RESUMO
The aim of this study was to investigate the effect of acid challenge on the activation of matrix metalloproteinases (MMPs) in the Dentinoenamel junction of primary and permanent teeth submitted to radiotherapy. For this purpose, a total of 178 dental fragments obtained from molars were used, and randomly divided into 2 groups (primary and permanent teeth) / 4 experimental subgroups (irradiated and non-irradiated, demineralized and non-demineralized). The fragments were exposed to radiation, with a dose fraction of 2 Gy, for 5 consecutive days, until a total dose of 60 Gy was reached, with a total of 30 cycles, for 6 weeks. To determine the activity of MMPs on the dentinoenamel junction (DEJ), in situ zymography assays on 0.6mm dental fragments were performed. To assess whether MMP activity would be impacted by an acidic environment, the fragments were placed in a demineralizing solution (pH of 4.8). The finding was that irradiation activated MMPs in DEJ and these effects were more evident in permanent when compared with primary teeth. When the effect of an acid challenge on MMPs activity was investigated, demineralization was observed not to increase MMPs activity in non-irradiated teeth, but it did increase MMPs activity in irradiated teeth. In conclusion, an acid challenge was found to exacerbate activation of MMPs in DEJ of permanent teeth submitted to irradiation, but not in primary teeth.
Assuntos
Metaloproteinases da Matriz , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz/efeitos da radiação , Metaloproteinases da Matriz/análise , Humanos , Fatores de Tempo , Dente Decíduo/efeitos da radiação , Dente Decíduo/efeitos dos fármacos , Dentina/efeitos da radiação , Dentina/efeitos dos fármacos , Dentina/enzimologia , Dentição Permanente , Distribuição Aleatória , Concentração de Íons de Hidrogênio , Desmineralização do Dente , Estatísticas não Paramétricas , Análise de Variância , Valores de Referência , Ativação Enzimática/efeitos da radiação , Ativação Enzimática/efeitos dos fármacosRESUMO
Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent 'on' state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.
Assuntos
Adenilil Ciclases , Proteínas de Bactérias , Glutamina , Oscillatoria , Adenilil Ciclases/química , Adenilil Ciclases/genética , Adenilil Ciclases/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Flavinas/química , Flavinas/efeitos da radiação , Luz , Mutação , Glutamina/genética , Domínios Proteicos/efeitos dos fármacos , Transporte de Elétrons , Ativação Enzimática/efeitos da radiação , Oscillatoria/enzimologiaRESUMO
Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.
Assuntos
Adenilil Ciclases , Proteínas de Bactérias , Luz , Transdução de Sinais , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Adenilil Ciclases/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Subunidades Proteicas , Ativação Enzimática/efeitos da radiação , MutaçãoRESUMO
Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to exert tight and reversible control over their activity for various biotechnological applications. Here, we apply a rational, computation-guided methodology to design and test a transferase-class enzyme allosterically regulated by light-oxygen-voltage 2 sensing domain. We utilize computational techniques to determine the intrinsic allosteric networks within N-acyltransferase (Orf11/∗Dbv8) and identify potential allosteric sites on the protein's surface. We insert light-oxygen-voltage 2 sensing domain at the predicted allosteric site, exerting reversible control over enzymatic activity. We demonstrate blue-light regulation of N-acyltransferase (Orf11/∗Dbv8) function. Our study for the first time demonstrates optogenetic regulation of a transferase-class enzyme as a proof-of-concept for controllable transferase design. This successful design opens the door for many future applications in metabolic engineering and cellular programming.
Assuntos
Aciltransferases , Proteínas Recombinantes de Fusão , Aciltransferases/química , Aciltransferases/genética , Regulação Alostérica , Sítio Alostérico , Luz , Oxigênio , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Ativação Enzimática/efeitos da radiaçãoRESUMO
Water vapor absorbs well in the infra-red region of the electromagnetic spectrum. Absorption of radiant energy by water or water droplets leads to formation of exclusion zone water that possesses peculiar physico-chemical properties. In the course of this study, normally functioning and damaged alkaline phosphatase, horseradish peroxidase and catalase were treated with humid air irradiated with infrared light with a wavelength in the range of 1270 nm and referred to as coherent humidity (CoHu). One-minute long treatment with CoHu helped to partially protect enzymes from heat inactivation, mixed function oxidation, and loss of activity due to partial unfolding. Authors suggest that a possible mechanism underlying the observed effects involves altering the physicochemical properties of aqueous media while treatment of the objects with CoHu where CoHu acts as an intermediary.
Assuntos
Ar , Fosfatase Alcalina/efeitos da radiação , Catalase/efeitos da radiação , Peroxidase do Rábano Silvestre/efeitos da radiação , Umidade , Raios Infravermelhos , Fosfatase Alcalina/metabolismo , Animais , Catalase/metabolismo , Bovinos , Ativação Enzimática/efeitos da radiação , Escherichia coli/enzimologia , Peroxidase do Rábano Silvestre/metabolismo , Oxirredução/efeitos da radiação , Desnaturação Proteica/efeitos da radiação , Especificidade por Substrato/efeitos da radiação , TemperaturaRESUMO
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Recently, we demonstrated the overexpression of both DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B) in RMS tumour biopsies and cell lines compared to normal skeletal muscle. Radiotherapy may often fail due to the abnormal expression of some molecules able to drive resistance mechanisms. The aim of this study was to analyse the involvement of DNMT3A and DNMT3B in radioresistance in RMS. RNA interference experiments against DNMT3A/3B were performed in embryonal RMS cells, upon ionizing radiation (IR) exposure and the effects of the combined treatment on RMS cells were analysed. DNMT3A and DNMT3B knocking down increased the sensitivity of RMS cells to IR, as indicated by the drastic decrease of colony formation ability. Interestingly, DNMT3A/3B act in two different ways: DNMT3A silencing triggers the cellular senescence program by up-regulating p16 and p21, whilst DNMT3B depletion induces significant DNA damage and impairs the DNA repair machinery (ATM, DNA-PKcs and Rad51 reduction). Our findings demonstrate for the first time that DNMT3A and DNMT3B overexpression may contribute to radiotherapy failure, and their inhibition might be a promising radiosensitizing strategy, mainly in the treatment of patients with metastatic or recurrent RMS tumours.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A/metabolismo , Tolerância a Radiação , Rabdomiossarcoma Embrionário/radioterapia , Ciclo Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Senescência Celular/efeitos da radiação , Células Clonais , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Dano ao DNA , DNA Metiltransferase 3A/genética , Ativação Enzimática/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Inativação Gênica/efeitos da radiação , Histonas/metabolismo , Humanos , Desenvolvimento Muscular/efeitos da radiação , Tolerância a Radiação/genética , Radiação Ionizante , Rabdomiossarcoma Embrionário/genética , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , DNA Metiltransferase 3BRESUMO
Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as â¼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.
Assuntos
Butanóis/metabolismo , Flavanonas/biossíntese , Ácido Láctico/biossíntese , Luz , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/efeitos da radiação , Optogenética/métodos , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos da radiação , Reatores Biológicos , Proteínas de Ligação a DNA/genética , Ativação Enzimática/efeitos da radiação , Fermentação/efeitos da radiação , Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Redes e Vias Metabólicas/genética , Microrganismos Geneticamente Modificados , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos da radiaçãoRESUMO
As the skin is the largest body organ and critically serves as a barrier, it is frequently exposed and could be physiologically affected by radiofrequency electromagnetic field (RF-EMF) exposure. In this study, we found that 1760 MHz RF-EMF (4.0 W/kg specific absorption rate for 2 h/day during 4 days) exposure could induce intracellular reactive oxygen species (ROS) production in HaCaT human keratinocytes using 2',7'-dichlorofluorescin diacetate fluorescent probe analysis. However, cell growth and viability were unaffected by RF-EMF exposure. Since oxidative stress in the skin greatly influences the skin-aging process, we analyzed the skin senescence-related factors activated by ROS generation. Matrix metalloproteinases 1, 3, and 7 (MMP1, MMP3, and MMP7), the main skin wrinkle-related proteins, were significantly increased in HaCaT cells after RF-EMF exposure. Additionally, the gelatinolytic activities of secreted MMP2 and MMP9 were also increased by RF-EMF exposure. FoxO3a (Ser318/321) and ERK1/2 (Thr 202/Tyr 204) phosphorylation levels were significantly increased by RF-EMF exposure. However, Bcl2 and Bax expression levels were not significantly changed, indicating that the apoptotic pathway was not activated in keratinocytes following RF-EMF exposure. In summary, our findings show that exposure to 1760 MHz RF-EMF induces ROS generation, leading to MMP activation and FoxO3a and ERK1/2 phosphorylation. These data suggest that RF-EMF exposure induces cellular senescence of skin cells through ROS induction in HaCaT human keratinocytes.
Assuntos
Campos Eletromagnéticos/efeitos adversos , Proteína Forkhead Box O3/metabolismo , Queratinócitos/efeitos da radiação , Metaloproteinases da Matriz/metabolismo , Envelhecimento da Pele/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Células HaCaT , Humanos , Queratinócitos/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Fosforilação/efeitos da radiação , Ondas de Rádio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Triple-negative breast cancers (TNBCs) are associated with poor survival mediated by treatment resistance. TNBCs are fibrotic, yet little is known regarding how the extracellular matrix (ECM) evolves following therapy and whether it impacts treatment response. Analysis revealed that while primary untreated TNBCs are surrounded by a rigid stromal microenvironment, chemotherapy-resistant residual tumors inhabit a softer niche. TNBC organoid cultures and xenograft studies showed that organoids interacting with soft ECM exhibit striking resistance to chemotherapy, ionizing radiation, and death receptor ligand TRAIL. A stiff ECM enhanced proapoptotic JNK activity to sensitize cells to treatment, whereas a soft ECM promoted treatment resistance by elevating NF-κB activity and compromising JNK activity. Treatment-resistant residual TNBCs residing within soft stroma had elevated activated NF-κB levels, and disengaging NF-κB activity sensitized tumors in a soft matrix to therapy. Thus, the biophysical properties of the ECM modify treatment response, and agents that modulate stiffness-dependent NF-κB or JNK activity could enhance therapeutic efficacy in patients with TNBC.
Assuntos
Matriz Extracelular/metabolismo , NF-kappa B/metabolismo , Neoplasias de Mama Triplo Negativas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/efeitos da radiação , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Terapia Neoadjuvante , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiaçãoRESUMO
Birinapant is a novel SMAC peptidomimetic molecule in clinical development. It suppresses the inhibitor of apoptosis proteins (IAPs) and promotes cytochrome-C/Apaf-1/caspase-9 activation to induce effective apoptosis. Because IAP inhibition has been shown to enhance the sensitivity of cancer cells to radiation, we investigated the role of birinapant in radiosensitization of glioblastoma cells in vitro and in vivo. Two glioblastoma cell lines, U-251 and U-87, were used to analyze radiosensitization in vitro with 7-AAD cell death/apoptosis and clonogenic assays. Subcutaneous flank (U-251 and U-87) and intracranial orthotopic (U-251) xenografts in nude mice were used to evaluate radiosensitization in vivo. TNF-α levels in media and serum were measured using electrochemiluminescence. Radiosensitization in vitro was more prominent for U-251 cells than for U-87 cells. In vivo, in both tumor models, significant tumor growth delay was observed with combination treatment compared to radiation alone. There was a survival benefit with combination treatment in the orthotopic U-251 model. TNF-α levels in media correlated directly with radiation dose in vitro. These findings show that birinapant can enhance the radiosensitivity of glioblastoma cell lines in cell-based assays and tumor models via radiation-induced TNF-α. Further study into the use of birinapant with radiation therapy is warranted.
Assuntos
Dipeptídeos/farmacologia , Glioblastoma/patologia , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Humanos , Camundongos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.
Assuntos
Celulases/metabolismo , Celulose/metabolismo , Ativação Enzimática/efeitos da radiação , Proteínas Fúngicas/metabolismo , Luz , Oxigenases de Função Mista/metabolismo , Sordariales/enzimologia , Biomassa , Catálise , Celulose/química , Proteínas Fúngicas/química , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Glucanos/química , Glucanos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/genética , Oxirredução , Filogenia , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo , Estereoisomerismo , Especificidade por Substrato , Difração de Raios X , Xilanos/química , Xilanos/metabolismoRESUMO
Ultraviolet B (UVB) exposure activates various inflammatory molecules of keratinocytes in the epidermis layer. Such UVB-mediated skin inflammation leaves post-inflammatory hyperpigmentation (PIH). Reports show a close relationship between PIH and high-mobility group box 1 (HMGB1) and its receptors. General clinical treatments of PIH, such as oral medication and laser treatment, have reported side effects. Recent studies reported the effects of radiofrequency (RF) irradiation on restoring dermal collagen, modulating the dermal vasculature, and thickening the basement membrane. To validate how RF regulates the inflammatory molecules from UVB-irradiated keratinocytes, we used UVB-radiated keratinocytes and macrophages, as well as animal skin. In addition, we examined two cases of RF-irradiated skin inflammatory diseases. We validated the effects of RF irradiation on keratinocytes by measuring expression levels of HMGB1, Toll-like receptors (TLRs), and other inflammatory factors. The results show that the RF modulates UVB-radiated keratinocytes to secrete fewer inflammatory factors and also modulates the expression of macrophages from HMGB1, TLRs, and inflammatory factors. RF irradiation could alleviate inflammatory skin diseases in patients. RF irradiation can regulate the macrophage indirectly through modulating the keratinocyte and inflammatory molecules of macrophages reduced in vitro and in vivo. Although the study is limited by the low number of cases, it demonstrates that RF irradiation can regulate skin inflammation in patients.
Assuntos
Dermatite/radioterapia , Ativação Enzimática/efeitos da radiação , Proteína HMGB1/metabolismo , Hiperpigmentação/radioterapia , Receptores Toll-Like/metabolismo , Animais , Proliferação de Células/efeitos da radiação , Citocinas/metabolismo , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Hiperpigmentação/complicações , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Raios UltravioletaRESUMO
Under physiological conditions, cells produce low basal levels of reactive oxygen species (ROS); however, in pathologic conditions ROS production increases dramatically, generating high concentrations of toxic unsaturated aldehydes. Aldehyde dehydrogenases (ALDHs) are responsible for detoxification of these aldehydes protecting the cell. Due to the physiological relevance of these enzymes, it is important to design strategies to modulate their activity. It was previously reported that omeprazole activation of ALDH1A1 protected Escherichia coli cells overexpressing this enzyme, from oxidative stress generated by H2 O2 . In this work, omeprazole cell protection potential was evaluated in eukaryotic cells. AS-30D cell or hepatocyte suspensions were subjected to a treatment with omeprazole and exposure to light (that is required to activate omeprazole in the active site of ALDH) and then exposed to H2 O2 . Cells showed viability similar to control cells, total activity of ALDH was preserved, while cell levels of lipid aldehydes and oxidative stress markers were maintained low. Cell protection by omeprazole was avoided by inhibition of ALDHs with disulfiram, revealing the key role of these enzymes in the protection. Additionally, omeprazole also preserved ALDH2 (mitochondrial isoform) activity, diminishing lipid aldehyde levels and oxidative stress in this organelle, protecting mitochondrial respiration and transmembrane potential formation capacity, from the stress generated by H2 O2 . These results highlight the important role of ALDHs as part of the antioxidant system of the cell, since if the activity of these enzymes decreases under stress conditions, the viability of the cell is compromised.
Assuntos
Família Aldeído Desidrogenase 1/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Omeprazol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Família Aldeído Desidrogenase 1/genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Luz , Oxidantes/farmacologia , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Radio frequency (RF) is an emerging technology applied in blanching treatment as alternative to conventional treatment. This study aimed to investigate the effects of RF heating rate on peroxidase (POD) inactivating efficiency, physiochemical properties (texture, color and vitamin C) and cell structure of stem lettuce. POD enzyme inactivation efficiency increased with increased RF heating rate. When POD activity was reduced to <5%, better physiochemical properties and less cell damage occurred in RF heating compared with conventional hot water (HW) blanching (HWB); Relative electrolyte leakage (REL) analysis and microscopic observation suggested that the loss of integrity of cell walls and membranes, the decrease in turgor pressure and the weakened connections between adjacent cells leaded to the deterioration of physiochemical properties. The degrees of cell destruction varied with RF heating rates, which provide a new idea for RF blanching for producing different types of fruits and vegetables products (Solid and juice products).
Assuntos
Fenômenos Químicos/efeitos da radiação , Temperatura Alta , Lactuca/citologia , Lactuca/efeitos da radiação , Peroxidase/metabolismo , Ondas de Rádio , Cor , Ativação Enzimática/efeitos da radiação , Manipulação de Alimentos , Lactuca/enzimologia , Lactuca/metabolismoRESUMO
In this work, the impact of APTES-modified TiO2 photocatalysts on antioxidant enzymes (catalase and superoxide dismutase) activity secreted by bacteria was presented. Microbial tests has been examined using Escherichia coli (ATCC 29425) and Staphylococcus epidermidis (ATCC 49461) as model organisms. It was found that APTES-TiO2 affected the activity of antioxidant enzymes. Additionally, obtained APTES-TiO2 photocatalysts were capable of total E. coli and S. epidermidis inactivation under artificial solar light irradiation. The sample modified with the concentration of APTES equals 300 mM (TiO2-4h-120°C-300mM) showed the strongest photocatalytic activity toward both bacteria species. The two-stage photocatalytic mechanism of bacteria response to photocatalysts was proposed.
Assuntos
Catalase/metabolismo , Escherichia coli/enzimologia , Propilaminas/química , Silanos/química , Staphylococcus epidermidis/enzimologia , Superóxido Dismutase/metabolismo , Titânio/química , Catálise/efeitos da radiação , Desinfecção , Ativação Enzimática/efeitos da radiação , Escherichia coli/citologia , Escherichia coli/efeitos da radiação , Luz , Viabilidade Microbiana/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Processos Fotoquímicos/efeitos da radiação , Staphylococcus epidermidis/citologia , Staphylococcus epidermidis/efeitos da radiaçãoRESUMO
Malignant tumors pose a major problem in the medical field. Millimeter wave (MMW) exposure have potential apoptosis-promoting effects on several types of tumors. Considering that the penetration depth of millimeter wave is usually several millimeters, we study the apoptosis-promoting effects of millimeter wave exposure on A375 human melanoma tumor cells in vitro, and this topic has not been explored in the previous literature. In this study, we use the A375 human melanoma cell line as an experimental model exposed to 35.2 GHz millimeter wave in vitro to determine any positive effect and further explore the underlying mechanisms. In this study, 2 groups namely, exposed and sham groups, were set. The exposed groups included 4 exposure time periods of 15, 30, 60, and 90 minutes. The cells in the sham group did not receive millimeter wave exposure. After millimeter wave exposure, the A375 cells in the exposed and sham groups were collected for further experimental procedures. The cell viability after exposure was determined using a cell counting kit, and the apoptosis of A375 cells was assessed by Annexin V/propidium iodide. Changes in the expression of apoptosis-related proteins, including cleaved-caspase-3, and -8, were examined by Western blot. We observed that the millimeter wave exposure could inhibit the viability and induce apoptosis in A375 cells, and the expression of cleaved caspase-3 and -8 were upregulated (P < .05). The results indicated that the millimeter wave at 35.2 GHz exerted apoptosis-promoting effects on the A375 cells via a pathway by activating of caspase-8 and -3.
Assuntos
Apoptose/efeitos da radiação , Radiação Eletromagnética , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , MelanomaRESUMO
Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is characterized by poor survival. Radiotherapy plays an important role in treating TNBC. The purpose of this study was to determine whether inhibiting the AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K) pathways alone or in combination potentiates radiotherapy in TNBC. AMPKα1 and AMPKα2 knockdown diminished cyclin D1 expression and induced G1 cell cycle arrest but did not induce apoptosis alone or in combination with radiotherapy. Next, we analyzed the role of PI3K p85α, p85ß, p110α, p110ß, Akt1, and Akt2 proteins on TNBC cell cycle progression and apoptosis induction. Akt1 and p110α knockdown diminished cyclin D1 expression and induced apoptosis. Silencing Akt1 promoted synergistic apoptosis induction during radiotherapy and further reduced survival after radiation. Treatment with the Akt inhibitor, MK-2206 48 h after radiotherapy decreased Akt1 levels and potentiated radiation-induced apoptosis. Together, our results demonstrate that AMPKα, p110α, and Akt1 promote TNBC proliferation and that Akt1 is a key regulator of radiosensitivity in TNBC. Importantly, combining radiotherapy with the pharmacological inhibition of Akt1 expression is a potentially promising approach for the treatment of TNBC.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Tolerância a Radiação , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/radioterapia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos com 3 Anéis/farmacologia , Xenoenxertos , Humanos , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Neoplasias de Mama Triplo Negativas/patologia , Raios XRESUMO
Thermococcus gammatolerans is anaerobic euryarchaeon which grows optimally at 88⯰C and its genome encodes a family B DNA polymerase (Tga PolB). Herein, we cloned the gene of Tga PolB, expressed and purified the gene product, and characterized the enzyme biochemically. The recombinant Tga PolB can efficiently synthesize DNA at high temperature, and retain 93% activity after heated at 95⯰C for 1.0â¯h, suggesting that the enzyme is thermostable. Furthermore, the optimal pH for the enzyme activity was measured to be 7.0-9.0. Tga PolB activity is dependent on a divalent cation, among which magnesium ion is optimal. NaCl at low concentration stimulates the enzyme activity but at high concentration inhibits enzyme activity. Interestingly, Tga PolB is able to efficiently bypass uracil in DNA, which is distinct from other archaeal family B DNA pols. By contrast, Tga PolB is halted by an AP site in DNA, as observed in other archaeal family B DNA polymerases. Furthermore, Tga PolB extends the mismatched ends with reduced efficiencies. The enzyme possesses 3'-5' exonuclease activity and this activity is inhibited by dNTPs. The DNA binding assays showed that Tga PolB can efficiently bind to ssDNA and primed DNA, and have a marked preference for primed DNA. Last, Tga PolB can be used in routine PCR.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Tolerância a Radiação , Temperatura , Thermococcus/fisiologia , Thermococcus/efeitos da radiação , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Fenômenos Químicos , Clonagem Molecular , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/isolamento & purificação , Ativação Enzimática/efeitos da radiação , Expressão Gênica , Ligação Proteica , Proteínas Recombinantes , Especificidade por SubstratoRESUMO
Enzymatic hydrolysis of starch is an important process in the food industry. In the present work, ultrasound was introduced in glucoamylase pretreatment, starch pretreatment and mixed reaction system treatment to enhance starch hydrolysis efficiency. These different processes were studied to explore the mechanism of ultrasound in promoting enzymatic reactions. The hydrolysis degree of starch was determined via measuring the reducing sugar yield. Ultrasound caused enzyme inactivation under high temperatures, high ultrasonic power and long-time treatment, especially at high temperatures exceeding 65 °C. Ultrasound pretreatment of starch before enzymolysis led to the furtherance of starch hydrolysis degree. Meanwhile, sonicating the mixed enzymatic reaction system below 65 °C promoted starch hydrolysis significantly, inducing more than five- fold growth in the degree of starch hydrolysis as much as the ultrasound pretreatment caused. Molecular weights analysis conducted by the MALLS system reflected the enormous damage of starch molecules caused by ultrasound. The amylose contents and chain length distributions of samples were separately analyzed by iodine binding method and size exclusion chromatography. The results of the two experiments illustrate that ultrasound could promote the enzymatic hydrolysis of amylopectin, which is harder for glucoamylase to hydrolyze compared to amylose.