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1.
Food Chem ; 300: 125194, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325749

RESUMO

The effects of near freezing temperature (NFT) storage at -1.9 °C on cell wall degradation of 'Shushanggan' apricot was studied comparing to 0 °C and 5 °C storage. Our results indicated that NFT storage strongly inhibited the solubilization of Na2CO3-soluble pectin and cellulose, by the suppression of cell wall modifying enzymes (polygalacturonase, ß-Galactosidase, pectin methyl esterase and cellulase) and related genes expressions. The loss of side chains was the main modification in CDTA (Cyclohexane-diamine-tetraacetic Acid)-soluble pectin during storage and made the main contribution to the softening of apricot, while the loss of side chain was suppressed by NFT storage. Microscopic observation showed that NFT storage delayed the degradation of pectin fraction and protected cell wall structure from loosing. This study proves that NFT storage is an effective technology to suppress the cell wall polysaccharides degradation and ultrastructure modification of apricot.


Assuntos
Parede Celular/ultraestrutura , Armazenamento de Alimentos/métodos , Polissacarídeos/química , Prunus armeniaca/química , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Celulose/química , Temperatura Baixa , Congelamento , Frutas/química , Frutas/citologia , Frutas/ultraestrutura , Pectinas/química , Células Vegetais/química , Células Vegetais/ultraestrutura , Poligalacturonase/química , Poligalacturonase/metabolismo , Polissacarídeos/metabolismo , Prunus armeniaca/citologia , Solubilidade , beta-Galactosidase/química , beta-Galactosidase/metabolismo
2.
Food Chem ; 297: 125005, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253325

RESUMO

Multiwalled carbon nanotubes molybdenum disulfide 3D nanocomposite (MWCNT-MoS2 NC) was successfully synthesized via eco-friendly hydrothermal method. The microstructural characterization of synthesized nanocomposite was carried out using different spectroscopic and microscopic techniques. Nanocomposite was activated using glutaraldehyde chemistry and used as a platform to immobilize Lens culinaris ß-galactosidase (Lsbgal) which resulted in 93% of immobilization efficiency. Attachment of Lsbgal onto nanocomposite was confirmed by AFM, FE-SEM, FTIR, and CLSM. The nanobiocatalyst showed broadening in operational pH and temperature working range. Remarkable increase in thermal stability was observed as compared to soluble enzyme. Nanobiocatalyst showed outstanding increase in storage stability, retained 92% of residual activity over a period of 8 months. This offers good reusability as it retained ∼50% residual activity up to 21 reuses and exhibited higher rate of lactose hydrolysis in whey. MWCNT-MoS2 NC conjugated to biomolecules can serve as a potential platform for fabrication of lactose biosensor.


Assuntos
Lactose/metabolismo , Lens (Planta)/enzimologia , Nanocompostos/química , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo , Biocatálise , Dissulfetos/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Molibdênio/química , Nanotubos de Carbono/química , Temperatura Ambiente , beta-Galactosidase/química
3.
Chem Commun (Camb) ; 55(50): 7175-7178, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31162503

RESUMO

The identification and removal of senescent cells is very important to improve human health and prolong life. In this study, we introduced a novel strategy of ß-galactosidase (ß-Gal) instructed peptide self-assembly to selectively form nanofibers and hydrogels in senescent cells. We demonstrated that the in situ formed nanofibers could alleviate endothelial cell senescence by reducing p53, p21, and p16INK4a expression levels. We also demonstrated that our strategy could selectively remove senescent endothelial cells by inducing cell apoptosis, with an increase in the BAX/BCL-2 ratio and caspase-3 expression. Our study reports the first example of enzyme-instructed self-assembly (EISA) by a sugar hydrolase, which may lead to the development of supramolecular nanomaterials for the diagnosis and treatment of many diseases, such as cancer, and for other applications, such as wound healing and senescence.


Assuntos
Senescência Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Nanofibras , beta-Galactosidase/metabolismo , Regulação da Expressão Gênica , Humanos , Hidrolases/metabolismo , Lipopolissacarídeos/toxicidade
4.
Vet Res ; 50(1): 40, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126325

RESUMO

Systemic infections caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide and are also potentially threatening to human health. Pathogens must be able to precisely modulate gene expression to facilitate their survival and the successful infection. The Cpx two-component signal transduction system (TCS) regulates surface structure assembly and virulence factors implicated in Gram-negative bacterial pathogenesis. However, the roles of the Cpx TCS in bacterial fitness and pathogenesis during APEC infection are not completely understood. Here, we show that the Cpx TCS response regulator CpxR is critical to the survival and virulence of APEC. Inactivation of cpxR leads to significant defects in the interbacterial competition activity, invasion and survival of APEC in vitro and in vivo. Moreover, activation of CpxR positive regulates the expression of the APEC type VI secretion system 2 (T6SS2). Further investigations revealed that phosphorylated CpxR directly bound to the T6SS2 hcp2B promoter region. Taken together, our results demonstrated that CpxR contributes to the pathogensis of APEC at least through directly regulating the expression and function of T6SS2. This study broadens understanding of the regulatory effect of Cpx TCS, thus elucidating the mechanisms through which Cpx TCS involved in bacterial virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Escherichia coli/veterinária , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Doenças das Aves Domésticas/microbiologia , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Patos , Ensaio de Desvio de Mobilidade Eletroforética , Infecções por Escherichia coli/microbiologia , Mutação , Regiões Promotoras Genéticas , Virulência , beta-Galactosidase/metabolismo
5.
Food Chem ; 294: 231-237, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126458

RESUMO

A fully mechanized Arduino-controlled multi-pumping flow analysis system and procedure for the determination of ß-galactosidase activity are proposed. The applied bioanalytical method is based on the determination of p-nitrophenol formed in the course of enzyme-catalyzed hydrolysis of p-nitrophenyl-galactopyranosides. The photometric detection is performed using dedicated flow-through optoelectronic detector made of paired light emitting diodes. The developed bioanalytical system was applied for evaluation of optimal enzyme detection conditions (pH, temperature and reaction time), selection of appropriate substrate for the assays, comparison of enzymes of different origins (isoenzymes), detection of ß-galactosidase inhibitor and finally to the determination of enzyme activity in some dietary supplements dedicated for people suffering from lactose intolerance. Depending on measurements conditions the developed bioanalytical system allows determination of ß-galactosidase in the wide range of activity (up to 15 U/mL at detection limit ca 0.01 U/mL) with high sample flowthroughput (up to 30 detections per hour). Additionally, the potential utility of the developed analytical system for amyloglucosidase activity assays has been demonstrated.


Assuntos
Galactose/metabolismo , beta-Galactosidase/metabolismo , Biocatálise , Ensaios Enzimáticos , Galactose/química , Concentração de Íons de Hidrogênio , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Nitrofenóis/química , Temperatura Ambiente , beta-Galactosidase/antagonistas & inibidores
6.
Science ; 364(6436)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30975858

RESUMO

Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted ß-galactosidase 1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O-glycan. In counter defense, P. syringae pathovars evade host immunity by using BGAL1-resistant O-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.


Assuntos
Flagelina/imunologia , Flagelina/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Polímeros/metabolismo , Pseudomonas syringae/imunologia , Tabaco/enzimologia , Tabaco/microbiologia , beta-Galactosidase/metabolismo , Glicosilação , Hidrólise , Polissacarídeos/química , Polissacarídeos/metabolismo , Pseudomonas syringae/patogenicidade , Tabaco/genética , Tabaco/imunologia , beta-Galactosidase/genética
7.
Artif Cells Nanomed Biotechnol ; 47(1): 1075-1084, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942622

RESUMO

In this study, an attempt has been made to evaluate the effect of products of ß-galactosidase (ßGS) catalyzed reaction i.e. glucose and galactose and their structurally related compound vitamin C (VC) on the catalytic activity of native and PANI-CS-NC and PANI-CS-Ag-NC adsorbed ßGS. Results indicated a decline in catalytic activity of soluble enzyme in the presence of all investigated compounds. The order of inhibition was found to be VC < glucose < galactose. However, the immobilized preparations were found more resistant to inactivation caused by the added compounds. About 48% activity was retained by PANI-CS-Ag-NC-ßGS in the presence of galactose (5%, w/v), while the native enzyme exhibited only 18% of its original activity. A significant decrease in absorbance and fluorescence intensity was evaluated in soluble enzyme incubated in the presence of all investigated compounds. Three-dimensional fluorescence graphs, CD and FT-IR spectroscopic studies illustrated noteworthy conformational changes in the secondary structure and microenvironment of the soluble enzyme in the presence of VC and tested sugars. These results suggest that both PANI-CS-NC and PANI-CS-Ag-NC bound ßGS are more resistant to the exposure caused by the higher concentration of added glucose, galactose, and VC and, therefore, can be effectively utilized for the production of a hassle-free lactose nano-biosensor.


Assuntos
Compostos de Anilina/química , Quitosana/química , Inibidores Enzimáticos/farmacologia , Nanocompostos/química , Prata/química , beta-Galactosidase/antagonistas & inibidores , beta-Galactosidase/química , Ácido Ascórbico/farmacologia , Catálise , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Galactose/farmacologia , Glucose/farmacologia , Ligação Proteica , beta-Galactosidase/metabolismo
8.
Photochem Photobiol Sci ; 18(6): 1447-1460, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957809

RESUMO

Fluorescence change systems that can respond to biological objects have attracted attention for use as biological probes and sensors. In this study, we report emission enhancement in a fluorescent aggregate composed of amphiphilic donor-acceptor dye molecules. The emission efficiency of the aggregate was reduced upon introducing a hydrophilic galactopyranose moiety, because of the decrease in the aggregate stability, which in turn was due to disruption of the hydrophilicity-hydrophobicity balance. In contrast, emission enhancement could be achieved by treatment with ß-galactosidase, as a result of the removal of the galactopyranose moiety. The change in aggregate stabilization based on the hydrophilicity-hydrophobicity balance leads to the emission enhancement into detectable ß-galactosidase activity.


Assuntos
Compostos de Anilina/química , Fluorescência , Corantes Fluorescentes/química , Tensoativos/química , Tiadiazóis/química , beta-Galactosidase/análise , Compostos de Anilina/síntese química , Corantes Fluorescentes/síntese química , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Espectrometria de Fluorescência , Tensoativos/síntese química , Tiadiazóis/síntese química , beta-Galactosidase/metabolismo
9.
Microb Pathog ; 131: 87-97, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30951817

RESUMO

Occasionally, endophytic fungal species cognize as a hidden prospective source of plant secondary metabolites. In this study, a potent Penicillium setosum sp. nov. was explored for its detailed antibacterial action on Escherichia coli and Staphylococcus aureus through different in vitro and in silico assays. Fluorescence based viability assay determined increase in the number of dead cells in course of time with the continual exposure of extract during a 4 h period. Scanning electron micrographs reflect the distinguishable morphological changes in treated cells, namely shortening of size, bubbles, and blisters on the surface of E. coli, as well as open holes and deep craters on the surface of S. aureus, ultimately leading to rupture of cells. Significant intracellular changes in bacteria were remarkably noticed through different membrane permeabilization assays. The rate of Na+ and K+ leakage with respect to time, intracellular material and cytoplasmic ß-galactosidase release were measured spectroscopically. The results indisputably prove that membrane disruption of S. aureus cells occurs within 2 h and in E.coli occurs in between 2 and 4 h of exposure. Crude extract of P. setosum was fractioned using semi-preparative HPLC and the separated antibacterial active fraction showed antibacterial efficacy with the minimum inhibitory concentration of 8 µg/mL against both organisms. Active fraction contains four well-known plant metabolite belongs to the polyphenolic group (Leucodelphinidin, dihydroquercetin, kaempferol, and quercetin) and one polyketide (patulin) familiar as fungal metabolite, identified through high resolution LC-MS. Interaction mechanisms of identified compounds with nine important antimicrobial drug targets showed highest binding affinity by leucodelphinidin followed by dihydroquercetin > kaempferol > quercetin. This is the first instance of using leucodelphinidin and dihydroquercetin for detailed interaction study with multiple targets, and it was found that they showed more effective interaction than quercetin, which was earlier utilized for antibacterial studies.


Assuntos
Antibacterianos/farmacologia , Simulação por Computador , Simulação de Acoplamento Molecular , Penicillium/metabolismo , Antibacterianos/biossíntese , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Flavonoides/biossíntese , Flavonoides/farmacologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Potássio/metabolismo , Quercetina/análogos & derivados , Quercetina/biossíntese , Quercetina/farmacologia , Metabolismo Secundário , Sódio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , beta-Galactosidase/metabolismo
10.
Front Biosci (Schol Ed) ; 11: 89-104, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844738

RESUMO

Human adult stem cells hold promise for regenerative medicine. They are usually expanded for multiple passages in vitro to increase cell yield prior to transplantation. Unfortunately, prolonged culture leads to cell senescence, a major drawback from successful outcomes in cell therapy approaches. Here, we show that an extract from early Zebrafish embryo (ZF1) counteracted senescence progression in human adipose-derived stem cells (hASCs) along multiple culture passages (from the 5th to the 20th). Exposure to ZF1 strongly reduced the expression of senescence marker beta-galactosidase. Both stemness (NANOG, OCT4, and MYC) and anti-senescence (BMI1, and telomerase reverse transcriptase - TERT) related genes were overexpressed at specific experimental points, without recruitment of the cyclin-dependent kinase Inhibitor 2A (CDKN2A, ali-as p16). Increased telomerase activity was associatt-ed with TERT overexpression. Both osteogenic and adipogenic abilities were enhanced. In conclusion, hASCs exposure to ZF1 is a feasible tool to counteract and reverse human stem cell senescence in long-term culturing conditions.


Assuntos
Extratos Celulares/química , Senescência Celular , Embrião não Mamífero/química , Células-Tronco/citologia , Peixe-Zebra/embriologia , Adipócitos/citologia , Adipogenia , Adulto , Animais , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Humanos , Osteogênese , Transplante de Células-Tronco , Telomerase/genética , beta-Galactosidase/metabolismo
11.
Food Chem ; 288: 102-107, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902269

RESUMO

A new bi-enzymatic catalyst has been produced by precipitation and crosslinking (combi-CLEAs) of ß-galactosidase and glucose isomerase for catalyzing the cascade reactions of lactose conversion into fructose, producing a lactose-fructose syrup (LFS). Glucose isomerase was chemically aminated to increase its reactive surface groups for favour the crosslinking step. The effect of ß-galactosidase to glucose isomerase activity ratio and glutaraldehyde to protein mass ratio in combi-CLEAs production was evaluated. The selected combi-catalyst was successfully used in the production of fructose syrup from lactose in a single reaction vessel. The biocatalyst could be used at least in five sequential batches of LFS production, remaining fully stable after a total of 50 h of reaction, obtaining a product of constant quality. A robust bi-enzymatic catalyst was produced that can be repeatedly used in LFS production, an attractive mild sweetener for the dairy food industry.


Assuntos
Frutose/metabolismo , Lactose/metabolismo , beta-Galactosidase/metabolismo , Aldose-Cetose Isomerases , Catálise , Glucose/metabolismo , Glutaral/metabolismo
12.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889901

RESUMO

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of ß-hexosaminidase (Hex, EC 3.2.1.52) and ß-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Membrana Celular/metabolismo , Curcumina/análogos & derivados , Curcumina/farmacologia , Hexosaminidases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , beta-Galactosidase/metabolismo , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Exocitose/efeitos dos fármacos , Humanos , Células Jurkat , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Fito-Hemaglutininas/farmacologia , Transporte Proteico/efeitos dos fármacos
13.
Photochem Photobiol Sci ; 18(5): 1280-1289, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30907896

RESUMO

Softening processes after ripening are a major factor contributing to the perishability of fleshy fruit and, together with mechanical damage, represent the onset of physiological decay. Softening involves multiple co-ordinated events leading to modifications of the cell wall architecture. Several studies described that UV-B radiation positively affects both the nutraceutical and aesthetical qualities of fruit. However, very few studies investigated the effect of UV-B irradiation on the activity of cell wall-related enzymes. This research aimed at studying how different UV-B treatments (10 min and 60 min) affect the activity of cell wall-modifying enzymes (pectin methylesterase, polygalacturonase and ß-galactosidase) together with the expression of some of their isoforms up to 36 h after UV-B treatment of peach (cv. Fairtime, melting phenotype) fruit. Results revealed that UV-B radiation did not affect the soluble solid content and the titratable acidity, two important parameters influencing consumers' choice and taste. In contrast, UV-B was effective at reducing the loss of firmness 24 h after the 60 min irradiation. Generally, a lower activity of the hydrolytic enzymes compared to untreated fruit was observed, regardless of the UV-B dose. However, gene expression did not reflect the corresponding enzymatic activity. Based on these results, UV-B irradiation might be a successful tool in reducing the loss of firmness of peach fruit during post-harvest, thus improving their quality and shelf-life.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/enzimologia , Frutas/metabolismo , Poligalacturonase/metabolismo , Prunus persica/metabolismo , beta-Galactosidase/metabolismo , Hidrolases de Éster Carboxílico/genética , Frutas/genética , Oxirredução , Fenótipo , Poligalacturonase/genética , Prunus persica/genética , RNA/genética , RNA/isolamento & purificação , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Raios Ultravioleta , beta-Galactosidase/genética
14.
Biosci Biotechnol Biochem ; 83(6): 986-995, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30836860

RESUMO

Our previous work has reported an anti-proliferative compound from moutan cortex, paeoniflorigenone which can induce cancer-selective apoptosis. However, its anti-proliferative mechanism is still unknown. According to morphology changes (hypertrophy and flattening), we hypothesized that PFG can induce senescence or inhibit cell mitosis. Here we show that PFG can induce cellular senescence, evidenced by the expression of senescence-associated ß-galactosidase, G0/G1 cell cycle arrest and permanent loss of proliferative ability, in normal TIG-1 diploid fibroblast but not cancerous HeLa cells. In cancerous HeLa cells, PFG inhibited proliferation by inducing S and G2/M cell cycle arrest and mitosis inhibition. DNA damage response was activated by PFG, interestingly the reactive oxygen species level was suppressed instead of escalated. To sum up, we report 3 new roles of PFG as, 1. inducer of premature senescence in normal TIG-1 cells, 2. inhibitor of mitosis in cancerous HeLa cells, 3. ROS scavenger. Abbreviations: PFG: Paeoniflorigenone; ROS: reactive oxygen species; ATM: ataxia telangiectasia mutated; t-BHP: tert-butyl hydroperoxide; SA-ß-gal: senescence-associatedß-galactosidase; DNA-PKcs: DNA-dependent protein kinase; γ-H2AX: H2AX phosphoryla-tion at Ser-139.


Assuntos
Senescência Celular/efeitos dos fármacos , Diploide , Fibroblastos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Monoterpenos/farmacologia , Paeonia/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/enzimologia , Citometria de Fluxo , Imunofluorescência , Depuradores de Radicais Livres/farmacologia , Células HeLa , Humanos , Monoterpenos/administração & dosagem , Monoterpenos/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , beta-Galactosidase/metabolismo
15.
Molecules ; 24(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889805

RESUMO

Senescence is an irreversible state of cell cycle arrest that can be triggered by multiple stimuli, such as oxygen reactive species and DNA damage. Growing evidence has proven that senescence is a tumor-suppressive approach in cancer treatment. Therefore, developing novel agents that modulate senescence may be an alternative strategy against cancer. In our study, we investigated the inhibitory effect of gypenoside L (Gyp-L), a saponin isolated from Gynostemma pentaphyllum, on cancer cell growth. We found that Gyp-L increased the SA-ß-galactosidase activity, promoted the production of senescence-associated secretory cytokines, and inhibited cell proliferation of human liver and esophageal cancer cells. Moreover, Gyp-L caused cell cycle arrest at S phase, and activated senescence-related cell cycle inhibitor proteins (p21 and p27) and their upstream regulators. In addition, Gyp-L activated p38 and ERK MAPK pathways and NF-κB pathway to induce senescence. Consistently, adding chemical inhibitors efficiently counteracted the Gyp-L-mediated senescence, growth inhibition, and cell cycle arrest in cancer cells. Furthermore, treatment with Gyp-L, enhanced the cytotoxicity of clinic therapeutic drugs, including 5-fluorouracil and cisplatin, on cancer cells. Overall, these results indicate that Gyp-L inhibits proliferation of cancer cells by inducing senescence and renders cancer cells more sensitive to chemotherapy.


Assuntos
Senescência Celular/efeitos dos fármacos , Neoplasias Esofágicas/patologia , Neoplasias Hepáticas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Gynostemma , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Regulação para Cima/efeitos dos fármacos , beta-Galactosidase/metabolismo
16.
Food Chem ; 286: 362-367, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30827619

RESUMO

The ß-galactosidase from Aspergillus oryzae produces galactooligosaccharides with beneficial prebiotic effects through the transglycosylation of lactose. However, its low galactooligosaccharide yield greatly limits its use in industrial applications. Rational design and site-directed mutagenesis were used to produce three mutants of A. oryzae ß-galactosidase: N140C, W806F, and N140C/W806F. The galactooligosaccharide yields of N140C (50.7%), W806F (49.3%), and N140C/W806F (59.8%) represent substantial improvements over that of the wild-type (35.7%). The galactooligosaccharide yield of N140C/W806F is the highest reported to date. Our rational design approach suggests novel strategies for further study of the ß-galactosidase reaction mechanism and has produced mutants may be more useful in industrial applications than wild-type A. oryzae ß-galactosidase.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Oligossacarídeos/metabolismo , beta-Galactosidase/metabolismo , Sítios de Ligação , Proteínas Fúngicas/genética , Glicosilação , Cinética , Lactose/química , Lactose/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase/genética
17.
Appl Biochem Biotechnol ; 189(1): 37-48, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30863986

RESUMO

This study evaluated the production of lignocellulose-degrading enzymes by solid-state fermentation (SSF) using a microbial consortium of Aspergillus fumigatus SCBM6 and A. niger SCBM1 (AFN extract). The fungal strains were cultivated in sugarcane bagasse (SCB) and wheat bran (WB) as lignocellulosic substrates for 7 days at 30 °C. After SSF, the highest peaks of enzyme production were 150 and 80 U g-1 for ß-xylosidase and ß-glucosidase at 48 h, 375 U g-1 for xylanase at 96 h, and 80 U g-1 for endoglucanase and 4 U g-1 for cellulase activity on filter paper (FPase) at 144 h. The efficiency of the produced AFN extract was investigated in the enzymatic hydrolysis of crude biomass sorghum (BS) and after the removal of extractives (ES). After saccharification, the glucose and xylose concentrations were 10× superior in ES than in BS hydrolysate (2.5 g L-1 after 12 h). The presence of inhibitors of alcoholic fermentation, such as formic acid, was also reduced in ES hydrolysates, indicating that the removal of extractives positively contributed to the effectiveness of enzymatic hydrolysis of biomass sorghum using AFN extract.


Assuntos
Aspergillus fumigatus/metabolismo , Aspergillus niger/metabolismo , Biomassa , Celulose/metabolismo , Sorghum/metabolismo , Açúcares/química , Xilosidases/metabolismo , beta-Galactosidase/metabolismo , Aspergillus fumigatus/enzimologia , Aspergillus niger/enzimologia , Hidrólise , Especificidade da Espécie
18.
J Sci Food Agric ; 99(8): 4003-4010, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30723911

RESUMO

BACKGROUND: Fruit dips in calcium ions solutions have been shown as an effective treatment to extend strawberries (Fragaria × ananassa, Duch) quality during storage. In the present work, strawberry fruit were treated with 10 g L-1 calcium chloride solution and treatment effects on cell wall enzymes activities and the expression of encoding genes, as well as enzymes involved in fruit defense responses were investigated. RESULTS: Calcium treatment enhanced pectin methylesterase activity while inhibited those corresponding to pectin hydrolases as polygalacturonase and ß-galactosidase. The expression of key genes for strawberry pectin metabolism was up-regulated (for FaPME1) and down-regulated (for FaPG1, FaPLB, FaPLC, FaßGal1 and FaAra1) by calcium dips. In agreement, a higher firmness level and ionically-bound pectins (IBPs) amount were detected in calcium-treated fruit compared with controls. The in vitro and in vivo growth rate of fungal pathogen Botrytis cinerea was limited by calcium treatment. Moreover, the activities of polyphenol oxidases, chitinases, peroxidases and ß-1,3-glucanases were enhanced by calcium ion dips. CONCLUSION: News insights concerning the biochemical and molecular basis of cell wall preservation and resistance to fungal pathogens on calcium-treated strawberries are provided. © 2019 Society of Chemical Industry.


Assuntos
Cloreto de Cálcio/farmacologia , Parede Celular/efeitos dos fármacos , Conservantes de Alimentos/farmacologia , Fragaria/efeitos dos fármacos , Parede Celular/enzimologia , Parede Celular/metabolismo , Fragaria/enzimologia , Fragaria/genética , Fragaria/metabolismo , Frutas/efeitos dos fármacos , Frutas/enzimologia , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Pectinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poligalacturonase/genética , Poligalacturonase/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
19.
Bioresour Technol ; 278: 296-302, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30708333

RESUMO

Lactulose synthesis from fructose and lactose in continuous packed-bed reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae ß-galactosidase is reported for the first time. Alternative strategies to conventional batch synthesis have been scarcely explored for lactulose synthesis. The effect of flow rate, substrates ratio and biocatalyst-inert packing material mass ratio (MB/MIM) were studied on reactor performance. Increase in any of these variables produced an increase in lactulose yield (YLu) being higher than obtained in batch synthesis at comparable conditions. Maximum YLu of 0.6 g·g-1 was obtained at 50 °C, pH 4.5, 50% w/w total sugars, 15 mL·min-1, fructose/lactose molar ratio of 12 and MB/MIM of 1/8 g·g-1; at such conditions yield of transgalactosylated oligosaccharides (YTOS) was 0.16 g·g-1, selectivity (lactulose/TOS molar ratio) was 5.4 and lactose conversion (XLactose) was 28%. Reactor operation with recycle had no significant effect on yield, producing only some decrease in productivity.


Assuntos
Aspergillus oryzae/enzimologia , Lactulose/biossíntese , beta-Galactosidase/metabolismo , Enzimas Imobilizadas/metabolismo , Frutose/metabolismo , Glioxilatos/metabolismo , Lactose/metabolismo , Oligossacarídeos/metabolismo , Sefarose/metabolismo
20.
J Biotechnol ; 294: 38-48, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30771444

RESUMO

Over the past decades, Bacillus megaterium has gained significant interest in the biotechnological industry due to its high capacity for protein production. Although many proteins have been expressed efficiently using the optimized xylose inducible system so far, there is a considerable demand for novel promoters with varying activities, particularly for the adjustment of protein levels in multi-enzyme cascades. Genome-wide microarray analyses of the industrially important B. megaterium strain MS941 were applied to identify constitutive and growth phase dependent promoters for the expression of heterologous proteins from the early exponential to the early stationary phase of bacterial growth. Fifteen putative promoter elements were selected based on differential gene expression profiles and signal intensities of the generated microarray data. The corresponding promoter activities were evaluated in B. megaterium via ß-galactosidase screening. ß-Galactosidase expression levels ranged from 15% to 130% compared to the optimized xylose inducible promoter. Apart from these constitutive promoters we also identified and characterized novel inducible promoters, which were regulated by the addition of arabinose, galactose and the commonly used allolactose analog IPTG. The potential application of the identified promoters for biotechnologically relevant processes was demonstrated by overexpression of the cholesterol oxidase II from Brevibacterium sterolicum, thus obtaining product yields of up to 1.13 g/l/d. The provided toolbox of novel promoters offers versatile promoter strengths and will significantly contribute to harmonize protein expression in synthetic metabolic pathways, thereby pushing forward the engineering of B. megaterium as microbial cell factory for the biosynthesis and conversion of valuable compounds.


Assuntos
Bacillus megaterium/genética , Regiões Promotoras Genéticas , Bacillus megaterium/metabolismo , Colesterol Oxidase , Genoma Bacteriano , Engenharia Metabólica , Análise de Sequência com Séries de Oligonucleotídeos , Pregnenolona/metabolismo , Progesterona/metabolismo , beta-Galactosidase/metabolismo
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