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1.
Small ; : e2312120, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38558528

RESUMO

The tunable properties of 2D transition-metal dichalcogenide (TMDs) materials are extensively investigated for high-performance and wavelength-tunable optoelectronic applications. However, the precise modification of large-scale systems for practical optoelectronic applications remains a challenge. In this study, a wafer-scale atomic assembly process to produce 2D multinary (binary, ternary, and quaternary) TMDs for broadband photodetection is demonstrated. The large-area growth of homogeneous MoS2, Ni0.06Mo0.26S0.68, and Ni0.1Mo0.9S1.79Se0.21 is carried out using a succinct coating of the single-source precursor and subsequent thermal decomposition combined with thermal evaporation of the chalcogen powder. The optoelectrical properties of the multinary TMDs are dependent on the combination of heteroatoms. The maximum photoresponsivity of the MoS2-, Ni0.06Mo0.26S0.68-, and Ni0.1Mo0.9S1.79Se0.21-based photodetectors is 3.51 × 10-4, 1.48, and 0.9 A W-1 for 532 nm and 0.063, 0.42, and 1.4 A W-1 for 1064 nm, respectively. The devices exhibited excellent photoelectrical properties, which is highly beneficial for visible and near-infrared (NIR) photodetection.

2.
FASEB J ; 37(2): e22763, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36625326

RESUMO

Diabetic retinopathy (DR) is caused by retinal vascular dysfunction and neurodegeneration. Intraocular delivery of C-peptide has been shown to be beneficial against hyperglycemia-induced microvascular leakage in the retina of diabetes; however, the effect of C-peptide on diabetes-induced retinal neurodegeneration remains unknown. Moreover, extraocular C-peptide replacement therapy against DR to avoid various adverse effects caused by intravitreal injections has not been studied. Here, we demonstrate that systemic C-peptide supplementation using osmotic pumps or biopolymer-conjugated C-peptide hydrogels ameliorates neurodegeneration by inhibiting vascular endothelial growth factor-induced pathological events, but not hyperglycemia-induced vascular endothelial growth factor expression, in the retinas of diabetic mice. C-peptide inhibited hyperglycemia-induced activation of macroglial and microglial cells, downregulation of glutamate aspartate transporter 1 expression, neuronal apoptosis, and histopathological changes by a mechanism involving reactive oxygen species generation in the retinas of diabetic mice, but transglutaminase 2, which is involved in retinal vascular leakage, is not associated with these pathological events. Overall, our findings suggest that systemic C-peptide supplementation alleviates hyperglycemia-induced retinal neurodegeneration by inhibiting a pathological mechanism, involving reactive oxygen species, but not transglutaminase 2, in diabetes.


Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Hiperglicemia , Animais , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peptídeo C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Retina/metabolismo , Fatores de Crescimento do Endotélio Vascular , Retinopatia Diabética/metabolismo , Hiperglicemia/metabolismo , Suplementos Nutricionais
3.
BMC Med ; 21(1): 49, 2023 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-36782199

RESUMO

BACKGROUND: Hyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin. METHODS: Insulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney. RESULTS: We found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage. CONCLUSIONS: Overall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.


Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Humanos , Camundongos , Animais , Peptídeo C , Espécies Reativas de Oxigênio , Células Endoteliais , Diabetes Mellitus Experimental/complicações , Retina , Transglutaminases/fisiologia , Insulina/farmacologia , Pulmão , Retinopatia Diabética/complicações
4.
FASEB J ; 36(12): e22643, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36331561

RESUMO

Dopamine is a neurotransmitter that mediates visual function in the retina and diabetic retinopathy (DR) is the most common microvascular complication of diabetes and the leading cause of blindness; however, the role of dopamine in retinal vascular dysfunction in DR remains unclear. Here, we report a mechanism of hyperglycemic memory (HGM)-induced retinal microvascular dysfunction and the protective effect of dopamine against the HGM-induced retinal microvascular leakage and abnormalities. We found that HGM induced persistent oxidative stress, mitochondrial membrane potential collapse and fission, and adherens junction disassembly and subsequent vascular leakage after blood glucose normalization in the mouse retinas. These persistent hyperglycemic stresses were inhibited by dopamine treatment in human retinal endothelial cells and by intravitreal injection of levodopa in the retinas of HGM mice. Moreover, levodopa supplementation ameliorated HGM-induced pericyte degeneration, acellular capillary and pericyte ghost generation, and endothelial apoptosis in the mouse retinas. Our findings suggest that dopamine alleviates HGM-induced retinal microvascular leakage and abnormalities by inhibiting persistent oxidative stress and mitochondrial dysfunction.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Camundongos , Animais , Humanos , Retinopatia Diabética/tratamento farmacológico , Dopamina , Vasos Retinianos , Células Endoteliais , Levodopa/farmacologia , Retina
5.
Int J Mol Sci ; 23(2)2022 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-35054938

RESUMO

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2-/- mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.


Assuntos
Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Células Endoteliais/metabolismo , Hiperglicemia/complicações , Glomérulos Renais/metabolismo , Midazolam/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase/antagonistas & inibidores , Animais , Biomarcadores , Cálcio/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Diabetes Mellitus Experimental , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
6.
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
7.
FASEB J ; 33(11): 12655-12667, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31462079

RESUMO

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.-Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.


Assuntos
Aorta/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Hiperglicemia/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transglutaminases/metabolismo , Animais , Aorta/patologia , Linhagem Celular , Endotélio Vascular/patologia , Proteínas de Ligação ao GTP/genética , Humanos , Hiperglicemia/genética , Hiperglicemia/patologia , Camundongos , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética
8.
FASEB J ; 33(1): 750-762, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30020832

RESUMO

C-peptide has a beneficial effect against diabetic complications, but its role in hyperglycemia-induced metastasis is unknown. We investigated hyperglycemia-mediated pulmonary vascular leakage and metastasis and C-peptide inhibition of these molecular events using human pulmonary microvascular endothelial cells (HPMVECs) and streptozotocin-induced diabetic mice. VEGF, which is elevated in the lungs of diabetic mice, activated transglutaminase 2 (TGase2) in HPMVECs by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels. VEGF also induced vascular endothelial (VE)-cadherin disruption and increased the permeability of endothelial cells, both of which were prevented by the TGase inhibitors monodansylcadaverine and cystamine or TGM2-specific small interfering RNA. C-peptide prevented VEGF-induced VE-cadherin disruption and endothelial cell permeability through inhibiting ROS-mediated activation of TGase2. C-peptide supplementation inhibited hyperglycemia-induced ROS generation and TGase2 activation and prevented vascular leakage and metastasis in the lungs of diabetic mice. The role of TGase2 in hyperglycemia-induced pulmonary vascular leakage and metastasis was further demonstrated in diabetic Tgm2-/- mice. These findings demonstrate that hyperglycemia induces metastasis, and C-peptide prevents the hyperglycemia-induced metastasis in the lungs of diabetic mice by inhibiting VEGF-induced TGase2 activation and subsequent vascular leakage.-Jeon, H.-Y., Lee, Y.-J., Kim, Y.-S., Kim, S.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.


Assuntos
Peptídeo C/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Hiperglicemia/complicações , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Animais , Apoptose , Feminino , Proteínas de Ligação ao GTP/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteína 2 Glutamina gama-Glutamiltransferase , Espécies Reativas de Oxigênio/metabolismo , Transglutaminases/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
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.

10.
Malar J ; 16(1): 133, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28351409

RESUMO

BACKGROUND: Although a number of Plasmodium vivax proteins have been identified, few have been investigated as potential vaccine candidates. This study characterized the Plasmodium vivax merozoite surface antigen 180 (PvMSA180, PVX_094920), a novel P. vivax antigenic protein. METHODS: The target gene was amplified as four overlapping domains (D1, D2, D3 and D4) to enable expression of the recombinant protein using cell-free and bacterial expression systems. The recombinant PvMSA180 proteins were used in protein microarrays to evaluate the humoral immune response of 72 vivax-infected patients and 24 vivax-naïve individuals. Antibodies produced in mice against the PvMSA180-D1 and -D4 domains were used to assess the subcellular localization of schizont-stage parasites with immunofluorescence assays. A total of 51 pvmsa180 sequences from 12 countries (41 sequences from PlasmoDB and 6 generated in this study) were used to determine the genetic diversity and genealogical relationships with DNAsp and NETWORK software packages, respectively. RESULTS: PvMSA180 consists of 1603 amino acids with a predicted molecular mass of 182 kDa, and has a signal peptide at the amino-terminus. A total of 70.8% of patients (51/72) showed a specific antibody response to at least one of the PvMSA180 domains, and 20.8% (15/72) exhibited a robust antibody response to at least three of the domains. These findings suggest that PvMSA180 is targeted by the humoral immune response during natural infection with P. vivax. Immunofluorescence analysis demonstrated that PvMSA180 is localized on the merozoite surface of schizont-stage parasites, and pvmsa180 sequences originating from various geographic regions worldwide showed low genetic diversity. Twenty-two haplotypes were found, and haplotype 6 (Hap_6, 77%) of pvmsa180 was detected in isolates from six countries. CONCLUSIONS: A novel P. vivax surface protein, PvMSA180, was characterized in this study. Most of P. vivax-infected patients had specific antibodies against particular antigenic domains, indicating that this protein is immunogenic in naturally exposed populations. Genetic analysis of worldwide isolates showed that pvmsa180 is less polymorphic than other well-known candidates and that some haplotypes are common to several countries. However, additional studies with a larger sample size are necessary to evaluate the antibody responses in geographically separated populations, and to identify the function of PvMSA180 during parasite invasion.


Assuntos
Antígenos de Protozoários/análise , Antígenos de Superfície/análise , Merozoítos/química , Plasmodium vivax/química , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Feminino , Variação Genética , Humanos , Masculino , Merozoítos/imunologia , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Peso Molecular , Filogeografia , Plasmodium vivax/imunologia , Sinais Direcionadores de Proteínas/genética , Adulto Jovem
11.
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
12.
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
13.
Infect Immun ; 83(8): 3083-95, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25987709

RESUMO

Tryptophan-rich antigens (TRAgs) are an antigen family that has been identified in human and rodent malaria parasites. TRAgs have been proposed as candidate antigens for potential vaccines. The Plasmodium vivax TRAg (PvTRAg) family includes 36 members. Each PvTRAg contains a tryptophan-rich (TR) domain in the C-terminal region. In this study, we recombinantly expressed all 36 PvTRAgs using a cell-free expression system, and, for the first time, profiled the IgG antibody responses against all PvTRAgs in the sera from 96 vivax malaria patients and 40 healthy individuals using protein microarray technology. The mean seropositive rate for all PvTRAgs was 60.3%. Among them, nine PvTRAgs were newly identified in this study and showed a seropositive rate of >50%. Five of them, PvTRAg_13, PvTRAg_15, PvTRAg_16, PvTRAg_26, and PvTRAg_29, produced higher levels of IgG antibody, even in low-endemicity countries. In addition, the results of an immunofluorescence analysis suggest that PvTRAgs are, at least in part, associated with caveola-vesicle complexes, a unique structure of P. vivax-infected erythrocytes. The mechanism of formation and the function of these abundant membrane structures are not known. Further investigation aimed at determining the functions of these proteins would lead to a better understanding of the blood-stage biology of P. vivax.


Assuntos
Antígenos de Protozoários/imunologia , Malária Vivax/parasitologia , Plasmodium vivax/imunologia , Triptofano/imunologia , Adolescente , Adulto , Idoso , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Criança , Sequência Conservada , Feminino , Humanos , Malária Vivax/imunologia , Masculino , Pessoa de Meia-Idade , Plasmodium vivax/química , Plasmodium vivax/genética , Triptofano/química , Triptofano/genética , Adulto Jovem
14.
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
15.
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
16.
Diabetes ; 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39024127

RESUMO

Diabetic retinopathy is the most common microvascular complication caused by chronic hyperglycemia and is a leading cause of blindness; however, the underlying molecular mechanism has not been clearly elucidated. Thus, we investigated whether regulation of AMPactivated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by transglutaminase 2 (TGase2) is important for hyperglycemia-induced microvascular leakage in the diabetic retina. In HRECs and diabetic mouse retinas, we found that TGase2, activated by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels, played an essential role in hyperglycemia-induced vascular leakage. ROS generation and TGsae2 activation were involved in hyperglycemia-induced AMPK dephosphorylation, which resulted in VE-cadherin disassembly and increased fluorescein isothiocyanate-dextran extravasation. Furthermore, high glucose-induced TGase2 activation suppressed GAPDH activity, determined by an on-chip activity assay, through inhibition of AMPK, which induced VE-cadherin disassembly and endothelial permeability in HRECs. Overall, our findings suggest that inhibition of AMPK and GAPDH by TGase2 plays a pivotal role in hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

17.
Biomed Pharmacother ; 172: 116232, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38310652

RESUMO

Proinsulin C-peptide, a biologically active polypeptide released from pancreatic ß-cells, is known to prevent hyperglycemia-induced microvascular leakage; however, the role of C-peptide in migration and invasion of cancer cells is unknown. Here, we investigated high glucose-induced migration and invasion of ovarian cancer cells and the inhibitory effects of human C-peptide on metastatic cellular responses. In SKOV3 human ovarian cancer cells, high glucose conditions activated a vicious cycle of reactive oxygen species (ROS) generation and transglutaminase 2 (TGase2) activation through elevation of intracellular Ca2+ levels. TGase2 played a critical role in high glucose-induced ovarian cancer cell migration and invasion through ß-catenin disassembly. Human C-peptide inhibited high glucose-induced disassembly of adherens junctions and ovarian cancer cell migration and invasion through inhibition of ROS generation and TGase2 activation. The preventive effect of C-peptide on high glucose-induced ovarian cancer cell migration and invasion was further demonstrated in ID8 murine ovarian cancer cells. Our findings suggest that high glucose conditions induce the migration and invasion of ovarian cancer cells, and human C-peptide inhibits these metastatic responses by preventing ROS generation, TGase2 activation, and subsequent disassembly of adherens junctions.


Assuntos
Neoplasias Ovarianas , Humanos , Animais , Camundongos , Feminino , Peptídeo C/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Neoplasias Ovarianas/patologia , Movimento Celular , Glucose/farmacologia
19.
Theranostics ; 13(8): 2424-2438, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37215567

RESUMO

Rationale: Neovascularization is a hallmark of the late stages of diabetic retinopathy (DR) leading to blindness. The current anti-DR drugs have clinical disadvantages including short circulation half-lives and the need for frequent intraocular administration. New therapies with long-lasting drug release and minimal side effects are therefore needed. We explored a novel function and mechanism of a proinsulin C-peptide molecule with ultra-long-lasting delivery characteristics for the prevention of retinal neovascularization in proliferative diabetic retinopathy (PDR). Methods: We developed a strategy for ultra-long intraocular delivery of human C-peptide using an intravitreal depot of K9-C-peptide, a human C-peptide conjugated to a thermosensitive biopolymer, and investigated its inhibitory effect on hyperglycemia-induced retinal neovascularization using human retinal endothelial cells (HRECs) and PDR mice. Results: In HRECs, high glucose conditions induced oxidative stress and microvascular permeability, and K9-C-peptide suppressed those effects similarly to unconjugated human C-peptide. A single intravitreal injection of K9-C-peptide in mice resulted in the slow release of human C-peptide that maintained physiological levels of C-peptide in the intraocular space for at least 56 days without inducing retinal cytotoxicity. In PDR mice, intraocular K9-C-peptide attenuated diabetic retinal neovascularization by normalizing hyperglycemia-induced oxidative stress, vascular leakage, and inflammation and restoring blood-retinal barrier function and the balance between pro- and anti-angiogenic factors. Conclusions: K9-C-peptide provides ultra-long-lasting intraocular delivery of human C-peptide as an anti-angiogenic agent to attenuate retinal neovascularization in PDR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Hiperglicemia , Neovascularização Retiniana , Humanos , Camundongos , Animais , Neovascularização Retiniana/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Peptídeo C/farmacologia , Peptídeo C/uso terapêutico , Células Endoteliais , Neovascularização Patológica/tratamento farmacológico , Hiperglicemia/tratamento farmacológico
20.
J Mol Endocrinol ; 68(4): 209-223, 2022 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-35266881

RESUMO

Proinsulin C-peptide has a protective effect against diabetic complications; however, its role in hyperglycemia-induced pulmonary fibrosis is unknown. In this study, we investigated the inhibitory effect of C-peptide on hyperglycemia-induced pulmonary fibrosis and the molecular mechanism of C-peptide action in the lungs of diabetic mice and in human pulmonary microvascular endothelial cells (HPMVECs). We found that, in the lungs of diabetic mice, C-peptide supplementation using osmotic pumps attenuated hyperglycemia-induced pulmonary fibrosis and expression of fibrosis-related proteins. In HPMVECs, C-peptide inhibited vascular endothelial growth factor-induced adherens junction disruption and endothelial cell permeability by inhibiting reactive oxygen species generation and transglutaminase (TGase) activation. In the lungs, C-peptide supplementation suppressed hyperglycemia-induced reactive oxygen species generation, TGase activation, and microvascular leakage. C-peptide inhibited hyperglycemia-induced inflammation and apoptosis, which are involved in the pathological process of pulmonary fibrosis. We also demonstrated the role of TGase2 in hyperglycemia-induced vascular leakage, inflammation, apoptosis, and pulmonary fibrosis in the lungs of diabetic TGase2-null (Tgm2-/-) mice. Furthermore, we demonstrated a long-term inhibitory effect of systemic delivery of C-peptide using K9-C-peptide hydrogels on hyperglycemia-induced fibrosis in diabetic lungs. Overall, our findings suggest that C-peptide alleviates hyperglycemia-induced pulmonary fibrosis by inhibiting TGase2-mediated microvascular leakage, inflammation, and apoptosis in diabetes.


Assuntos
Diabetes Mellitus Experimental , Hiperglicemia , Fibrose Pulmonar , Animais , Peptídeo C/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/metabolismo , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Glutamina gama-Glutamiltransferase , Fibrose Pulmonar/complicações , Fibrose Pulmonar/etiologia , Espécies Reativas de Oxigênio/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
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