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1.
ACS Omega ; 6(47): 31945-31954, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34870017

RESUMO

Hyperpigmentation is induced by the overactivation of tyrosinase, which is a rate-limiting enzyme in melanogenesis. The defatted extract of hemp (Cannabis sativa L.) seed is known to have inhibitory effects on melanogenesis; however, effective compounds in the extract have not been identified yet. In this study, three phenethyl cinnamamides present in hemp seed extract were prepared by purification and chemical synthesis and were assessed for their inhibitory effect on melanogenesis in B16F10 melanoma cells. A comparison of the anti-melanogenesis and anti-tyrosinase activity of hemp seed solvent fractions revealed that the ethyl acetate fraction possessed the greatest potential for suppressing melanogenesis in melanoma cells by decreasing tyrosinase activity. We tentatively identified 26 compounds in the ethyl acetate fraction by comparing spectroscopic data with the literature. Three phenethyl cinnamamides such as N-trans-caffeoyltyramine, N-trans-coumaroyltyramine, and N-trans-feruloyltyramine present abundantly in the ethyl acetate fraction were prepared and their anti-melanogenesis and anti-tyrosinase activities in melanoma cells were evaluated. We found that N-trans-caffeoyltyramine and N-trans-feruloyltyramine inhibited alpha melanocyte stimulating hormone (α-MSH)-induced melanogenesis without cytotoxicity, while N-trans-coumaroyltyramine inhibited melanogenesis with cytotoxicity. IC50 values of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and N-trans-coumaroyltyramine for inhibition of α-MSH-mediated tyrosinase activation were 0.8, 20.2, and 6.3 µM, respectively. Overall, N-trans-caffeoyltyramine possessed the strongest anti-melanogenesis activity among the three phenethyl cinnamamides evaluated. The inhibitory effect of N-trans-caffeoyltyramine was verified by determining the melanin content and tyrosinase activity in melanoma after treating the cells with synthetic compounds. Thus, N-trans-caffeoyltyramine isolated from hemp seed extract could be useful in cosmetics as a skin-whitening agent.

2.
Molecules ; 26(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069624

RESUMO

Velutin, one of the flavones contained in natural plants, has various beneficial activities, such as skin whitening, as well as anti-inflammatory, anti-allergic, antioxidant, and antimicrobial activities. However, the relationship between the structure of velutin and its anti-melanogenesis activity is not yet investigated. In this study, we obtained 12 velutin derivatives substituted at C5, C7, C3', and C4' of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities by chemical synthesis, to perform SAR analysis of velutin structural analogues. The SAR study revealed that the substitution of functional groups at C5, C7, C3', and C4' of the flavone backbone affects biological activities related to melanin synthesis. The coexistence of hydroxyl and methoxy at the C5 and C7 position is essential for inhibiting tyrosinase activity. However, 1,2-diol compounds substituted at C3' and C4' of flavone backbone induce apoptosis of melanoma cells. Further, substitution at C3' and C4' with methoxy or hydrogen is essential for inhibiting melanogenesis. Thus, this study would be helpful for the development of natural-derived functional materials to regulate melanin synthesis.


Assuntos
Flavonas/farmacologia , Melaninas/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Flavonas/química , Melaninas/biossíntese , Camundongos , Simulação de Acoplamento Molecular , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-Atividade
3.
ACS Omega ; 5(48): 30946-30955, 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33324802

RESUMO

Hyperpigmentation resulting from the overactivation of tyrosinase leads to darker spots or patches on the human skin. Although these phenomena are harmless, there is still great demand for melanogenesis inhibitors to prevent hyperpigmentation by inhibiting the tyrosinase, a rate-limiting enzyme in melanogenesis. Although Lepisorus thunbergianus has been used in folk remedies as a diuretic and hemostatic agent, its effect on melanogenesis has not yet been reported. In this study, we prepared an L. thunbergianus extract and its solvent fractions and evaluated their biological activity against free radical and melanin synthesis. The extract of L. thunbergianus inhibited mushroom tyrosinase activity more efficiently than, and with similar antioxidant activity to, arbutin in vitro. Comparative evaluation of the anti-melanogenesis and anti-tyrosinase activity of L. thunbergianus solvent fractions demonstrated that, by inhibiting tyrosinase activity, the butanol fraction has the highest potential for the inhibition of melanogenesis in melanoma cells. We found by structural analysis using high-performance liquid chromatography (HPLC) and NMR spectroscopy that the major compounds in butanol fraction were three caffeoylquinic acid derivatives. The three derivatives had similar radical scavenging and anti-tyrosinase activities in vitro, while only 5-caffeoylquinic acid had an inhibitory effect on α-MSH-induced melanogenesis. The inhibitory effect of 5-caffeoylquinic acid was verified by the determination of the melanin content and tyrosinase activity in melanoma after treating the cells with a commercial compound. Further, we revealed that 5-caffeoylquinic acid inhibited melanogenesis by chelating a copper cation from a copper-tyrosinase complex. Thus, 5-caffeoylquinic acid or butanol fraction isolated from L. thunbergianus might be useful in cosmetics as a skin-whitening agent.

4.
Biochem Biophys Res Commun ; 524(3): 750-755, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32035617

RESUMO

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications.


Assuntos
Diabetes Mellitus Experimental/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Masculino , Camundongos Endogâmicos C57BL
5.
Life Sci ; 233: 116711, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31374233

RESUMO

AIMS: Insulin is a central peptide hormone required for carbohydrate metabolism; however, its role in diabetes-associated pulmonary disease is unknown. Here, we investigated the preventative effect of insulin against hyperglycemia-induced pulmonary vascular leakage and its molecular mechanism of action in the lungs of diabetic mice. MAIN METHODS: Vascular endothelial growth factor (VEGF) activated transglutaminase 2 (TGase2) by sequentially elevating intracellular Ca2+ and reactive oxygen species (ROS) levels in primary human pulmonary microvascular endothelial cells (HPMVECs). KEY FINDINGS: Insulin inhibited VEGF-induced TGase2 activation, but did not affect intracellular Ca2+ elevation and ROS generation. Insulin prevented VEGF-induced vascular leakage by inhibiting TGase2-mediated c-Src phosphorylation, disassembly of VE-cadherin and ß-catenin, and stress fiber formation. Insulin replacement therapy prevented hyperglycemia-induced TGase2 activation, but not ROS generation, in the lungs of diabetic mice. Insulin also prevented vascular leakage and cancer metastasis in the diabetic lung. Notably, vascular leakage was not detectable in the lungs of TGase2-null (Tgm2-/-) diabetic mice. SIGNIFICANCE: These findings demonstrate that insulin prevents hyperglycemia-induced pulmonary vascular leakage in diabetic mice by inhibiting VEGF-induced TGase2 activation rather than ROS generation.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Hemorragia/prevenção & controle , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Pneumopatias/prevenção & controle , Transglutaminases/antagonistas & inibidores , Animais , Proteínas de Ligação ao GTP/fisiologia , Hemorragia/etiologia , Hemorragia/patologia , Humanos , Pneumopatias/etiologia , Pneumopatias/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/fisiologia , Células Tumorais Cultivadas
6.
Molecules ; 24(14)2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336931

RESUMO

The abnormal regulation of melanin synthesis leads to a wide range of pigmentary disorders. Although various melanin biosynthesis inhibitors have been developed, their efficacy and long-term safety needs to be further improved, and thus the goal of this study is to develop promising natural compound inhibitors of melanin biosynthesis. Here, we obtained aglycone flavonoid extract through the microwave-assisted hydrolysis of glycoside extract from Korean mistletoe in acidic condition. The aglycone extract inhibited tyrosinase activity more efficiently with better antioxidant activity than glycoside extract in vitro. The microwave-assisted aglycone extract of mistletoe was further analyzed for in vivo activity, and the results showed the aglycone extract inhibited both early melanocyte development and melanin synthesis more efficiently in zebrafish embryo in a dose-dependent manner. Our in vivo toxicity assay quantitatively measured cell death in zebrafish embryos and showed that the microwave-assisted aglycone extract of mistletoe had no significant effect on cell death (p < 0.001), indicating that aglycone extract is more biocompatible than glycoside extract. Furthermore, our in vitro and in vivo analyses successfully identified and characterized velutin, an aglycone of a homoflavoyadorinin B glycoside, as a major inhibitory component in the microwave-assisted mistletoe extract. Ultimately, this study showed that the novel natural compound inhibitor velutin, which was generated through microwave-assisted extraction from mistletoe, improved the efficacy of melanin biosynthesis inhibition with little toxicity.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Flavonas/farmacologia , Melaninas/biossíntese , Erva-de-Passarinho/química , Extratos Vegetais/farmacologia , Animais , Linhagem Celular Tumoral , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Flavonas/química , Flavonas/isolamento & purificação , Flavonoides/química , Flavonoides/farmacologia , Glicosídeos/química , Hidrólise , Melanócitos/metabolismo , Micro-Ondas , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Peixe-Zebra
7.
Macromol Biosci ; 19(9): e1900129, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31310433

RESUMO

C-peptide has emerged as a potential drug for treating diabetic complications. However, clinical application of C-peptide is limited by its short half-life during circulation and costly synthesis methods. To overcome these limitations, a biocompatible and thermosensitive biopolymer-C-peptide conjugate composed of human C-peptide genetically conjugated at the C-terminus of nine repeats of lysine-containing elastin-like polypeptide (K9-C-peptide) is generated. K9-C-peptide exhibits reversible thermal phase behavior with a transition temperature dependent on polypeptide concentration. Degradation of K9-C-peptide hydrogel depends on the concentration of four cleavage enzymes as well as the reaction time and frequency of treatments with elastase-2. The preventive effect of K9-C-peptide against high glucose-induced human aortic endothelial cell dysfunction is further investigated. K9-C-peptide inhibits high glucose-induced intracellular reactive oxygen species generation, transglutaminase 2 activation, and apoptosis, similar to the inhibitory effects of human C-peptide. Thus, K9-C-peptide is a potential drug depot for the sustained delivery of C-peptide to treat diabetic complications.


Assuntos
Biopolímeros/farmacologia , Peptídeo C/farmacologia , Células Endoteliais/patologia , Glucose/toxicidade , Temperatura , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Peptídeo C/química , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Humanos , Peptídeo Hidrolases/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Proteólise/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transglutaminases/metabolismo
8.
Anal Biochem ; 559: 62-70, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30165045

RESUMO

Transglutaminase 2 (TGase2) kinase has emerged as an important regulator of apoptosis as well as chromatin structure and function; however, details about the pathophysiological functions of TGase2 kinase have been limited because of the lack of a suitable activity assay for systematic investigation of TGase2 kinase regulation in a high-throughput manner. Thus, we developed a novel on-chip TGase2 kinase activity assay using a cysteine-modified insulin-like growth factor-binding protein-3-derived peptide (CMI peptide) on an array platform. This peptide array-based activity assay was reproducible, with a detection limit of 2.127 µg/ml. We successfully applied this assay to investigate the effects of thiol-reactive compounds and divalent cations on TGase2 kinase by determining the half maximal inhibitory concentrations (IC50). Thiol-reactive compounds inhibited TGase2 kinase activity in a concentration-dependent manner, with IC50 values ranging from 0.125 to 5.550 mM. Divalent metal cations also showed a concentration-dependent inhibition, with IC50 values ranging from 0.005 to 1.937 mM; however, Ca2+ had no effect on TGase2 kinase activity. Thus, this novel kinase activity assay using the CMI peptide array described here is suitable for systematic investigation of TGase2 kinase regulation and may be useful for investigating the roles of TGase2 kinase in pathogenesis of kinase-mediated diseases.


Assuntos
Cisteína/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ensaios de Triagem em Larga Escala , Peptídeos/metabolismo , Transglutaminases/metabolismo , Cisteína/química , Proteínas de Ligação ao GTP/química , Humanos , Peptídeos/química , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/química
9.
Anal Chim Acta ; 1027: 92-100, 2018 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-29866275

RESUMO

Transglutaminase 2 (TGase2), a multifunctional enzyme exhibiting both transamidase and kinase activity, is involved in a variety of cellular processes and diseases. However, details of the regulation of TGase2 have not been reported due to the lack of a suitable assay to examine both activities on the same platform under near-physiologic conditions. Thus, we developed an on-chip dual enzyme activity assay for TGase2 to simultaneously monitor the transamidase and kinase activities. Reaction mixtures specific for each enzyme activity were applied onto osteopontin arrays, and enzyme activity was monitored by sequential probing with Cy5-strepavidin and Pro-Q Diamond stain. This approach was used to determine the optimal concentrations of ATP, Mg2+, and Ca2+ for dual-activity assays. The optimized assay was then used to investigate regulation of TGase2 transamidase and kinase activities by various cofactors that could potentially affect its conformation. Monothiol- and disulfide-containing compounds differentially regulated TGase2 transamidase and kinase activities. Acetylation of TGase2 activated the kinase activity but had no effect on the transamidase activity. Phosphorylation and dephosphorylation of TGase2 reciprocally regulated the transamidase and kinase activities. The new approach described here is thus useful for screening potential regulators of TGase2 transamidase and kinase and investigating the pathogenesis of TGase2-associated diseases.


Assuntos
Aminoaciltransferases/análise , Ensaios Enzimáticos/métodos , Proteínas de Ligação ao GTP/análise , Fosfotransferases/análise , Análise Serial de Proteínas/métodos , Transglutaminases/análise , Regulação Alostérica , Animais , Carbocianinas/química , Dissulfetos/química , Proteínas de Ligação ao GTP/química , Glicerol/análogos & derivados , Glicerol/química , Cobaias , Humanos , Osteopontina/química , Fosforilação , Proteína 2 Glutamina gama-Glutamiltransferase , Estreptavidina/química , Transglutaminases/química
10.
FASEB J ; : fj201800014RR, 2018 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-29782207

RESUMO

We investigated the beneficial effects of midazolam against vascular endothelial growth factor (VEGF)-induced vascular leakage and its molecular mechanism of action in human retinal endothelial cells (HRECs) and the retinas of diabetic mice. Midazolam inhibited VEGF-induced elevation of intracellular Ca2+, generation of reactive oxygen species (ROS), and transglutaminase activation in HRECs; these effects were reversed by the GABA, type A (GABAA) receptor antagonist flumazenil but not by the translocator protein antagonist PK11195. Midazolam also prevented VEGF-induced disassembly of adherens junctions and in vitro permeability. Intravitreal injection of midazolam prevented hyperglycemia-induced ROS generation, transglutaminase activation, and subsequent vascular leakage in the retinas of diabetic mice, and those effects were reversed by flumazenil. The roles of flumazenil were further supported by identifying GABAA receptors in mouse retinas. Thus, midazolam prevents hyperglycemia-induced vascular leakage by inhibiting VEGF-induced intracellular events in the retinas of diabetic mice.-Lee, Y.-J., Kim, M., Lee, J.-Y., Jung, S.-H., Jeon, H.-Y., Lee, S.-A., Kang, S., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

11.
J Endocrinol ; 235(1): 39-48, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28751454

RESUMO

Cysteamine (an aminothiol), which is derived from coenzyme A degradation and metabolized into taurine, has beneficial effects against cystinosis and neurodegenerative diseases; however, its role in diabetic complications is unknown. Thus, we sought to determine the preventive effect of cysteamine against hyperglycemia-induced vascular leakage in the retinas of diabetic mice. Cysteamine and ethanolamine, the sulfhydryl group-free cysteamine analogue, inhibited vascular endothelial growth factor (VEGF)-induced stress fiber formation and vascular endothelial (VE)-cadherin disruption in endothelial cells, which play a critical role in modulating endothelial permeability. Intravitreal injection of the amine compounds prevented hyperglycemia-induced vascular leakage in the retinas of streptozotocin-induced diabetic mice. We then investigated the potential roles of reactive oxygen species (ROS) and transglutaminase (TGase) in the cysteamine prevention of VEGF-induced vascular leakage. Cysteamine, but not ethanolamine, inhibited VEGF-induced ROS generation in endothelial cells and diabetic retinas. In contrast, VEGF-induced TGase activation was prevented by both cysteamine and ethanolamine. Our findings suggest that cysteamine protects against vascular leakage through inhibiting VEGF-induced TGase activation rather than ROS generation in diabetic retinas.


Assuntos
Cisteamina/administração & dosagem , Retinopatia Diabética/prevenção & controle , Retina/metabolismo , Vasos Retinianos/enzimologia , Transglutaminases/metabolismo , Animais , Retinopatia Diabética/enzimologia , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/enzimologia , Vasos Retinianos/efeitos dos fármacos , Transglutaminases/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Analyst ; 142(12): 2239-2246, 2017 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-28536714

RESUMO

Protein kinases play a critical role in a wide variety of cellular processes through post-translational protein phosphorylation and identification of their substrate proteins is important for understanding the enzymes' mechanism of action and elucidating their functions in physiological and disease processes. However, there have been few systematic investigations of protein kinase substrates due to a lack of high-throughput kinetic assays. Thus, we designed an on-chip protein kinase assay for profiling kinase kinetic parameters by introducing the phosphorylation rate (Vp) under physiological conditions, instead of the maximal velocity (Vmax), in a high-throughput manner. We applied this method to the kinetic analysis of protein kinase A (PKA) for 28 nuclear, cytosolic, plasma membrane, and extracellular target proteins to determine the substrate affinity (Km), Vp (FIATP10/s), and substrate preference (Vp/Km). We then constructed a map of PKA's kinetic parameters with respect to the 28 proteins based on subcellular localization. Thus, the on-chip protein kinase kinetic profiling is an ideal approach for systematically investigating substrate proteins of protein kinases and fully characterizing the enzymes' physiological functions.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Análise Serial de Proteínas , Fluorescência , Humanos , Cinética , Fosforilação , Especificidade por Substrato
13.
J Endocrinol ; 231(1): 97-108, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27554111

RESUMO

C-peptide exerts protective effects against diabetic complications; however, its role in inhibiting hyperglycemic memory (HGM) has not been elucidated. We investigated the beneficial effect of C-peptide on HGM-induced vascular damage in vitro and in vivo using human umbilical vein endothelial cells and diabetic mice. HGM induced apoptosis by persistent generation of intracellular ROS and sustained formation of ONOO(-) and nitrotyrosine. These HGM-induced intracellular events were normalized by treatment with C-peptide, but not insulin, in endothelial cells. C-peptide also inhibited persistent upregulation of p53 and activation of mitochondrial adaptor p66(shc) after glucose normalization. Further, C-peptide replacement therapy prevented persistent generation of ROS and ONOO(-) in the aorta of diabetic mice whose glucose levels were normalized by the administration of insulin. C-peptide, but not insulin, also prevented HGM-induced endothelial apoptosis in the murine diabetic aorta. This study highlights a promising role for C-peptide in preventing HGM-induced intracellular events and diabetic vascular damage.


Assuntos
Apoptose/efeitos dos fármacos , Peptídeo C/farmacologia , Endotélio Vascular/efeitos dos fármacos , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/farmacologia , Substâncias Protetoras/farmacologia , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Genes p53/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Hiperglicemia/etiologia , Insulina/farmacologia , Camundongos , Ácido Peroxinitroso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/efeitos dos fármacos
14.
Diabetes ; 65(8): 2414-28, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27207524

RESUMO

Diabetic retinopathy is predominantly caused by vascular endothelial growth factor (VEGF)-induced vascular leakage; however, the underlying mechanism is unclear. Here we designed an in vivo transglutaminase (TGase) activity assay in mouse retina and demonstrated that hyperglycemia induced vascular leakage by activating TGase2 in diabetic retina. VEGF elevated TGase2 activity through sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) concentrations in endothelial cells. The TGase inhibitors cystamine and monodansylcadaverin or TGase2 small interfering RNA (siRNA) prevented VEGF-induced stress fiber formation and vascular endothelial (VE)-cadherin disruption, which play a critical role in modulating endothelial permeability. Intravitreal injection of two TGase inhibitors or TGase2 siRNA successfully inhibited hyperglycemia-induced TGase activation and microvascular leakage in the retinas of diabetic mice. C-peptide or ROS scavengers also inhibited TGase activation in diabetic mouse retinas. The role of TGase2 in VEGF-induced vascular leakage was further supported using diabetic TGase2(-/-) mice. Thus, our findings suggest that ROS-mediated activation of TGase2 plays a key role in VEGF-induced vascular leakage by stimulating stress fiber formation and VE-cadherin disruption.


Assuntos
Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Transglutaminases/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Western Blotting , Peptídeo C/metabolismo , Caderinas/genética , Caderinas/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Experimental/genética , Proteínas de Ligação ao GTP/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transglutaminases/genética , beta Catenina/genética , beta Catenina/metabolismo
15.
Biosens Bioelectron ; 82: 40-8, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-27040940

RESUMO

Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34µg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions.


Assuntos
Técnicas Biossensoriais/instrumentação , Proteínas de Ligação ao GTP/metabolismo , Análise Serial de Proteínas/instrumentação , Proteínas Quinases/metabolismo , Transglutaminases/metabolismo , Apoptose , Ensaios Enzimáticos/instrumentação , Desenho de Equipamento , Humanos , Cinética , Fosforilação , Proteína 2 Glutamina gama-Glutamiltransferase , Especificidade por Substrato
16.
Anal Chem ; 88(10): 5398-405, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27109435

RESUMO

The objective of this study was to utilize an on-chip degradation assay to evaluate polymer depots and the predicted drug release from the depots. We conjugated four silk-elastinlike protein (SELP) polymers including SELP-815K, SELP-815K-RS1, SELP-815K-RS2, and SELP-815K-RS5 with a Cy5-NHS ester and fabricated SELP arrays by immobilizing the conjugated polymers onto well-type amine arrays. SELP polymer degradation rates were investigated by calculating the half-maximal effective concentration (EC50). Eight cleavage enzymes were applied, all of which exhibited distinctive EC50 values for SELP-815K and its three analogues. We successfully utilized this assay to study the in vitro release of the Cy5-conjugated C-peptide from SELP-815K hydrogel arrays. Additionally, cumulative C-peptide release from the SELP-815K depots was also demonstrated using repetitive elastase treatments. Therefore, this array-based on-chip degradation assay could potentially be used for evaluating depot degradation and controlled drug release from polymer depots at the molecular level.


Assuntos
Peptídeo C/análise , Análise em Microsséries/métodos , Peptídeo Hidrolases/metabolismo , Seda/metabolismo , Sequência de Aminoácidos , Peptídeo C/química , Peptídeo C/metabolismo , Carbocianinas/química , Ensaios de Triagem em Larga Escala , Hidrogéis/química , Dispositivos Lab-On-A-Chip , Análise em Microsséries/instrumentação , Seda/química , Espectroscopia de Infravermelho com Transformada de Fourier
17.
Biosens Bioelectron ; 79: 930-7, 2016 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-26802575

RESUMO

Glucose-6-phosphate dehydrogenase (G6PD) regulates nicotinamide adenine dinucleotide phosphate (NADPH) levels and is related to the pathogenesis of various diseases, including G6PD deficiency, type 2 diabetes, aldosterone-induced endothelial dysfunction, and cancer. Therefore, a highly sensitive array-based assay for determining quantitative G6PD activity is required. Here, we developed an on-chip G6PD activity assay using liquid droplet fluorescence arrays. Quantitative G6PD activity was determined by calculating reduced resorufin concentrations in liquid droplets. The limit of detection (LOD) of this assay was 0.162 mU/ml (2.89 pM), which is much more sensitive than previous assays. We used our activity assay to determine kinetic parameters, including Michaelis-Menten constants (Km) and maximum rates of enzymatic reaction (Vmax) for NADP(+) and G6P, and half-maximal inhibitory concentrations (IC50). We successfully applied this new assay to determine G6PD activity in human plasma from normal healthy individuals (n=30) and patients with inflammation (n=30). The inflammatory group showed much higher G6PD activities than did the normal group (p<0.001), with a high area under the curve value of 0.939. Therefore, this new activity assay has the potential to be used for diagnosis of G6PD-associated diseases and utilizing kinetic studies.


Assuntos
Ensaios Enzimáticos/instrumentação , Glucosefosfato Desidrogenase/sangue , Dispositivos Lab-On-A-Chip , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Feminino , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , NADP/metabolismo , Oxazinas/química , Oxirredução
18.
Clin Biochem ; 49(1-2): 127-31, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26327563

RESUMO

OBJECTIVES: We investigated the effect of cPKAα conformational states during protein immobilization on an array platform for cPKA autoantibody assays for sensitive and high-throughput profiling of protein kinase A (PKA) autoantibody levels in human sera. DESIGN AND METHODS: We prepared activated human cPKAα protein arrays by addition of cofactors including ATP, MgCl2, and Triton X-100 to incubation buffer. Anti-human cPKAα antibody or PKA autoantibody levels in human sera were analyzed using activated human cPKAα protein arrays. RESULTS: Activation of cPKAα with ATP, Mg(2+), and Triton X-100 enhanced the sensitivity of the assay by increasing the signal/noise ratio and lowering the limit of detection. cPKAα activation also enhanced the sensitivity of cPKA autoantibody detection in human sera. We successfully applied this assay to determine cPKA autoantibody levels in human sera from normal individuals (n=30) and hepatic cancer patients (n=30). CONCLUSIONS: Our results demonstrate that cPKAα activation enhanced the sensitivity of array-based PKA autoantibody assays, and that this assay is suitable for high-throughput analyses of cPKA autoantibodies in human sera.


Assuntos
Autoanticorpos/sangue , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Adulto , Idoso , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade
19.
Phytother Res ; 29(12): 1910-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26400610

RESUMO

Diabetic retinopathy is a major diabetic complication predominantly caused by vascular endothelial growth factor (VEGF)-induced vascular permeability in the retina; however, treatments targeting glycemic control have not been successful. Here, we investigated the protective effect of dammarenediol-II, a precursor of triterpenoid saponin biosynthesis, on VEGF-induced vascular leakage using human umbilical vein endothelial cells (HUVECs) and diabetic mice. We overproduced the compound in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase gene and purified using column chromatography. Analysis of the purified compound using a gas chromatography-mass spectrometry system revealed identical retention time and fragmentation pattern to those of authentic standard dammarenediol-II. Dammarenediol-II inhibited VEGF-induced intracellular reactive oxygen species generation, but it had no effect on the levels of intracellular Ca(2+) in HUVECs. We also found that dammarenediol-II inhibited VEGF-induced stress fiber formation and vascular endothelial-cadherin disruption, both of which play critical roles in modulating endothelial permeability. Notably, microvascular leakage in the retina of diabetic mice was successfully inhibited by intravitreal dammarenediol-II injection. Our results suggest that the natural drug dammarenediol-II may have the ability to prevent diabetic microvascular complications, including diabetic retinopathy.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Alquil e Aril Transferases/genética , Animais , Cálcio/metabolismo , Diabetes Mellitus Experimental/complicações , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Plantas Geneticamente Modificadas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/fisiopatologia , Saponinas/biossíntese , Nicotiana/genética , Nicotiana/metabolismo
20.
Analyst ; 140(19): 6588-94, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26307918

RESUMO

Protein kinase A (PKA) plays a crucial role in several biological processes; however, there is no assay with sufficient sensitivity and specificity to determine serological PKA (sPKA) activity. Here we present an on-chip activity assay that employs cysteine-modified kemptide arrays to determine specific sPKA activity in human sera that eliminates the potential contributions of other kinases with a protein kinase peptide inhibitor. The sensitivity of the on-chip sPKA activity assay was greatly enhanced by Triton X-100, with a 0.01 U mL(-1) detection limit. sPKA activity was determined by subtracting nonspecific sPK activity from total sPK activity. Our assay provided greater sensitivity and specificity and more accurate area under the curve values for gastric cancer compared to the total sPK activity assay. sPKA activities in human sera from patients with hepatic (n = 30), gastric (n = 30), lung (n = 30), and colorectal (n = 30) cancers were significantly higher than those in controls (n = 30, p < 10(-4)), but no significant difference in sPKA activities between normal and inflammation groups was observed. These results demonstrate that the on-chip assay accurately measures sPKA activity in human sera and that the sPKA activity may be a potential biomarker for cancer diagnosis.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/sangue , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ensaios Enzimáticos/métodos , Neoplasias/diagnóstico , Oligopeptídeos/metabolismo , Análise Serial de Proteínas/métodos , Sequência de Aminoácidos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Cisteína/química , Humanos , Neoplasias/sangue , Neoplasias/enzimologia , Octoxinol/química , Oligopeptídeos/química , Espectrometria de Fluorescência
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