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1.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1568-1577, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924355

RESUMO

Catalase catalyzes the decomposition of H2O2 to H2O and O2, and has a wide range of industrial applications. However, most catalases used in the textile and paper industries are often subjected to high-alkaline challenges which makes it necessary to develop alkaline catalase. In this study, a catalase from Corynebacterium glutamicum was expressed in Escherichia coli, and the expression conditions were optimized. The recombinant catalase was purified by Ni-chelating affinity chromatography, and the recombinant enzyme was characterized. The optimal conditions of producing the recombinant catalase were: an IPTG concentration of 0.2 mmol/L, a culturing temperature of 25 °C and a culturing time of 11 h. The purified catalase had a specific activity of 55 266 U/mg, and it had a high activity in the pH range of 4.0 to11.5, with the highest activity at pH 11.0. When treated in pH 11.0 for 3 h, the enzyme retained 93% of its activity, indicating that the enzyme was qualified with a favorable stability under high-alkaline condition. The recombinant catalase had maximal activity at 30 °C, and showed a satisfactory thermal stability at a range of 25 °C to 50 °C. The apparent Km and Vmax values of purified catalase were 25.89 mmol/L and 185.18 mmol/(minmg), respectively. Besides, different inhibitors, such as sodium dodecyl sulfate (SDS), urea, NaN2, ß-mercaptoethanol, and EDTA had different degrees of inhibition on enzyme activity. The catalase from C. glutamicum shows high catalytic efficiency and high alkaline stability, suggesting its potential utilization in industrial production.


Assuntos
Catalase , Corynebacterium glutamicum , Regulação Enzimológica da Expressão Gênica , Catalase/genética , Catalase/isolamento & purificação , Catalase/metabolismo , Corynebacterium glutamicum/enzimologia , Ativação Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
2.
PLoS Pathog ; 16(8): e1008131, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866196

RESUMO

Invasion of hepatocytes by Plasmodium sporozoites initiates the pre-erythrocytic step of a malaria infection. Subsequent development of the parasite within hepatocytes and exit from them is essential for starting the disease-causing erythrocytic cycle. Identification of signaling pathways that operate in pre-erythrocytic stages provides insight into a critical step of infection and potential targets for chemoprotection from malaria. We demonstrate that P. berghei homologs of Calcium Dependent Protein Kinase 1 (CDPK1), CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP can compensate for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome loss of CDPK4. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages implicate these kinases in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays initiation of the erythrocytic cycle without affecting infectivity of hepatic merozoites. These data suggest that CDPK5 may be required for the rupture of merosomes. Our work provides evidence that sporozoite invasion requires CDPK1 and CDPK5, and suggests that CDPK5 participates in the release of hepatic merozoites.


Assuntos
Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Malária/epidemiologia , Merozoítos/enzimologia , Plasmodium berghei/enzimologia , Proteínas Quinases/biossíntese , Proteínas de Protozoários/biossíntese , Esporozoítos/enzimologia , Animais , Eritrócitos/enzimologia , Eritrócitos/parasitologia , Feminino , Células Hep G2 , Humanos , Fígado/enzimologia , Fígado/parasitologia , Malária/patologia , Camundongos
4.
PLoS One ; 15(7): e0236597, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735634

RESUMO

INTRODUCTION: McArdle disease presents clinical and genetic heterogeneity. There is no obvious association between genotype and phenotype. PYGM (muscle glycogen phosphorylase gene) mRNA expression and its association with clinical, morphological, and genetic aspects of the disease as a set have not been studied previously. METHODS: We investigated genetic variation in PYGM considering the number of PTCs (premature termination codon) per sample and compared mRNA expression in skeletal muscle samples from 15 patients with McArdle disease and 16 controls to PTCs number and different aspects of the disease. RESULTS: The main variant found was c.148C>T (PTC-premature termination codon). Patients with two PTCs showed 42% mRNA expression compared to the control group. Most cases showed an inversely proportional relation among PTCs and mRNA expression. Association between mRNA expression and other aspects of the disease showed no statistically significant difference (p> 0.05). DISCUSSION: mRNA expression is not useful as a predictor factor for the prognosis and severity of the disease. Different mechanisms as post-transcriptional events, epigenetics factors or protein function may be involved.


Assuntos
Demografia , Regulação Enzimológica da Expressão Gênica , Glicogênio Fosforilase Muscular/genética , Doença de Depósito de Glicogênio Tipo V/genética , Adulto , Códon sem Sentido/genética , Estudos Transversais , Feminino , Doença de Depósito de Glicogênio Tipo V/epidemiologia , Doença de Depósito de Glicogênio Tipo V/patologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Adulto Jovem
5.
Chem Biol Interact ; 330: 109216, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810488

RESUMO

In our previous study, an antimutagenic compound from spinach (Spinacea oleracea L.), ethoxy-substituted phylloquinone (ESP) was isolated and characterized. The current study deals with elucidation of the possible mechanism of antimutagenicity of ESP against ethyl methanesulfonate (EMS) deploying model systems such as human lymphoblast (TK+/- or TK6) cell line (thymidine kinase gene mutation assay) and Escherichia coli MG1655 (rifampicin resistance assay). Findings of the study ruled out the possibility of direct inactivation of EMS by ESP. DAPI competitive binding assay indicated the DNA minor groove binding activity of ESP. Interestingly, ESP did not display major groove binding or intercalating abilities. Further, proteomics study using 2-D gel electrophoresis in E. coli and subsequent studies involving single gene knockout strains revealed the possible role of tnaA (tryptophanase) and dgcP (diguanylate cyclase) genes in observed antimutagenicity. These genes have been reported to be involved in indole and cyclic-di-GMP biosynthesis, respectively, which eventually lead to cell division inhibition. In case of TK+/- cell line system, ADCY genes (adenylate cyclase), a functional analogue of dgcP gene, were found to be transcriptionally up-regulated. The generation/doubling time were significantly higher in E. coli or TK+/- cells treated with ESP than control cells. The findings indicated inhibition of cell proliferation by ESP through gene regulation as a possible mechanism of antimutagenicity across the biological system. Cell division inhibition actually provides additional time for the repair of damaged DNA leading to antimutagenicity.


Assuntos
Mutagênese/efeitos dos fármacos , Spinacia oleracea/química , Vitamina K 1/química , Vitamina K 1/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Liases/metabolismo , Vitamina K 1/metabolismo
6.
Int J Nanomedicine ; 15: 5005-5016, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764932

RESUMO

Background and Aim: With the wide applications of chitosan and gold nanoparticles in drug delivery and many consumer products, there is limited available information about their effects on drug-metabolizing enzymes (DMEs). Changes in DMEs could result in serious drug interactions. Therefore, this study aimed to investigate the effects of exposure to chitosan or gold nanoparticles on hepatic Phase I and II DMEs, liver function and integrity, oxidative damage and liver architecture in male rats. Methods: Animals were divided into three equal groups: a control group, a group treated with chitosan nanoparticles (200 mg/kg, 50±5 nm) and a group treated with gold nanoparticles (4 mg/kg, 15±5 nm). Rats were orally administered their respective doses daily for 10 days. Results: Both chitosan and gold nanoparticles decreased the body weights by more than 10%. Gold nanoparticles reduced the activities of antioxidants (superoxide dismutase and catalase), and reduced glutathione level and elevated the malondialdehyde level in the liver. Gold nanoparticles caused significant reductions in CYP1A1, CYP2E1, quinone oxidoreductase1, and glutathione S-transferase and elevated CYP2D6 and N-acetyl transferase2. Chitosan elevated CYP2E1 and CYP2D6 and reduced UDP-glucuronosyltransferase 1A1. Both nanoparticles disturbed the architecture of the liver, but the deleterious effects after gold nanoparticles treatment were more prominent. Conclusion: Taken together, gold nanoparticles severely perturbed the DMEs and would result in serious interactions with many drugs, herbs, and foods.


Assuntos
Antioxidantes/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inativação Metabólica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Animais , Catalase/genética , Catalase/metabolismo , Quitosana/química , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Interações Medicamentosas , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Ouro/química , Ouro/farmacocinética , Fígado/metabolismo , Fígado/patologia , Masculino , Malondialdeído/metabolismo , Nanopartículas Metálicas/química , Ratos Wistar , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(33): 20316-20324, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737163

RESUMO

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Glucanos/biossíntese , Glucosiltransferases/metabolismo , Células Vegetais/metabolismo , Xilanos/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Mutação , Filogenia
8.
Nat Commun ; 11(1): 3532, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669539

RESUMO

Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/enzimologia , Proteína Fosfatase 1/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Proliferação de Células , GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Fosfatidilcolinas/química , Domínios Proteicos , Proteoma , Ubiquitina-Proteína Ligases/metabolismo
9.
PLoS Negl Trop Dis ; 14(7): e0008447, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730343

RESUMO

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.


Assuntos
Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Helminto/metabolismo , Diester Fosfórico Hidrolases/genética , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Animais , Linhagem Celular , Deleção de Genes , Perfilação da Expressão Gênica , Genoma Helmíntico , Proteínas de Helminto/genética , Masculino , Camundongos , Filogenia , Trypanosoma brucei brucei , Leveduras
11.
Proc Natl Acad Sci U S A ; 117(33): 20180-20189, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32719134

RESUMO

Two-component signal transduction systems (TCSs) represent a major mechanism that bacteria use to sense and respond to their environment. Prototypical TCSs are composed of a membrane-embedded histidine kinase, which senses an environmental stimulus and subsequently phosphorylates a cognate partner protein called a response regulator that regulates gene expression in a phosphorylation-dependent manner. Vibrio cholerae uses the hybrid histidine kinase ChiS to activate the expression of the chitin utilization program, which is critical for the survival of this facultative pathogen in its aquatic reservoir. A cognate response regulator for ChiS has not been identified and the mechanism of ChiS-dependent signal transduction remains unclear. Here, we show that ChiS is a noncanonical membrane-embedded one-component system that can both sense chitin and directly regulate gene expression via a cryptic DNA binding domain. Unlike prototypical TCSs, we find that ChiS DNA binding is diminished, rather than stimulated, by phosphorylation. Finally, we provide evidence that ChiS likely activates gene expression by directly recruiting RNA polymerase. This work addresses the mechanism of action for a major transcription factor in V. cholerae and highlights the versatility of signal transduction systems in bacterial species.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/genética , Quitina/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/genética , Ligação Proteica , Vibrio cholerae/metabolismo
12.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 1018-1022, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701246

RESUMO

OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 µg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury (P < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma (P < 0.05) and increased further at 2 days (P < 0.01); the water content of the brain did not change significantly at 2 h (P > 0.05) but increased significantly 2 d after the injury (P < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma (P < 0.01). CONCLUSIONS: Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.


Assuntos
Edema Encefálico , Lesões Encefálicas Traumáticas , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz , Animais , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Indazóis/farmacologia , Indazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
13.
Nat Commun ; 11(1): 2689, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483206

RESUMO

The antiandrogen enzalutamide (Enz) has improved survival in castration resistant prostate cancer (CRPC) patients. However, most patients eventually develop Enz resistance that may involve inducing the androgen receptor (AR) splicing variant 7 (ARv7). Here we report that high expression of monoamine oxidase-A (MAO-A) is associated with positive ARv7 detection in CRPC patients following Enz treatment. Targeting MAO-A with phenelzine or clorgyline, the FDA-approved drugs for antidepression, resensitize the Enz resistant (EnzR) cells to Enz treatment and further suppress EnzR cell growth in vitro and in vivo. Our findings suggest that Enz-increased ARv7 expression can transcriptionally enhance MAO-A expression resulting in Enz resistance via altering the hypoxia HIF-1α signals. Together, our results show that targeting the Enz/ARv7/MAO-A signaling with the antidepressants phenelzine or clorgyline can restore Enz sensitivity to suppress EnzR cell growth, which may indicate that these antidepression drugs can overcome the Enz resistance to further suppress the EnzR CRPC.


Assuntos
Clorgilina/farmacologia , Inibidores da Monoaminoxidase/farmacologia , Fenelzina/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Estabilidade Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Monoaminoxidase/química , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Células Neoplásicas Circulantes/metabolismo , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Toxicol Appl Pharmacol ; 401: 115111, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32553695

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6 h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b > Tnfa> Ifng) within 6 h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.


Assuntos
Células da Medula Óssea/metabolismo , Citocromo P-450 CYP1B1/biossíntese , Regulação Enzimológica da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Células CHO , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Cricetinae , Cricetulus , Citocromo P-450 CYP1B1/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos
15.
PLoS One ; 15(6): e0234780, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579589

RESUMO

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.


Assuntos
Serina Proteases/metabolismo , Ganho de Peso , Animais , Fezes/enzimologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Células MCF-7 , Camundongos , Mitocôndrias/metabolismo , Serina Proteases/deficiência , Serina Proteases/genética , Transdução de Sinais
16.
Nat Commun ; 11(1): 2813, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499479

RESUMO

5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.


Assuntos
5-Aminolevulinato Sintetase/química , Regulação Enzimológica da Expressão Gênica , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Acil Coenzima A/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Protoporfiria Eritropoética/genética , Especificidade por Substrato
17.
PLoS One ; 15(6): e0234039, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555710

RESUMO

Sepsis is characterized by organ dysfunction due to a dysregulated immune response to infection. Currently, no effective treatment for sepsis exists. Platelets are recognized as mediators of the immune response and may be a potential therapeutic target for the treatment of sepsis. We previously demonstrated that NLRP3 inflammasome activation in sepsis-induced activated platelets was associated with multi-organ injury in the cecal-ligation puncture (CLP) rat model of sepsis. In this study, we tested the hypothesis that inhibition of NLRP3 would inhibit platelet activation and attenuate multi-organ injury in the CLP rat. CLP (n = 10) or Sham (n = 10) surgery were performed in male and female Sprague-Dawley rats. A subset of CLP rats were treated with MCC950 (50mg/kg/d), a specific NLRP3 inhibitor (CLP+MCC950, n = 10). At 72 hrs. post-CLP, blood and organs were harvested for analysis of platelet activation, NLRP3 activation, inflammation and end organ damage. Platelet activation increased from 8±0.8% in Sham to 16±1% in CLP, and was reduced to 9±1% in CLP+M rats (p<0.05). NLRP3 activation was also increased in platelets of CLP vs Sham. NLRP3 expression was unchanged in kidney and lung after CLP, but Caspase 1 expression and IL-1ß were increased. MCC950 treatment attenuated NLRP3 activation in platelets. Plasma, kidney, and lung levels of NLRP3 inflammasome associated cytokines, IL-1ß and IL-18, were significantly increased in CLP compared to Sham rats. Inhibition of NLRP3 normalized cytokine levels. Glomerular injury, pulmonary edema, and endothelial dysfunction markers were increased in CLP rats vs Sham. MCC950 treatment significantly decreased renal and pulmonary injury and endothelial dysfunction in CLP+M. Our results demonstrate a role for NLRP3 in contributing to platelet activation and multi-organ injury in sepsis.


Assuntos
Ceco/cirurgia , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Ativação Plaquetária/efeitos dos fármacos , Punções/efeitos adversos , Sepse/tratamento farmacológico , Sepse/fisiopatologia , Animais , Caspase 1/metabolismo , Citocinas/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/fisiopatologia , Ligadura/efeitos adversos , Masculino , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Sepse/metabolismo
18.
Am J Physiol Cell Physiol ; 319(2): C250-C257, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579474

RESUMO

The classic view of the red blood cell (RBC) presents a biologically inert cell that upon maturation has limited capacity to alter its physical properties. This view developed largely because of the absence of translational machinery and inability to synthesize or repair proteins in circulating RBC. Recent developments have challenged this perspective, in light of observations supporting the importance of posttranslational modifications and greater understanding of ion movement in these cells, that each regulate a myriad of cellular properties. There is thus now sufficient evidence to induce a step change in understanding of RBC: rather than passively responding to the surrounding environment, these cells have the capacity to actively regulate their physical properties and thus alter flow behavior of blood. Specific evidence supports that the physical and rheological properties of RBC are subject to active modulation, primarily by the second-messenger molecules nitric oxide (NO) and calcium-ions (Ca2+). Furthermore, an isoform of nitric oxide synthase is expressed in RBC (RBC-NOS), which has been recently demonstrated to have an active role in regulating the physical properties of RBC. Mechanical stimulation of the cell membrane activates RBC-NOS, leading to NO generation, which has several intracellular effects, including the S-nitrosylation of integral membrane components. Intracellular concentration of Ca2+ is increased upon mechanical stimulation via the recently identified mechanosensitive cation channel piezo1. Increased intracellular Ca2+ modifies the physical properties of RBC by regulating cell volume and potentially altering several important intracellular proteins. A synthesis of recent advances in understanding of molecular processes within RBC thus challenges the classic view of these cells and rather indicates a highly active cell with self-regulated mechanical properties.


Assuntos
Eritrócitos/metabolismo , Canais Iônicos/genética , Mecanotransdução Celular/genética , Óxido Nítrico Sintase/genética , Cálcio/metabolismo , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática/genética , Eritrócitos/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Canais Iônicos/sangue , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo
20.
Invest Ophthalmol Vis Sci ; 61(5): 29, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32421147

RESUMO

Purpose: Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) maintenance and remodeling. The present study aimed to determine whether transforming growth factor (TGF)-ß2 regulates MMP-2 and MMP-9 levels and activities in astrocytes derived from the optic nerve head (ONH) and the role of statins in such modulation. Methods: Primary astrocytes cultured from the lamina cribrosa of human donor ONHs were incubated with three types of statins (5 µg/mL) for 1 hour followed by recombinant TGF-ß2 (5 ng/mL) for various periods to test their effects. Levels and activities of MMP-2 and MMP-9 in astrocytes in vitro were determined by western blotting and zymography, respectively. Levels of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in astrocyte lysates were determined by western blotting, and those of phosphorylated myosin light chain (MLC) were determined by western blotting and immunocytochemistry. Results: MMP-2 and MMP-9 levels were upregulated by TGF-ß2 in human ONH astrocytes. Prior incubation with simvastatin, lovastatin, and atorvastatin inhibited TGF-ß2-mediated MMP-2 and MMP-9 expression and activities. Prior incubation with statins downregulated the TGF-ß2-induced phosphorylation of MYPT1 and MLC, which are downstream substrates of RhoA and ROCKs. Conclusions: Statins inhibited the TGF-ß2-mediated regulation of MMP-2 and MMP-9 by inhibiting the RhoA/ROCK signaling pathway. Considering the role of MMP in ECM remodeling, the present findings support the notion that statins positively impact ECM remodeling within the ONH.


Assuntos
Astrócitos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Adulto , Astrócitos/enzimologia , Atorvastatina/farmacologia , Western Blotting , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Lovastatina/farmacologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Disco Óptico/citologia , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , Fator de Crescimento Transformador beta2/farmacologia
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