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1.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204265

RESUMO

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.


Assuntos
ADP Ribose Transferases , Antineoplásicos Imunológicos/farmacologia , Toxinas Bacterianas , Exotoxinas , Imunotoxinas/farmacologia , Proteínas Ligantes de Maltose , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética
2.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198626

RESUMO

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.


Assuntos
Fator de Células-Tronco/biossíntese , Sequência de Aminoácidos , Ciclo Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fator de Células-Tronco/química
3.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067755

RESUMO

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.


Assuntos
Cromatografia em Gel/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Isomerases de Dissulfetos de Proteínas/metabolismo , Diferenciação Celular , Eletroforese em Gel de Poliacrilamida/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas Ligantes de Maltose/metabolismo , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/fisiologia , Transporte Proteico , Solubilidade
4.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073063

RESUMO

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.


Assuntos
Meios de Cultura/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Pluripotentes , Proteína Desglicase DJ-1/metabolismo , Técnicas de Cultura de Células , Proliferação de Células , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
5.
Nat Metab ; 3(3): 410-427, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33758420

RESUMO

TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca2+-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB-GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB-GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Resistência à Insulina , Lisossomos/metabolismo , Obesidade/prevenção & controle , Estresse Fisiológico , Tecido Adiposo/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Obesidade/metabolismo
6.
Sci Rep ; 10(1): 9438, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523015

RESUMO

Fluorescence-based assays should be feasible in aqueous media for effectively detecting the biological factors. However, numerous sensors have limited signal transductions and low fluorescence quantum yields due to the ingerently reduced excited state energy of fluorophores in aqueous solution, which reduces their sensitivity. This necessitates a smart sensing approach with an amplified fluorescence response for analytes in aqueous solution. Herein, a new building block which self-assembles in aqueous media, giving a micellar sturcuture with the hydrophobic π-extended conjugated system at the core and hydrophilic groups at the periphery, was devised for the first time. We demonstrated that the aggregated fluorophores in a micelle induce amplified fluorescence quenching, in which the excited electron efficiently migrates through π-extended conjugated system in a micelle, as in a polymeric system. Such feature differentiates this sensing approach from the numerous fluorescence-based tools previously developed for sensitive detection. This new system exhibited highly sensitive signal transduction for specific analytes even under actual bioanalytical conditions.


Assuntos
Corantes Fluorescentes/química , Heparina/análise , Imagem Óptica/métodos , Fluorescência , Heparina/sangue , Humanos , Interações Hidrofóbicas e Hidrofílicas , Micelas , Polímeros/química , Espectrometria de Fluorescência/métodos , Água/química
7.
Int J Obes (Lond) ; 44(3): 697-706, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31965068

RESUMO

BACKGROUND: This study investigated depot-specific mRNA expression of uncoupling protein 1 (UCP1) in human white adipose tissue (WAT) and its association with obesity-related markers. METHODS: We recruited 39 normal-weight, 41 nondiabetic obese, and 22 diabetic obese women. We measured UCP1 mRNA expression in abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and investigated the associations between UCP1 mRNA expression in VAT and SAT, and obesity-related markers including mRNA expression of leptin, adiponectin, CCAAT-enhancer-binding protein homologous protein (CHOP), and positive regulatory domain-containing protein 16 (PRDM16). We also evaluated UCP1 mRNA expression in differentiated human white adipocytes after treatment with various stressors and metabolic improvement agents in vitro. RESULTS: UCP1 mRNA in VAT was significantly higher than in SAT in all groups. UCP1 mRNA in SAT was negatively correlated with BMI, total abdominal fat area, visceral fat area, blood pressure, fasting glucose, insulin, HOMA-IR score, triglyceride, hsCRP, fasting leptin levels, and adipocyte size. UCP1 mRNA in SAT was positively correlated with fasting adiponectin levels. UCP1 mRNA in VAT was negatively correlated with visceral-to-subcutaneous fat ratio (VSR), fasting glucose, and triglyceride levels. In SAT, UCP1 mRNA was negatively correlated with mRNA expression of leptin and CHOP, and positively correlated with mRNA expression of adiponectin. The expression of PRDM16 was positively correlated with UCP1 mRNA in both VAT and SAT. UCP1 mRNA expression in differentiated human white adipocytes was significantly reduced after incubation with thapsigargin, tunicamycin, homocysteine, TNF-α, or IL-ß, and significantly increased after incubation with exendin 4, dapagliflozin, and telmisartan. CONCLUSIONS: This study demonstrated depot-specific mRNA expression of UCP1 and its association with obesity-related markers in human WAT. UCP1 mRNA in human white adipocytes was suppressed by inflammatory agents and enhanced by metabolic improvement agents. UCP1 in human WAT might participate in the pathogenesis of obesity-related metabolic diseases.


Assuntos
Tecido Adiposo Branco/metabolismo , Obesidade/metabolismo , Proteína Desacopladora 1 , Adipócitos/metabolismo , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático/fisiologia , Feminino , Humanos , Inflamação/metabolismo , Resistência à Insulina/fisiologia , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Proteína Desacopladora 1/análise , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
8.
J Clin Endocrinol Metab ; 105(1)2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31642491

RESUMO

CONTEXT: Adrenomedullin 2 (AM2) plays protective roles in the renal and cardiovascular systems. Recent studies in experimental animals demonstrated that AM2 is an adipokine with beneficial effects on energy metabolism. However, there is little information regarding AM2 expression in human adipose tissue. OBJECTIVE: To investigate the pattern and regulation of the expression of AM2 and its receptor component in human adipose tissue, in the context of obesity and type 2 diabetes. METHODS: We measured metabolic parameters, serum AM2, and expression of ADM2 and its receptor component genes in abdominal subcutaneous and visceral adipose tissue in obese (with or without type 2 diabetes) and normal-weight women. Serum AM2 was assessed before and 6 to 9 months after bariatric surgery. Expression/secretion of AM2 and its receptor were assessed in human adipocytes. RESULTS: ADM2 mRNA in both fat depots was higher in obese patients, whether diabetic or not. Although serum AM2 was significantly lower in obese patients, it was not changed after bariatric surgery. AM2 and its receptor complex were predominantly expressed by adipocytes, and the expression of CALCRL, encoding a component of the AM2 receptor complex, was lower in both fat depots of obese patients. Incubating adipocytes with substances mimicking the microenvironment of obese adipose tissue increased ADM2 mRNA but reduced both AM2 secretion into culture media and CALCRL mRNA expression. CONCLUSIONS: Our data indicate that AM2 signaling is suppressed in adipose tissue in obesity, involving lower receptor expression and ligand availability, likely contributing to insulin resistance and other aspects of the pathophysiology associated with obesity.


Assuntos
Tecido Adiposo/metabolismo , Proteína Semelhante a Receptor de Calcitonina/genética , Obesidade/genética , Hormônios Peptídicos/genética , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Adulto , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Resistência à Insulina/genética , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/patologia , Hormônios Peptídicos/metabolismo , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Transdução de Sinais/genética , Adulto Jovem
9.
Protein Expr Purif ; 167: 105530, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31698036

RESUMO

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.


Assuntos
Albumina Sérica Humana/biossíntese , Cromatografia em Gel , Escherichia coli/genética , Escherichia coli/metabolismo , Etiquetas de Sequências Expressas/metabolismo , Humanos , Proteínas Ligantes de Maltose/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/isolamento & purificação , Albumina Sérica Humana/metabolismo , Solubilidade
10.
Sci Rep ; 9(1): 13706, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31548569

RESUMO

Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b'a' domain of PDI (PDIb'a'), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.


Assuntos
Oncostatina M/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Escherichia coli , Expressão Gênica , Engenharia Genética , Histidina , Humanos , Proteínas Ligantes de Maltose/metabolismo , Oligopeptídeos , Oncostatina M/genética , Proteínas Recombinantes de Fusão/genética , Solubilidade
11.
BMB Rep ; 52(8): 496-501, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30670149

RESUMO

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins. [BMB Reports 2019; 52(8): 496-501].


Assuntos
ADP Ribose Transferases/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Exotoxinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Fatores de Virulência/farmacologia , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antineoplásicos/química , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Exotoxinas/química , Exotoxinas/metabolismo , Feminino , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo
12.
Sci Rep ; 8(1): 13976, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30228336

RESUMO

Angiopoietin-like protein 2 has been proposed to be a key mediator linking obesity and insulin resistance. However, no detailed study of ANGPTL2 expression in human adipose tissues has yet been reported. To investigate the pattern and regulation of ANGPTL2 expression in human adipose tissues in obesity and its related diseases, we recruited 32 non-diabetic and 13 type 2 diabetic obese women and 32 normal-weight women. ANGPTL2 mRNA was expressed at a similar level in visceral and subcutaneous adipose tissues. Adipose tissue ANGPTL2 mRNA was much higher in obese patients. Adipose tissue ANGPTL2 mRNA and serum ANGPTL2 levels showed strong associations with metabolic parameters associated with insulin resistance. In adipose tissue, ANGPTL2 mRNA was closely correlated with the expression of genes involved in inflammation and ER stress. ANGPTL2 mRNA was principally expressed in adipocytes, and its expression was markedly higher in the adipocyte but non-adipocyte fraction of obese adipose tissues. Culture of human adipocytes under conditions mimicking the microenvironment of obese adipose tissue (especially, increased ER stress) stimulated ANGPTL2 gene expression and secretion. In addition, co-culture of adipocytes and macrophages suggested that ANGPTL2 excessively produced by adipocytes, may contribute inflammation and remodeling in obese adipose tissues, thereby promoting insulin resistance.


Assuntos
Tecido Adiposo/patologia , Proteínas Semelhantes a Angiopoietina/metabolismo , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/patologia , Inflamação/diagnóstico , Resistência à Insulina , Obesidade/patologia , Tecido Adiposo/metabolismo , Adulto , Proteínas Semelhantes a Angiopoietina/genética , Estudos de Casos e Controles , Estudos de Coortes , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Pessoa de Meia-Idade , Obesidade/metabolismo , Prognóstico
13.
Sci Rep ; 7(1): 16139, 2017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29170489

RESUMO

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a ß-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Escherichia coli/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Humanos , Proteínas Ligantes de Maltose/genética , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
14.
J Microbiol Biotechnol ; 27(12): 2156-2164, 2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29032646

RESUMO

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/µg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC50 and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.


Assuntos
Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Antineoplásicos/farmacologia , Apoptose , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Proteínas Ligantes de Maltose/genética , Isomerases de Dissulfetos de Proteínas/genética , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF/genética
15.
Sci Rep ; 6: 30609, 2016 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-27465988

RESUMO

Extracellular matrix (ECM) remodeling dynamically occurs to accommodate adipose tissue expansion during obesity. One non-fibrillar component of ECM, biglycan, is released from the matrix in response to tissue stress; the soluble form of biglycan binds to toll-like receptor 2/4 on macrophages, causing proinflammatory cytokine secretion. To investigate the pattern and regulatory properties of biglycan expression in human adipose tissues in the context of obesity and its related diseases, we recruited 21 non-diabetic obese women, 11 type 2 diabetic obese women, and 59 normal-weight women. Regardless of the presence of diabetes, obese patients had significantly higher biglycan mRNA in both visceral and subcutaneous adipose tissue. Biglycan mRNA was noticeably higher in non-adipocytes than adipocytes and significantly decreased during adipogenesis. Adipose tissue biglycan mRNA positively correlated with adiposity indices and insulin resistance parameters; however, this relationship disappeared after adjusting for BMI. In both fat depots, biglycan mRNA strongly correlated with the expression of genes related to inflammation and endoplasmic reticulum stress. In addition, culture of human preadipocytes and differentiated adipocytes under conditions mimicking the local microenvironments of obese adipose tissues significantly increased biglycan mRNA expression. Our data indicate that biglycan gene expression is increased in obese adipose tissues by altered local conditions.


Assuntos
Tecido Adiposo/fisiologia , Biglicano/genética , Obesidade/genética , Gordura Abdominal/fisiologia , Adipócitos/patologia , Adipócitos/fisiologia , Adulto , Biglicano/metabolismo , Estudos de Casos e Controles , Diferenciação Celular/genética , Tamanho Celular , Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/genética , Feminino , Expressão Gênica , Humanos , Hiperglicemia/genética , Pessoa de Meia-Idade , Gordura Subcutânea/fisiologia
16.
Health Care Women Int ; 37(3): 288-300, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25424487

RESUMO

We used the job-demand-control model to answer our two research questions concerning the effects of working conditions on self-rated health and gender differences and the association between these working conditions and health among Korean manual workers. Since a disproportionate representation of women in nonstandard work positions is found in many countries, including Korea, it is important to examine how working conditions explain gender inequality in health. We used data from the 2008-2009 Korean National Health and Nutrition Examination Survey and analyzed a total sample of 1,482 men and 1,350 women using logistic regression. We found that job control was positively related to self-rated health, while both physical and mental job demands were negatively related to self-rated health. We also found significant interaction effects of job demands, control, and gender on health. Particularly, female workers' health was more vulnerable to mentally demanding job conditions. We discussed theoretical and practice implications based on these findings.


Assuntos
Emprego/psicologia , Nível de Saúde , Fatores Sexuais , Estresse Psicológico/complicações , Carga de Trabalho/psicologia , Local de Trabalho/normas , Adolescente , Adulto , Idoso , Família , Feminino , Inquéritos Epidemiológicos , Humanos , Satisfação no Emprego , Masculino , Saúde Mental , Pessoa de Meia-Idade , Saúde do Trabalhador , República da Coreia , Distribuição por Sexo , Fatores Socioeconômicos , Adulto Jovem
17.
Obes Surg ; 26(4): 825-32, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26231823

RESUMO

BACKGROUND: Bariatric surgery has beneficial effects on weight loss and metabolic profiles. Recent evidence suggests that liver-derived hepatokines play a role in the pathophysiology of metabolic diseases. However, few studies have reported longitudinal changes in hepatokines after gastric bypass surgery. We investigated changes in the serum levels of angiopoietin-like protein 6 (Angptl6) and selenoprotein P after gastric bypass surgery. METHODS: We followed 10 patients who were treated with gastric bypass for weight loss. We measured metabolic parameters and the serum levels of Angptl6 and selenoprotein P before, 1 month after, and 9 months after surgery. We investigated the changes in those hepatokines after surgery and the associations between changes in Angptl6 and selenoprotein P, respectively, and metabolic parameters. RESULTS: Body mass index decreased linearly. Levels of hemoglobin A1c (HbA1c), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), total cholesterol, triglyceride, LDL cholesterol, and Angptl6 were significantly lower 1 and 9 months after surgery. Fasting plasma glucose was normal throughout the study. Fasting insulin decreased 1 month after surgery but increased 9 months post-surgery. Levels of selenoprotein P increased linearly. Significant correlations were detected between the levels of Angptl6 and LDL cholesterol and fasting insulin. Changes in Angptl6 levels were significantly correlated with changes in total cholesterol and LDL cholesterol. Selenoprotein P levels were inversely correlated with GGT, and changes in selenoprotein P were inversely correlated with changes in homeostasis model assessment for insulin resistance (HOMA-IR). CONCLUSIONS: Our results suggest that gastric bypass may alter the serum levels of hepatokines independent of weight loss, and these changes are related to certain hepatic metabolic changes.


Assuntos
Angiopoietinas/sangue , Derivação Gástrica , Selenoproteína P/sangue , Adulto , Alanina Transaminase/sangue , Proteínas Semelhantes a Angiopoietina , Aspartato Aminotransferases/sangue , Feminino , Hemoglobina A Glicada/análise , Humanos , Insulina/sangue , Resistência à Insulina/fisiologia , Masculino , Pessoa de Meia-Idade , Triglicerídeos/sangue , gama-Glutamiltransferase/sangue
18.
Pflugers Arch ; 467(8): 1689-97, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25196539

RESUMO

Diabetes mellitus and hypertension are common diseases frequently coexisting. Although augmentation of L-type Ca(2+) channel (ICaL) activity has been reported in vascular smooth muscle cells (VSMCs) of a spontaneously hypertensive rat model, no study on ICaL has been conducted for coexisting hypertension and diabetes. Sprague Dawley rats were assigned to four groups: a sham-operated control group (CG), a unilateral nephrectomy group (UNG), a streptozotocin (STZ)-induced type 1 diabetic group (SDG) and a coexisting hypertension and diabetes group (DHG), which underwent nephrectomy and received STZ injection. Blood pressure (BP) was significantly lower in the CG than in the other three groups. The membrane capacitance of VSMCs was nearly doubled in the SDG and DHG but not in the UNG. The ICaL was increased approximately 2-fold in both the UNG and SDG and approximately 4-fold in the DHG. The current density of ICaL was increased approximately 2-fold in the UNG and DHG, while no significant increase was seen in the SDG. The rate of Ca(2+) removal was inhibited significantly, by ~33 %, in the DHG. In conclusion, the effects of hypertension and diabetes on ICaL were apparently additive, and the vascular consequences of combined diabetes and hypertension may be caused by an elevated ICaL and slowed Ca(2+) removal.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hipertensão/metabolismo , Mesentério/irrigação sanguínea , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Nefrectomia , Estreptozocina , Animais , Arteríolas/metabolismo , Pressão Sanguínea , Cálcio/metabolismo , Sinalização do Cálcio , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/fisiopatologia , Hipertensão/etiologia , Hipertensão/fisiopatologia , Potenciais da Membrana , Músculo Liso Vascular/fisiopatologia , Ratos Sprague-Dawley , Fatores de Tempo
19.
Mol Biol Rep ; 42(3): 651-63, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25391768

RESUMO

Human chemokine (C-C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags-Trx, GST, MBP, NusA, His8, PDI, and PDIb'a'-for their ability to allow the soluble overexpression of hCCL2. Three tags-MBP, His8, and PDI-solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18 °C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor.


Assuntos
Quimiocina CCL2/genética , Escherichia coli/genética , Expressão Gênica , Proteínas Ligantes de Maltose/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Linhagem Celular , Quimiocina CCL2/metabolismo , Escherichia coli/metabolismo , Ordem dos Genes , Humanos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
J Clin Endocrinol Metab ; 99(7): E1263-71, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24712570

RESUMO

OBJECTIVES: IL-34 is a recently identified alternative ligand for colony-stimulating factor-1 (CSF-1) receptor. IL-34 and CSF-1 are regulators of differentiation, proliferation, and survival in mononuclear phagocytes. Here, we investigated the IL-34 serum concentration and expression in human adipose tissues and any associations with insulin resistance. METHODS: We recruited 19 nondiabetic obese women, 9 type 2 diabetic women, and 27 normal-weight women. Metabolic parameters, abdominal fat distribution, serum IL-34 concentration, and IL-34 mRNA expression were measured in abdominal sc adipose tissue (SAT) and visceral adipose tissue (VAT). In addition, the expression/secretion and putative effects of IL-34 were assessed in human differentiated adipocytes. Serum IL-34 concentration was measured before and 5 to 9 months after laparoscopic Roux-en-Y gastric bypass surgery was performed on the 20 obese patients. RESULTS: Regardless of diabetes status, obese patients demonstrated significantly higher serum IL-34 concentrations than controls. Serum IL-34 was significantly and positively correlated with insulin resistance-related metabolic parameters. IL-34 mRNA was significantly higher in VAT than SAT. IL-34 was expressed in adipocytes as well as nonadipocytes, and expression was significantly higher during adipogenesis. In differentiated adipocytes, the expression/secretion of IL-34 was enhanced by TNFα and IL-1ß. In addition, IL-34 augmented fat accumulation and inhibited the stimulatory effects of insulin on glucose transport. Moreover, serum IL-34 was significantly decreased after Roux-en-Y gastric bypass-induced weight loss. CONCLUSION: The present study demonstrates, for the first time, that IL-34 is expressed in human adipose tissues and the circulating concentration is significantly elevated in obese patients. This suggests that IL-34 is associated with insulin resistance.


Assuntos
Inflamação/sangue , Resistência à Insulina , Interleucinas/sangue , Obesidade/sangue , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Adulto , Células Cultivadas , Doença Crônica , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Feminino , Humanos , Inflamação/complicações , Inflamação/genética , Resistência à Insulina/genética , Interleucinas/genética , Interleucinas/farmacologia , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/genética , Adulto Jovem
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