., known as Yerba mate (YM), is a native tree of South America whose polyphenols in its leaves are described to exhibit photochemoprotective effect and are employed in the treatment of cancer. Additionally, the polyphenols are used to prevent lipid peroxidation and reduce the UV-induced damage, which ultimately decreases the oxidative stress. Thus, the present study aimed to characterize a new YM extract, evaluate the extract cytotoxicity and develop a formulation containing YM extract to prevent UVB-induced damage in mice skin. The YM extract showed high levels of polyphenols, flavonoids, and tannins and exhibited excellent antioxidant activity. Its main components were suggested as chlorogenic acid (1.92%) and caffeic acid (0.41%). Besides, YM extract did not exhibit cytotoxicity in fibroblasts and decreased the activity of myeloperoxidase and metalloproteinase-2 after acute UVB exposure. As a result, the formulation containing the YM extract showed a potential photochemoprotective.
Assuntos
Ilex paraguariensis/química , Metaloendopeptidases/efeitos dos fármacos , Peroxidase/efeitos dos fármacos , Extratos Vegetais/farmacologia , Raios Ultravioleta , Administração Tópica , Animais , Ácidos Cafeicos , Ácido Clorogênico , Metaloendopeptidases/metabolismo , Camundongos , Peroxidase/metabolismo , Polifenóis , Substâncias ProtetorasRESUMO
AIMS/HYPOTHESIS: This multicentre randomised double-blind placebo-controlled clinical trial assessed the efficacy and safety of a methionine aminopeptidase 2 (MetAP2) inhibitor, beloranib, in individuals with obesity (BMI ≥30 kg/m2) and type 2 diabetes (HbA1c 53-97 mmol/mol [7-11%] and fasting glucose <15.6 mmol/l). METHODS: Participants were randomised (via a centralised interactive web response system) to placebo, 1.2 or 1.8 mg beloranib s.c. twice weekly for 26 weeks. Participants, investigators and the sponsor were blinded to group assignment. The primary endpoint was the change in weight from baseline to week 26. The trial was terminated early when beloranib development was stopped because of an imbalance of venous thromboembolism events in beloranib-treated individuals vs placebo that became evident during late-stage development of the drug. RESULTS: In total, 153 participants were randomised, 51 to placebo, 52 to 1.2 mg beloranib and 50 to 1.8 mg beloranib. In participants who completed week 26, the least squares mean ± SE weight change (baseline 111 kg) was -3.1 ± 1.2% with placebo (n = 22) vs -13.5 ± 1.1% and -12.7 ± 1.3% with 1.2 and 1.8 mg beloranib, respectively (n = 25; n = 19; p < 0.0001). The change in HbA1c (baseline 67 mmol/mol [8.3%]) was -6.6 ± 2.2 mmol/mol (-0.6 ± 0.2%) with placebo vs -21.9 ± 2.2 mmol/mol (-2.0 ± 0.2%) or -21.9 ± 3.3 mmol/mol (-2.0 ± 0.3%) with 1.2 or 1.8 mg beloranib (p < 0.0001), respectively. The most common beloranib adverse events were sleep related. One beloranib-treated participant experienced a non-fatal pulmonary embolism. CONCLUSIONS/INTERPRETATION: MetAP2 inhibitors represent a novel mechanism for producing meaningful weight loss and improvement in HbA1c. TRIAL REGISTRATION: ClinicalTrials.gov NCT02324491 FUNDING: The study was funded by Zafgen, Inc.
Assuntos
Aminopeptidases/antagonistas & inibidores , Cinamatos/uso terapêutico , Cicloexanos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Compostos de Epóxi/uso terapêutico , Metaloendopeptidases/antagonistas & inibidores , Sesquiterpenos/uso terapêutico , Adolescente , Adulto , Idoso , Aminopeptidases/metabolismo , Fármacos Antiobesidade/uso terapêutico , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Método Duplo-Cego , Feminino , Glucose/metabolismo , Hemoglobinas Glicadas/metabolismo , Glicoproteínas , Humanos , Hipoglicemiantes/uso terapêutico , Masculino , Metaloendopeptidases/metabolismo , Metionil Aminopeptidases , Pessoa de Meia-Idade , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Redução de Peso/efeitos dos fármacos , Adulto JovemRESUMO
Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin-streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.
Assuntos
Metaloendopeptidases/metabolismo , Rutênio/metabolismo , Tri-Iodotironina/metabolismo , Biotina/química , Biotina/metabolismo , Biotinilação , Catálise , Células HEK293 , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/genética , Estrutura Molecular , Mutação Puntual , Rutênio/química , Estereoisomerismo , Estreptavidina/química , Estreptavidina/metabolismo , Tri-Iodotironina/genéticaRESUMO
Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Ferro/metabolismo , Fatores de Transcrição/metabolismo , Vibrio vulnificus/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Amidas/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Grupo dos Citocromos b/genética , Citocromos c/metabolismo , DNA Bacteriano , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Hemina/metabolismo , Hemoglobinas/metabolismo , Humanos , Hidrogenase/genética , Transferases Intramoleculares/metabolismo , Metaloendopeptidases/metabolismo , Oxazóis/metabolismo , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Sequência , Deleção de Sequência , Fatores de Transcrição/genética , Transcrição Gênica , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).