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1.
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
2.
Chem Biol Interact ; 325: 109088, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32360554

RESUMO

Osteoarthritis (OA) is one of the most common degenerative joint diseases in aging people. The activation of chondrocytes and their dysregulation are closely related to the pathogenesis of OA. GPR55 is an unique orphan G-receptor which binds to cannabinoids. In this study, we explored the role of GPR55 in advanced glycation end productions (AGEs)- induced chondrocytes activation in cultured cells. We showed that AGEs dose dependently induced GPR55 expression in ATDC5 chondrocytes. The blockage of GPR55 by its newly discovered antagonist-CID16020046 mitigated AGEs- induced increase in cellular ROS and decrease in antioxidant NRF2. Moreover, CID16020046 showed a dose-response suppressive effect on AGEs- induced expression of the major inflammatory mediators, including COX-2 and iNOS, and the production of NO and PGE2. CID16020046 also dose responsively inhibited AGEs- induced key effectors of cartilage degradation such as MMP-3 and MMP-13. In consequence, CID16020046 showed robust inhibition on AGEs- induced type II collagen degradation. Mechanistically, our data demonstrated that CID16020046 mediated GPR55 blockage ameliorated AGEs- induced NF-κB activation as revealed by its inhibition on IκBα, nuclear p65 translocation and NF-κB promoter activity. Collectively, our study demonstrates that GPR55 signaling mediates AGEs- induced chondrocyte activation, and the targeted blockage of GPR55 pathway could be therapeutic choice in the treatment of osteoarthritis.


Assuntos
Compostos Azabicíclicos/farmacologia , Benzoatos/farmacologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Receptores de Canabinoides/metabolismo , Linhagem Celular , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Proteólise/efeitos dos fármacos
3.
Chem Biol Interact ; 325: 109115, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32380060

RESUMO

UDP-glucuronosyltransferases (UGTs) are a family of phase II drug metabolizing enzymes that catalyze glucuronidation of numerous endogenous and exogenous substrates. Carbon tetrachloride (CCl4) is widely used to develop liver injuries mimicking human liver diseases. However, effects of CCl4 on the expression and activities of UGTs and the mechanism have not been fully elucidated. The present study aims to elucidate the dysregulation patterns of major UGTs induced by CCl4. Biochemical and histopathological results showed that CCl4 exerted hepatotoxicity in rats. The mRNA levels of UGTs were all significantly reduced in acute liver injury rats. However, mRNA levels of UGT1A1, 1A6, 2B1 and 2B2 were up-regulated while the UGT2B3, 2B6 and 2B12 levels were reduced in chronic CCl4-induced liver fibrosis rats. The protein expression of UGT1A1, 1A6 and 2B were decreased in acute liver injury rats. UGT1A1 and 1A6 proteins were increased, whereas UGT2B protein was reduced in liver fibrosis rats. In addition, CCl4 inhibited the enzyme activities of UGTs in rats. Moreover, the dysregulation of UGTs was accompanied by the decreased mRNA expression of Nrf2, CAR, FXR, PXR, PPAR-α and their corresponding target genes, except for Nrf2, HO-1, AhR and CYP1A1 in liver fibrosis rats. These findings suggest that dysregulation of UGTs under CCl4 exposure is isoform-specific, which could have a complex impact on drug efficacy and endogenous metabolism. Different exposure durations of CCl4 (single vs multiple doses) could have differential effects on rat hepatic UGTs expression.


Assuntos
Tetracloreto de Carbono/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Glucuronosiltransferase/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fibrose , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismo
4.
PLoS One ; 15(5): e0232880, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401761

RESUMO

The southern king crab (SKC) Lithodes santolla is an important commercial species in southern South America. Fishing pressure has caused the deterioration of its stocks. Currently, culture techniques are being developed for producing SKC juveniles to enhance the natural population and to recover the fishing stock. Therefore, it is necessary to know about physiology, energetic and nutritional requirements for SKC maintenance in hatchery. Thus, this study aims to evaluate the biochemical and physiological changes in the midgut gland, muscle and hemolymph of juveniles, pre-adults and adults of wild SKC. The energetic reserves, digestive enzymes activity, amino acid profile and energy were quantified in twelve juveniles, ten pre-adult, and ten adult crabs. Juveniles showed high glycogen and low lipids in the midgut gland, and low proteins and low lactate in muscle. In the hemolymph, juveniles had high lipids. Pre-adults had high glycogen and lipids in the midgut gland, and both high protein and lactate in muscle. In the hemolymph, pre-adults had high lipids. Adults had low glycogen and high lipids in midgut gland, and both high proteins and high lactate in muscle. In hemolymph, adults had high glucose and lactate. Juveniles and pre-adults had high proteinase activity, whereas adults had high lipase activity. Major essential amino acids of SKC were arginine, methionine, and tryptophan, and the non-essential amino acids were glycine, aspartic acid and glutamic acid. On another hand, SKC had similar energy in the midgut gland and muscle, regardless of the ontogenetic stage. Moreover, we demonstrated that the biochemical energy calculation underestimates the actual measured values by a calorimeter. Thus, our results help to understand the physiological changes, energetic and nutritional requirements of L. santolla, and this study is a baseline for research on diet formulation for maintaining this species under culture conditions.


Assuntos
Anomuros/fisiologia , Aquicultura/métodos , Proteínas de Artrópodes/genética , Optogenética/métodos , Aminoácidos/análise , Ração Animal , Animais , Anomuros/citologia , Anomuros/crescimento & desenvolvimento , Dieta , Metabolismo Energético , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hemolinfa , Masculino , Músculos/química
5.
PLoS One ; 15(5): e0233208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428030

RESUMO

To facilitate functional investigation of the role of NADPH oxidase 1 (NOX1) and associated reactive oxygen species in cancer cell signaling, we report herein the development and characterization of a novel mouse monoclonal antibody that specifically recognizes the C-terminal region of the NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human colorectal cancer cell lines, and correlated protein expression with NOX1 mRNA expression and superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1 mRNA levels could not be demonstrated in colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200 formalin-fixed, paraffin-embedded tissue cores from human epithelial tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal adenocarcinomas, as well as adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the protein level in tumors versus histologically normal tissues in prostate, lung, ovarian, or breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of colon cancer and in normal and malignant human tissues using a thoroughly evaluated monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small intestinal cancer.


Assuntos
Adenocarcinoma/enzimologia , Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Intestino Delgado/enzimologia , NADPH Oxidase 1/biossíntese , Proteínas de Neoplasias/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Células CACO-2 , Neoplasias do Colo/genética , Células HT29 , Humanos , Intestino Delgado/patologia , Modelos Biológicos , NADPH Oxidase 1/genética , Proteínas de Neoplasias/genética
6.
PLoS One ; 15(5): e0233130, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469892

RESUMO

Low temperature is one of the abiotic factors limiting germination, growth and distribution of the plant in current plant-products industry, especially for the tropical vegetables in non-tropical area or other fields under cold temperature. Screening the plant with ability against cold temperature captured worldwide attention and exerted great importance. In our previous work, the anti-cold specie of Momordica Charantia L. seedlings was screened out. Yet, the molecular and physiological mechanisms underlying this adaptive process still remain unknown. This study was aimed to investigate adaption mechanism of anti-cold species of Momordica Charantia L. seedlings in genetical and metabolomics levels. Two species, cold-susceptible group (Y17) and cold-resistant group (Y54), were evaluated containing the indexes of malondialdehyde (MDA), hydrogen peroxide (H2O2), proline content, activities of antioxidant enzymes, metabolites changes and genes differentiation in plant tissues after cold treatment. It found that low temperature stress resulted in increased accumulation of MDA, H2O2 and proline content in two species, but less expressions in cold-resistant species Y54. As compared to Y17, cold-resistant species Y54 presented significantly enhanced antioxidant enzyme activities of POD (peroxidase), CAT (cataalase) and SOD (superoxide dismutase). Meanwhile, higher expressed genes encoded antioxidant enzymes and transcription factors when exposure to the low temperature were found in cold-resistant species Y54, and core genes were explored by Q-PCR validation, including McSOD1, McPDC1 and McCHS1. Moreover, plant metabolites containing amino acid, sugar, fatty acid and organic acid in Y54 were higher than Y17, indicating their important roles in cold acclimation. Meanwhile, initial metabolites, including amimo acids, polypeptides, sugars, organic acids and nucleobases, were apparently increased in cold resistant species Y54 than cold susceptible species Y17. Our results demonstrated that the Momordica Charantia L. seedlings achieved cold tolerance might be went through mobilization of antioxidant systems, adjustment of the transcription factors and accumulation of osmoregulation substance. This work presented meaning information for revealing the anti-cold mechanism of the Momordica Charantia L. seedlings and newsight for further screening of anti-cold species in other plant.


Assuntos
Resposta ao Choque Frio , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Momordica charantia/metabolismo , Oxirredutases/biossíntese , Proteínas de Plantas/biossíntese , Plântula/metabolismo , Antioxidantes/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo
7.
Mol Genet Genomics ; 295(4): 911-922, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32367255

RESUMO

Tyrosinase (TYR) converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA into L-dopaquinone, which can produce melanin pigment. The abrogation of the functional activity of TYR can result in albino skin and eye diseases because of a deficiency in melanin pigment production. In this study, we developed and characterized an inducible knockout TYR platform comprising the heat-inducible heat-shock-promoter-70-driving CRISPR/Cas9 system and a zU6-promoter-driving tyr single guide RNA (sgRNA) system to investigate the temporal expression of TYR genes. To overcome the difficulty of identifying zebrafish germline integrations and facilitate the observation of Cas9 expression, heart-specific cmlc2:enhanced green fluorescent protein (EGFP; used to confirm tyr sgRNA expression) and two selectable markers (P2A-mCherry and internal ribosomal entry site-EGFP) were applied in our system. Heat shock treatment administered to Cas9 transgenic embryos induced mCherry or EGFP fluorescence expression throughout the embryos' bodies, and Cas9 protein was detected 1 h after heat shock treatment. Mutations were created by direct injection and line crossing, which led to mosaic and complete depigmentation phenotypes in approximately 50% and 100% of the embryos, respectively. Using our system, conditional TYR knockout in zebrafish was achieved efficiently and simply.


Assuntos
Sistemas CRISPR-Cas/genética , Resposta ao Choque Térmico/genética , Monofenol Mono-Oxigenase/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Embrião não Mamífero , Desenvolvimento Embrionário , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Mutação em Linhagem Germinativa , Proteínas de Fluorescência Verde/genética , Melaninas/biossíntese , Melaninas/genética , Cadeias Leves de Miosina/genética , Regiões Promotoras Genéticas/genética , RNA/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
9.
J Autoimmun ; 112: 102463, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32303424

RESUMO

It has been reported that SARS-CoV-2 may use ACE2 as a receptor to gain entry into human cells, in a way similar to that of SARS-CoV. Analyzing the distribution and expression level of ACE2 may therefore help reveal underlying mechanisms of viral susceptibility and post-infection modulation. In this study, we utilized previously uploaded information on ACE2 expression in various conditions including SARS-CoA to evaluate the role of ACE2 in SARS-CoV and extrapolate that to COVID-19. We found that the expression of ACE2 in healthy populations and patients with underlying diseases was not significantly different. However, based on the elevated expression of ACE2 in cigarette smokers, we speculate that long-term smoking may be a risk factor for COVID-19. Analysis of ACE2 in SARS-CoV infected cells suggests that ACE2 is not only a receptor but is also involved in post-infection regulation, including immune response, cytokine secretion, and viral genome replication. Moreover, we constructed Protein-protein interaction (PPI) networks and identified hub genes in viral activity and cytokine secretion. Our findings may help clinicians and researchers gain more insight into the pathogenesis of SARS-CoV-2 and design therapeutic strategies for COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/enzimologia , Regulação Enzimológica da Expressão Gênica , Pulmão/enzimologia , Peptidil Dipeptidase A/biossíntese , Pneumonia Viral/enzimologia , Fumar/efeitos adversos , Infecções por Coronavirus/patologia , Humanos , Pandemias , Pneumonia Viral/patologia , Mapas de Interação de Proteínas
10.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G816-G826, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32146834

RESUMO

The gastrointestinal tract houses a reservoir of bacterial-derived enzymes that can directly catalyze the metabolism of drugs, dietary elements and endogenous molecules. Both host and environmental factors may influence this enzymatic activity, with the potential to dictate the availability of the biologically-active form of endogenous molecules in the gut and influence inter-individual variation in drug metabolism. We aimed to investigate the influence of the microbiota, and the modulation of its composition, on fecal enzymatic activity. Intrinsic factors related to the host, including age, sex and genetic background, were also explored. Fecalase, a cell-free extract of feces, was prepared and used in a colorimetric-based assay to quantify enzymatic activity. To demonstrate the functional effects of fecal enzymatic activity, we examined ß-glucuronidase-mediated cleavage of serotonin ß-d-glucuronide (5-HT-GLU) and the resultant production of free 5-HT by HPLC. As expected, ß-glucuronidase and ß-glucosidase activity were absent in germ-free mice. Enzymatic activity was significantly influenced by mouse strain and animal species. Sex and age significantly altered metabolic activity with implications for free 5-HT. ß-Glucuronidase and ß-glucosidase activity remained at reduced levels for nearly two weeks after cessation of antibiotic administration. This effect on fecalase corresponded to significantly lower 5-HT levels as compared with incubation with pre-antibiotic fecalase from the same mice. Dietary targeting of the microbiota using prebiotics did not alter ß-glucuronidase or ß-glucosidase activity. Our data demonstrate that multiple factors influence the activity of bacterial-derived enzymes which may have potential clinical implications for drug metabolism and the deconjugation of host-produced glucuronides in the gut.NEW & NOTEWORTHY This article explores a comprehensive range of host and environmental factors that introduce variability in the expression of bacterial-derived metabolic enzymes. Our results demonstrate that altered ß-glucuronidase activity has implications for the bioavailability of luminal serotonin. The experimental approach employed, fecalase, provides a mechanistic basis and translational platform to further delineate the functional outputs of altered metabolic activity, and the associated physiological effects of microbiota-targeted interventions on host response to drugs and host-produced glucuronides.


Assuntos
Fezes/química , Glucuronidase/metabolismo , Serotonina/metabolismo , beta-Glucosidase/metabolismo , Animais , Antibacterianos , Caspase 1/genética , Caspase 1/metabolismo , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Vida Livre de Germes , Glucuronidase/química , Glucuronidase/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prebióticos , Ratos , Ratos Sprague-Dawley , Serotonina/química , Fatores Sexuais , Suínos , beta-Glucosidase/química , beta-Glucosidase/genética
11.
Ann Parasitol ; 66(1): 13-18, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32198991

RESUMO

Two predominant forms of cutaneous leishmaniosis are anthroponotic CL (ACL) and zoonotic CL (ZCL) caused by Leishmania (L.) tropica and L. major in Iran and many countries, respectively. Since differential gene expression play an important role in outcome of the infection, we compared relative gene expression value of pyruvate kinase (PyrK) and tryparedoxin peroxidase (TryP) in metacyclic forms of Iranian isolates of L. major and L. tropica. Clinical isolates of CL patients were sampled in endemic foci of Iran and identified by PCR-RFLP. Then, we employed real-time PCR to evaluation of the expression level of PyrK and Tryp genes in L. major and L. tropica. By this comparison, up-regulation of PyrK and Tryp genes was observed in metacyclic stage. Moreover, the average mRNA expression of PyrK and Tryp genes in L. major was 1.69 and 3.72 folds of its expression in L. tropica isolates. The results of this study could open the new window for further investigations of the correspondence between parasite gene expression level and disease pathology. Species-specific parasite factors contributing to virulence and pathogenicity in the host may be mostly due to the some of the differential regulation of conserved genes between species.


Assuntos
Regulação Enzimológica da Expressão Gênica , Leishmania tropica , Peroxidases , Proteínas de Protozoários , Piruvato Quinase , Humanos , Irã (Geográfico) , Leishmania tropica/enzimologia , Leishmania tropica/genética , Leishmaniose Cutânea/parasitologia , Estágios do Ciclo de Vida/genética , Peroxidases/genética , Proteínas de Protozoários/genética , Piruvato Quinase/genética , Reação em Cadeia da Polimerase em Tempo Real
12.
Plant Mol Biol ; 103(4-5): 391-407, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32193788

RESUMO

Mitogen-activated protein kinases (MAPKs) are important in regulating plant development as well as stress response. In this study, we genome-widely identified 56 MAPK genes in upland cotton. These MAPK genes unequally distribute on 22 chromosomes of cotton genome, but no MAPK gene is located on At_Chr6, Dt_Chr6, At_Chr13 and Dt_Chr13. The exons and introns in GhMAPK gene family vary widely at the position, number and length. Furthermore, GhMAPK family can be divided into 4 groups (A, B, C and D), and the TEY type of T-loop exists in three groups (A, B and C), but the TDY type of T-loop is only in group D. Further study revealed that some GhMAPK genes (including GhMPK6) are preferentially expressed in elongating fibers. GhMPK6 maintains a high phosphorylation level in elongating fibers, and its phosphorylation was enhanced in fibers by phytohormones brassinosteroid (BR), ethylene and indole-3-acetic acid (IAA). Additionally, GhMPK6 could interact with GhMKK2-2 and GhMKK4, suggesting that GhMKK2-2/4-GhMPK6 module may be involved in phosphorylation of its downstream proteins for regulating fiber elongation of cotton.


Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Genoma de Planta , Gossypium/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Plantas/metabolismo , Fibra de Algodão , Regulação Enzimológica da Expressão Gênica , Estudo de Associação Genômica Ampla , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Plantas/genética
13.
Plant Mol Biol ; 103(3): 355-371, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32193789

RESUMO

KEYMESSAGE: Biphasic starch granules in maize ae mutant underwent the weak to strong SBEIIb-defective effect during endosperm development, leading to no birefringence in their exterior due to extended long branch-chains of amylopectin. Biphasic starch granules are usually detected regionally in cereal endosperm lacking starch branching enzyme (SBE). However, their molecular structure, formation mechanism, and regional distribution are unclear. In this research, biphasic starch granules were observed in the inner region of crown endosperm of maize ae mutant, and had poorly oriented structure with comb-like profiles in their exterior. The inner endosperm (IE) rich in biphasic starch granules and outer endosperm (OE) without biphasic starch granules were investigated. The starch had lower amylose content and higher proportion of long branch-chains of amylopectin in IE than in OE, and the exterior of biphasic starch granules had less amylose and more long branch-chains of amylopectin than the interior. Compared with OE, the expression pattern of starch synthesis related enzymes changed significantly in IE. The granule-bound starch synthase I activity within biphasic starch granules decreased slightly. The IE experienced more severe hypoxic stress than OE, and the up-regulated anaerobic respiration pathway indicated an increase in carbon consumption. The starch in IE underwent the SBEIIb-defective effect from weak to strong due to the lack of sufficient carbon inflow, leading to the formation of biphasic starch granules and their regional distribution in endosperm. The results provided information for understanding the biphasic starch granules.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Amido/metabolismo , Zea mays/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/classificação , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Endosperma/enzimologia , Endosperma/ultraestrutura , Amido/ultraestrutura
14.
Life Sci ; 250: 117546, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32184125

RESUMO

AIM: The enzyme 3-phosphoinositide-dependent protein kinase-1 (PDK1) is associated with cardiac and pathological remodeling and ion channel function regulation. However, whether it regulates hyperpolarization-activated cyclic nucleotide-modulated channels (HCNs) remains unclear. MAIN METHODS: In the atrial myocytes of heart-specific PDK1 "knockout" mouse model and neonatal mice, protein kinase B (AKT)-related inhibitors or agonists as well as knockdown or overexpression plasmids were used to study the relationship between PDK1 and HCNs. KEY FINDINGS: HCN1 expression and AKT phosphorylation at the Thr308 site were significantly decreased in atrial myocytes after PDK1 knockout or inhibition; in contrast, HCN2 and HCN4 levels were significantly increased. Also, a similar trend of HCNs expression has been observed in cultured atrial myocytes after PDK1 inhibition, as further demonstrated via immunofluorescence and patch-clamp experiments. Moreover, these results of PDK1 overexpression indicate an opposite trend compared with the previous experimental results. However, the results of PDK1 inhibition or overexpression could be reversed by activating or inhibiting AKT, respectively. SIGNIFICANCE: These results indicate that the PDK1-AKT signaling pathway is involved in the regulation of HCN mRNA transcription, protein expression, HCN current density, and cell membrane location.


Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Regulação Enzimológica da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio/metabolismo , Transdução de Sinais , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Células Cultivadas , Feminino , Deleção de Genes , Átrios do Coração/citologia , Masculino , Camundongos , Camundongos Knockout , Células Musculares/citologia , Técnicas de Patch-Clamp , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tirosina/metabolismo
15.
Gene ; 741: 144523, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32142858

RESUMO

Chitinases play an important role in many biological processes in crustaceans, including molting, digestion, and immunity. In order to further explore the immune defense mechanism of chitinase in Portunus trituberculatus, the PtCht-1 gene was cloned by RACE (rapid-amplification of cDNA ends). This cDNA with a full length of 1910 bp, and an ORF (open reading frame) 1749 bp, coded for 582 amino acid residues and was classified into P. trituberculatus chitinase GH18-group4. It had the typical structural characteristics of GH18 chitinase family. Real-time PCR was used to analyze the expression of PtCht-1 in different tissues, molting stages, after pathogen infection, and low salinity (11‰). PtCht-1 was expressed in all tissues, with the highest expression in the hepatopancreas. In the hepatopancreas of different molting stages, the expression level decreased successively during post-molt stages (A/B), pre-molt stage (D) and inter-molt stage (C). Under normal circumstances, after artificial infection with WSSV and Vibrio parahaemolyticus, the expression of PtCht-1 in hepatopancreas reached the maximum at 48 h, and in hemolymph at 72 h and 24 h, respectively. Overall PtCht-1 expression was up-regulated compared with the control group. Low salinity stress significantly inhibited the expression of PtCht-1, up to 42 folds. Under low salinity stress, the time when WSSV infection reached the peak was markedly delayed by at least 24 h. The results of this study indicate that PtCht-1, as an immune factor, is likely involved in pathogen defense of P. trituberculatus, the immune function of which may be inhibited to some extent after low salinity stress.


Assuntos
Braquiúros/genética , Quitinases/genética , Sistema Imunitário , Estresse Fisiológico/imunologia , Animais , Organismos Aquáticos/genética , Organismos Aquáticos/imunologia , Braquiúros/imunologia , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Filogenia , Salinidade , Alinhamento de Sequência
16.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G781-G792, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32090605

RESUMO

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of newborns. Although incompletely understood, NEC is associated with intestinal barrier dysfunction. E-cadherin, an adherens junction, is a protein complex integral in maintaining normal barrier homeostasis. Rho-associated protein kinase-1 (ROCK1) is a kinase that regulates the E-cadherin complex, and p120-catenin is a subunit of the E-cadherin complex that has been implicated in stabilizing the cadherin complex at the plasma membrane. We hypothesized that E-cadherin is decreased in NEC and that inhibition of ROCK1 would protect against adherens junction disruption. To investigate this, a multimodal approach was used: In vitro Caco-2 model of NEC (LPS/TNFα), rap pup model (hypoxia + bacteria-containing formula), and human intestinal samples. E-cadherin was decreased in NEC compared with controls, with relocalization from the cell border to an intracellular location. ROCK1 exhibited a time-dependent response to disease, with increased early expression in NEC and decreased expression at later time points and disease severity. Administration of ROCK1 inhibitor (RI) resulted in preservation of E-cadherin expression at the cell border, preservation of intestinal villi on histological examination, and decreased apoptosis. ROCK1 upregulation in NEC led to decreased association of E-cadherin to p120 and increased intestinal permeability. RI helped maintain the stability of the E-cadherin-p120 complex, leading to improved barrier integrity and protection from experimental NEC.NEW & NOTEWORTHY This paper is the first to describe the effect of ROCK1 on E-cadherin expression in the intestinal epithelium and the protective effects of ROCK inhibitor on E-cadherin stability in necrotizing enterocolitis.


Assuntos
Amidas/uso terapêutico , Caderinas/metabolismo , Infecções por Enterobacteriaceae/tratamento farmacológico , Enterocolite Necrosante/tratamento farmacológico , Piridinas/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Células CACO-2 , Cronobacter sakazakii , Indutores das Enzimas do Citocromo P-450 , Infecções por Enterobacteriaceae/microbiologia , Enterocolite Necrosante/microbiologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Intestinos/patologia , Ratos
17.
Exp Hematol ; 82: 43-52.e4, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32014431

RESUMO

Aged hematopoietic stem cells (HSCs) undergo biased lineage priming and differentiation toward production of myeloid cells. A comprehensive understanding of gene regulatory mechanisms causing HSC aging is needed to devise new strategies to sustainably improve immune function in aged individuals. Here, a focused short hairpin RNA screen of epigenetic factors reveals that the histone acetyltransferase Kat6b regulates myeloid cell production from hematopoietic progenitor cells. Within the stem and progenitor cell compartment, Kat6b is highly expressed in long-term (LT)-HSCs and is significantly decreased with aging at the transcript and protein levels. Knockdown of Kat6b in young LT-HSCs causes skewed production of myeloid cells at the expense of erythroid cells both in vitro and in vivo. Transcriptome analysis identifies enrichment of aging and macrophage-associated gene signatures alongside reduced expression of self-renewal and multilineage priming signatures. Together, our work identifies KAT6B as a novel epigenetic regulator of hematopoietic differentiation and a target to improve aged immune function.


Assuntos
Envelhecimento/metabolismo , Diferenciação Celular , Células Eritroides/enzimologia , Regulação Enzimológica da Expressão Gênica , Histona Acetiltransferases/biossíntese , Células Progenitoras Mieloides/enzimologia , Envelhecimento/genética , Envelhecimento/patologia , Animais , Epigênese Genética , Células Eritroides/patologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Histona Acetiltransferases/genética , Masculino , Camundongos , Camundongos Transgênicos , Células Progenitoras Mieloides/patologia , Transcriptoma
18.
Proc Natl Acad Sci U S A ; 117(8): 4053-4060, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041867

RESUMO

Small molecules can affect many cellular processes. The disambiguation of these effects to identify the causative mechanisms of cell death is extremely challenging. This challenge impacts both clinical development and the interpretation of chemical genetic experiments. CX-5461 was developed as a selective RNA polymerase I inhibitor, but recent evidence suggests that it may cause DNA damage and induce G-quadraplex formation. Here we use three complimentary data mining modalities alongside biochemical and cell biological assays to show that CX-5461 exerts its primary cytotoxic activity through topoisomerase II poisoning. We then show that acquired resistance to CX-5461 in previously sensitive lymphoma cells confers collateral resistance to the topoisomerase II poison doxorubicin. Doxorubicin is already a frontline chemotherapy in a variety of hematopoietic malignancies, and CX-5461 is being tested in relapse/refractory hematopoietic tumors. Our data suggest that the mechanism of cell death induced by CX-5461 is critical for rational clinical development in these patients. Moreover, CX-5461 usage as a specific chemical genetic probe of RNA polymerase I function is challenging to interpret. Our multimodal data-driven approach is a useful way to detangle the intended and unintended mechanisms of drug action across diverse essential cellular processes.


Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Naftiridinas/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Interferência de RNA , Sensibilidade e Especificidade
19.
Proc Natl Acad Sci U S A ; 117(8): 4071-4077, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041886

RESUMO

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Šresolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.


Assuntos
Cobre/química , Cristalografia/métodos , Geobacillus/enzimologia , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Domínio Catalítico , Cristalização , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Geobacillus/genética , Geobacillus/metabolismo , Modelos Moleculares , Nitrito Redutases/genética , Conformação Proteica
20.
PLoS One ; 15(2): e0229171, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084182

RESUMO

Glutathione peroxidase (GPx) has been the focus of increased research because of its important role as an antioxidant and in reactive oxygen species (ROS) induced damage repair. Studies on GPxs have relevance with Macrobrachium nipponense because it has poor tolerance to hypoxia in Macrobrachium nipponense. The two subunits named as MnGPx-3 and MnGPx-4 according to the glutathione peroxidase nomenclature system. Both full-length cDNAs were cloned from the hepatopancreas. In this study, we analyzed the expression of two GPxs in Macrobrachium nipponense in response to changes in environmental oxygen. Expression levels of MnGPx-3 and MnGPx-4 indicated that both have strong responses to hypoxia. In situ hybridization showed that MnGPx-3 and MnGPx-4 were located in secretory and storage cells in hepatopancreas. These results suggest that GPx gene is expressed and released by secretory cells and released response to hypoxia. In the gill tissue, however, GPxs are located in blood cells, suggesting that they perform different functions in different tissues or organs. The results of in situ hybridization were consistent with those of quantitative Real-time PCR. This study provides a basis for understanding the oxidative stress response in M. nipponense under hypoxia.


Assuntos
Regulação Enzimológica da Expressão Gênica , Glutationa Peroxidase/genética , Oxigênio/metabolismo , Palaemonidae/genética , Palaemonidae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Glutationa Peroxidase/química , Hibridização In Situ , Palaemonidae/enzimologia , Filogenia , RNA Mensageiro/genética , Distribuição Tecidual
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