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1.
Cell Rep Med ; 4(8): 101133, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37586317

RESUMO

New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cell receptor (TCR) T cell therapy is effective in tumors with NY-ESO-1 expression, but a safe and effective TCR-T cell therapeutic protocol remains to be improved. Here, we report a phase 1 investigational new drug clinical trial with TCR affinity-enhanced specific T cell therapy (TAEST16001) for targeting NY-ESO-1. Enrolled patients receive TAEST16001 cell infusion after dose-reduced lymphodepletion with cyclophosphamide (15 mg/kg/day × 3 days) combined with fludarabine (20 mg/m2/day × 3 days), and the TCR-T cells are maintained with low doses of interleukin-2 injection post-adoptive transfer. Analysis of 12 patients treated with the regimen demonstrates no treatment-related serious adverse events. The overall response rate is 41.7%. The median progression-free survival is 7.2 months, and the median duration of response is 13.1 months. The protocol of TAEST16001 cells delivers a safe and highly effective treatment for patients with advanced soft tissue sarcoma (ClinicalTrials.gov: NCT04318964).


Assuntos
Imunoterapia Adotiva , Sarcoma , Neoplasias de Tecidos Moles , Humanos , Antígenos HLA-A/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Sarcoma/metabolismo , Neoplasias de Tecidos Moles/terapia , Linfócitos T
2.
Molecules ; 28(8)2023 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-37110776

RESUMO

The effects of variations in the heat treatment process of milk on its quality and flavor are inevitable. This study investigated the effect of direct steam injection and instantaneous ultra-high-temperature (DSI-IUHT, 143 °C, 1-2 s) sterilization on the physicochemical properties, whey protein denaturation (WPD) rate, and volatile compounds (VCs) of milk. The experiment compared raw milk as a control with high-temperature short-time (HTST, 75 °C 15 s and 85 °C 15 s) pasteurization and indirect ultra-high-temperature (IND-UHT, 143 °C, 3-4 s) sterilization. The results showed no significant differences (p > 0.05) in physical stability between milk samples with different heat treatments. The DSI-IUHT and IND-UHT milks presented smaller particle sizes (p < 0.05) and more concentrated distributions than the HTST milk. The apparent viscosity of the DSI-IUHT milk was significantly higher than the other samples (p < 0.05) and is consistent with the microrheological results. The WPD of DSI-IUHT milk was 27.52% lower than that of IND-UHT milk. Solid-phase microextraction (SPME) and solvent-assisted flavor evaporation (SAFE) were combined with the WPD rates to analyze the VCs, which were positively correlated with ketones, acids, and esters and negatively associated with alcohols, heterocycles, sulfur, and aldehydes. The DSI-IUHT samples exhibited a higher similarity to raw and HTST milk than the IND-UHT samples. In summary, DSI-IUHT was more successful in preserving the milk's quality due to its milder sterilization conditions compared to IND-UHT. This study provides excellent reference data for the application of DSI-IUHT treatment in milk processing.


Assuntos
Temperatura Alta , Leite , Animais , Leite/química , Vapor , Temperatura , Esterilização , Proteínas do Soro do Leite/análise
3.
J Sci Food Agric ; 102(14): 6668-6675, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35608931

RESUMO

BACKGROUND: Enzyme-modified butter is used as a common raw material to obtain a natural milk flavor. Butter protein is a by-product in butter processing that can be used as substrate to produce taste-active peptides, which can create additional value and new application opportunities, making the method more environmentally friendly. RESULTS: Putative kokumi peptides from hydrolysates of protein by-products were isolated by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The isolated peptide fraction with the most pronounced kokumi taste was screened by sensory evaluation and electronic tongue analysis. Eleven peptides were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Six peptides were synthesized to verify their taste characteristics. Five synthetic peptides (FTKK, CKEVVRNANE, EELNVPG, VPNSAEER and YPVEPFTER) showed different intensity levels of kokumi taste. Of these peptides, the decapeptide CKEVVRNANE had the highest kokumi intensity. CONCLUSION: The newly identified kokumi peptides increased the kokumi taste intensity and showed some synergistic effect with umami taste. Both termini of the peptides seem to play an important role in taste characteristic. Glu residue at both termini can increase the kokumi taste intensity. This work indicated that it was feasible to produce kokumi peptides by enzymatic hydrolysis of the protein by-products of butter. © 2022 Society of Chemical Industry.


Assuntos
Manteiga , Peptídeos , Manteiga/análise , Cromatografia em Gel , Nariz Eletrônico , Peptídeos/química , Paladar
4.
Mol Cancer ; 11: 61, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22917467

RESUMO

BACKGROUND: The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion. RESULTS: We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting. CONCLUSION: We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/farmacologia
5.
J Pharmacol Exp Ther ; 329(1): 94-101, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19131585

RESUMO

Bovine aortic endothelial cells (ECs) respond to nitric oxide (NO) donors by activating the redox-sensitive NF-E2-related factor 2/antioxidant response element pathway and up-regulating heme oxygenase (HO)-1 expression. EC exposure to steady laminar shear stress causes a sustained increase in NO, a transient increase in reactive oxygen species (ROS), and activation of the HO-1 gene. Because steady laminar flow increases the mitochondrial superoxide (O(2)(*-)) production, we hypothesized that mitochondria-derived ROS play a role in shear-induced HO-1 expression. Flow (10 dynes/cm(2), 6 h)-induced expression of HO-1 protein was abolished when BAECs were preincubated and sheared in the presence of either N(G)-nitro-L-arginine methyl ester or N-acetyl-L-cysteine, suggesting that either NO or ROS up-regulates HO-1. Ebselen and diphenylene iodonium blocked HO-1 expression, and uric acid had no effect. The mitochondrial electron transport chain inhibitors, myxothiazol, rotenone, or antimycin A, and the mitochondria-targeted antioxidant peptide, Szeto-Schiller (SS)-31, which scavenges O(2)(*-), hydrogen peroxide (H(2)O(2)), peroxynitrite, and hydroxyl radicals, markedly inhibited the increase in HO-1 expression. These data collectively suggest that mitochondrial H(2)O(2) mediates the HO-1 induction. MitoSOX and 2',7'-dichlorofluorescin (DCF) fluorescence showed that mitochondrial O(2)(*-) levels and intracellular peroxides, respectively, are higher in sheared ECs compared with static controls and, in part, dependent on NO. SS-31 significantly inhibited both the shear-induced MitoSOX and DCF fluorescence signals. Either phosphatidylinositol 3-kinase or mitogen-activated protein kinase cascade inhibitors blocked the HO-1 induction. In conclusion, under shear, EC mitochondria-derived H(2)O(2) diffuses to the cytosol, where it initiates oxidative signaling leading to HO-1 up-regulation and maintenance of the atheroprotective EC status.


Assuntos
Células Endoteliais/metabolismo , Heme Oxigenase-1/biossíntese , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Western Blotting , Bovinos , Transporte de Elétrons/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Espectrometria de Fluorescência
6.
Am J Physiol Cell Physiol ; 295(1): C180-91, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18480296

RESUMO

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.


Assuntos
Células Endoteliais/metabolismo , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Ácido Peroxinitroso/biossíntese , Animais , Aorta/citologia , Bovinos , Respiração Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/metabolismo , Consumo de Oxigênio , Pressão Parcial , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Resistência ao Cisalhamento , Estresse Mecânico , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo
7.
Ann Biomed Eng ; 35(5): 683-93, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17340195

RESUMO

Exposure of vascular endothelial cells (ECs) to steady laminar shear stress activates the NF-E2-related factor 2 (Nrf2) which binds to the antioxidant response element (ARE) and upregulates the expression of several genes. The onset of shear is known to increase the EC reactive oxygen species (ROS) production, and oxidative stress can activate the ARE. ARE-regulated genes include phase 2 enzymes, such as glutathione-S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and antioxidants, such as glutathione reductase (GR), glutathione peroxidase (GPx) and catalase. We examined how shear stress affects the antioxidant/phase 2 enzyme activities and whether ROS mediate these effects. ROS production, measured by dichlorofluorescin fluorescence, depended on level and time of shear exposure and EC origin, and was inhibited by either an endothelial nitric oxide synthase (eNOS) inhibitor or a superoxide dismutase (SOD) mimetic and peroxynitrite (ONOO-) scavenger. Shear stress (10 dynes/cm2, 16 h) significantly increased the NQO1 activity, did not change significantly the glutathione (GSH) content, and significantly decreased the GR, GPx, GST and catalase activities in human umbilical vein ECs. Either eNOS inhibition or superoxide radical (O2*-)/ONOO- scavenging differentially modulated the shear effects on enzyme activities suggesting that the intracellular redox status coordinates the shear-induced expression of cytoprotective genes.


Assuntos
Antioxidantes/metabolismo , Células Endoteliais/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Cardiovasculares , Oxirredutases/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , Simulação por Computador , Ativação Enzimática , Humanos , Estresse Oxidativo/fisiologia , Resistência ao Cisalhamento
8.
Am J Physiol Cell Physiol ; 292(3): C1103-12, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17020931

RESUMO

There is evidence that nitric oxide (NO), superoxide (O(2)(*-)), and their associated reactive nitrogen species (RNS) produced by vascular endothelial cells (ECs) in response to hemodynamic forces play a role in cell signaling. NO is known to impair mitochondrial respiration. We sought to determine whether exposure of human umbilical vein ECs (HUVECs) to steady laminar shear stress and the resultant NO production modulate electron transport chain (ETC) enzymatic activities. The activities of respiratory complexes I, II/III, and IV were dependent on the presence of serum and growth factor supplement in the medium. EC exposure to steady laminar shear stress (10 dyn/cm(2)) resulted in a gradual inhibition of each of the complexes starting as early as 5 min from the flow onset and lasting up to 16 h. Ramp flow resulted in inhibition of the complexes similar to that of step flow. When ECs were sheared in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 100 microM), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; 100 microM), or the peroxynitrite (ONOO(-)) scavenger uric acid (UA; 50 microM), the flow-inhibitory effect on mitochondrial complexes was attenuated. In particular, L-NAME and UA abolished the flow effect on complex IV. Increased tyrosine nitration was observed in the mitochondria of sheared ECs, and UA blocked the shear-induced nitrotyrosine staining. In summary, shear stress induces mitochondrial RNS formation that inhibits the electron flux of the ETC at multiple sites. This may be a critical mechanism by which shear stress modulates EC signaling and function.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Células Endoteliais/fisiologia , Mecanotransdução Celular/fisiologia , Mitocôndrias/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Células Cultivadas , Humanos , Estresse Oxidativo/fisiologia , Pressão , Resistência ao Cisalhamento , Estresse Mecânico
9.
Am J Respir Cell Mol Biol ; 33(5): 513-20, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16100081

RESUMO

We reported that cigarette smoke extract (CSE) causes decreases in the activity and expression of endothelial nitric oxide synthase (eNOS) and calpain activity in pulmonary artery endothelial cells (PAECs). Calpains are a family of calcium-dependent endopeptidases, and their specific endogenous inhibitor is calpastatin. In this study, we evaluated the role of calpain-calpastatin in CSE-induced decrease in eNOS gene expression. PAEC were incubated with 5-10% CSE for 2-24 h. eNOS gene transcription rate, eNOS messenger ribonucleic acid (mRNA) half-life, and the activity and protein contents of calpain and calpastatin were measured. Incubation of PAEC with CSE caused significant decreases in eNOS gene transcription and calpain activity and an increase in calpastatin protein content. eNOS mRNA half-life was not significantly altered by CSE. To investigate whether CSE-induced inhibition of eNOS gene expression is caused by decreased calpain activity due to an increase in calpastatin protein content, we cloned calpastatin gene from PAEC and constructed adenovirus vectors containing calpastatin. Overexpression of calpastatin mimics the inhibitory effects of CSE on calpain activity and on the activity, protein, and mRNA of eNOS. The cell-permeable calpain inhibitor, calpastatin peptide, inhibits acetylcholine-induced endothelium-dependent relaxation of the pulmonary artery. Incubation of PAEC with an antisense oligodeoxyribonucleotide of calpastatin prevented CSE-induced increases in calpastatin protein and CSE-induced decreases in calpain activity, eNOS gene transcription, activity and protein content of eNOS, and NO release. These results indicate that CSE-induced inhibition of eNOS expression in PAEC is caused by calpain inhibition due to an increase in calpastatin protein content.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Calpaína/fisiologia , Pulmão/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fumar/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Células Cultivadas , Endotélio/efeitos dos fármacos , Endotélio/enzimologia , Meia-Vida , Pulmão/citologia , Pulmão/efeitos dos fármacos , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo III/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Peptídeos/farmacologia , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Fumar/genética , Suínos , Transcrição Gênica
10.
Am J Physiol Lung Cell Mol Physiol ; 287(4): L794-800, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15180919

RESUMO

Angiogenesis is an integral part of both the pulmonary inflammatory response to chronic exposure to cigarette smoke and the lung tissue remodeling associated with cigarette smoke-induced chronic obstructive pulmonary disease (COPD). To investigate the role of angiogenesis in the pathogenesis of COPD, we evaluated the effect of cigarette smoke extract (CSE) on angiogenesis of pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 2.5-10% CSE resulted in a dose-dependent inhibition of endothelial monolayer wound repair. CSE also caused inhibition of tube formation on Matrigel, migration in a Boyden chamber, and proliferation of PAEC. Because calpain, a family of calcium-dependent intracellular proteases, mediates cytoskeletal signaling in endothelial motility, we explored the role of calpain in the CSE-induced inhibition of endothelial angiogenesis. Incubation of CSE resulted in a dose-dependent decrease in calpain activity. Calpain inhibitor-1, a specific inhibitor of calpain, potentiates inhibitory effect of CSE on the endothelial monolayer wound repair, tube formation, cell migration, and cell proliferation. Transfection of PAEC with antisense oligodeoxyribonucleotides of calpastatin, the major endogenous calpain inhibitor, prevented CSE-induced increase in calpastatin protein content and CSE-induced decreases in calpain activity. It also prevented CSE-induced decreases in monolayer wound repair, tube formation, and migration. These results suggest that CSE attenuates angiogenesis of PAEC and the mechanism involves inhibition of calpain. Impaired angiogenesis may impede the repair process in the lungs of cigarette smokers and contribute to the altered structural remodeling observed in the lungs of patients with cigarette smoke-related COPD.


Assuntos
Calpaína/metabolismo , Endotélio Vascular/fisiologia , Neovascularização Patológica/fisiopatologia , Artéria Pulmonar/fisiologia , Fumar/efeitos adversos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/genética , Calpaína/antagonistas & inibidores , Divisão Celular , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Modelos Animais , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Suínos , Transfecção , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
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