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CONTEXT: J-DISCOVER is a prospective observational cohort study aiming to understand the current management of patients with early-stage type 2 diabetes mellitus (T2DM) in Japan, enrolling patients initiating second-line treatment. OBJECTIVE: The current analysis examined the change in treatment satisfaction during the study period and factors affecting this change among patients in J-DISCOVER. METHODS: We used data from the J-DISCOVER study, in which 1798 patients with T2DM agedâ ≥â 20 years were enrolled from 142 sites across Japan. Treatment satisfaction was assessed using the Diabetes Treatment Satisfaction Questionnaire (DTSQ). RESULTS: The mean DTSQ treatment satisfaction score increased from 25.9 points at baseline to 27.3 points at 6 months, which was maintained through 36 months. Among the baseline characteristics examined, higher baseline DTSQ treatment satisfaction scores (Pâ <â 0.0001), older age (≥ 75 vsâ <â 65 years, Pâ =â 0.0096), living alone (Pâ =â 0.0356), and type of facility (clinics vs hospitals, Pâ =â 0.0044) had a significantly negative impact on the changes in DTSQ treatment satisfaction scores. Improvement in mean glycated hemoglobin (HbA1c) from baseline (7.7%) to 36 months (7.1%) was associated with positive changes in the DTSQ treatment satisfaction score (Pâ =â 0.0003). CONCLUSION: Changes in DTSQ treatment satisfaction scores were related to HbA1c improvement, suggesting that the management strategy was appropriately planned for each patient. The results also suggest that the availability of social support for patients with T2DM who are elderly or living alone may be an important factor affecting treatment satisfaction, adherence, and clinical outcomes.
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Diabetes Mellitus Tipo 2 , Anciano , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hemoglobina Glucada/análisis , Humanos , Hipoglucemiantes/uso terapéutico , Satisfacción del Paciente , Satisfacción Personal , Estudios ProspectivosRESUMEN
INTRODUCTION: J-DISCOVER is a prospective, observational cohort study that aimed to understand characteristics, glycaemic control, comorbidities and real-world management of patients with early-stage type 2 diabetes mellitus (T2DM) in Japan, by enrolling patients initiating second-line treatment from both diabetes specialist and non-specialist care settings. METHODS: As part of the global DISCOVER programme, J-DISCOVER enrolled 1798 patients with T2DM aged at least 20 years old from 142 sites across Japan, from September 2014 to December 2015, and followed these patients for 3 years. Glycaemic control, body mass index (BMI), blood pressure, lipid profiles, treatment patterns, and prevalence of CKD and retinopathy were examined from baseline to 6, 12, 24 and 36 months, stratified by class of second-line treatment. RESULTS: At baseline, the median time after T2DM diagnosis was 3.1 years and mean glycated haemoglobin (HbA1c) was 7.7%. The mean individualized HbA1c target was 6.7 ± 0.5%, and 55.3% of patients were set the target of < 7.0%. HbA1c reductions were noted from 6 months and mean HbA1c was 7.1% at 36 months. The proportion of patients with HbA1c < 7.0% increased from 28.8% at baseline to 53.3% at 36 months, and the achievement rate of individualized HbA1c targets increased from 6.1% to 30.3%. Only two cases of severe hypoglycaemia occurred during the study. No major changes in BMI, blood pressure, lipid profile or prescription of antihypertensive or dyslipidaemia medications were observed. The frequencies of screening to detect retinopathy and chronic kidney disease (CKD) were 17.0-21.0% and 14.5-16.0%, respectively, during the follow-up period. The prevalence of CKD, but not retinopathy, increased over the follow-up period. CONCLUSIONS: This study provided an overview of the 3-year management of early-stage T2DM in patients initiating second-line treatment. Contemporary management improved glycaemic control with an acceptable risk-benefit balance, although hurdles remain to sufficient implementation of guideline-recommended treatments in current clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02226822.
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INTRODUCTION: J-DISCOVER aims to research the treatment reality of Japanese patients with type 2 diabetes mellitus who begin second-line treatment. Here we report baseline characteristics and factors associated with selection of second-line treatment. METHODS: J-DISCOVER is a prospective, observational, multicenter, cohort study in patients with type 2 diabetes (aged ≥ 20 years) beginning second-line treatment after first-line oral monotherapy. Baseline characteristics and treatment patterns were descriptively summarized. Logistic regression models were used to identify factors associated with specific second-line treatments. RESULTS: A total of 1806 patients (mean age 61.7 years) were enrolled between September 2014 and December 2015. Mean ± standard deviation of baseline glycated hemoglobin (HbA1c) and body mass index (BMI) were 7.7 ± 1.3% and 25.5 ± 4.6 kg/m2, respectively. The most prescribed medication as first-line treatment was dipeptidyl peptidase 4 inhibitors (53.7% of patients) followed by biguanides (21.4%), sulfonylureas (7.2%), and alpha-glucosidase inhibitors (6.8%). Second-line treatments included dipeptidyl peptidase 4 inhibitors (31.0%), biguanides (27.9%), sodium-glucose cotransporter 2 inhibitors (12.2%), and sulfonylureas (10.9%). First- and second-line treatments had different modes of action in 76.3% of patients. Those receiving first-line dipeptidyl peptidase 4 inhibitors were more likely to receive second-line biguanides and vice versa. Selection of second-line treatment was also associated with age, BMI, HbA1c, and renal function. CONCLUSIONS: This study showed the treatment reality and factors associated with choice of second-line treatment in Japanese patients with type 2 diabetes mellitus. The choice of second-line treatment was associated with age, BMI, HbA1c, renal function, and the mode of action of the first-line treatment. TRIAL REGISTRATION: ClinicalTrials.gov NCT02226822.
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Cancer cells surviving in ascites exhibit cancer stem cell (CSC)-like features. This study analyzed the expression of the CSC marker CD133 in the ascites-derived exosomes obtained from patients with unresectable pancreatic cancer. In addition, inverse correlation of CD133 expression with prognosis was examined. Of the 133 consecutive patients, 19 patients were enrolled in the study. Exosomes derived from the malignant ascites demonstrated higher density and wider variation in size than those from non-malignant ascites. Western blot revealed enhanced expression of CD133 in exosomes obtained from patients with pancreatic cancer compared to those obtained from patients with gastric cancer or liver cirrhosis. A xenograft mouse model with malignant ascites was established by intraperitoneal inoculation of human pancreatic cancer cells in nude mice. Results obtained from the human study were reproduced in the mouse model. Statistically significant equilateral correlation was identified between the band intensity of CD133 in western blot and overall survival of patients. Lectin microarray analyses revealed glycosylation of CD133 by sialic acids as the major glycosylation among diverse others responsible for the glycosylation of exosomal CD133. These findings suggest that highly glycosylated CD133 in ascites-derived exosomes as a potential biomarker for better prognosis of patients with advanced pancreatic cancer.
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Antígeno AC133/metabolismo , Ascitis/metabolismo , Biomarcadores de Tumor/metabolismo , Exosomas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Línea Celular Tumoral , Glicosilación , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células PC-3 , Pronóstico , Neoplasias Gástricas/metabolismoRESUMEN
CD44v9 is expressed in cancer stem cells (CSC) and stabilizes the glutamate-cystine transporter xCT on the cytoplasmic membrane, thereby decreasing intracellular levels of reactive oxygen species (ROS). This mechanism confers ROS resistance to CSC and CD44v9-expressing cancer cells. The aims of the present study were to assess: (i) expression status of CD44v9 and xCT in hepatocellular carcinoma (HCC) tissues, including those derived from patients treated with hepatic arterial infusion chemoembolization (HAIC) therapy with cisplatin (CDDP); and (ii) whether combination of CDDP with sulfasalazine (SASP), an inhibitor of xCT, was more effective on tumor cells than CDDP alone by inducing ROS-mediated apoptosis. Twenty non-pretreated HCC tissues and 7 HCC tissues administered HAIC therapy with CDDP before surgical resection were subjected to immunohistochemistry analysis of CD44v9 and xCT expression. Human HCC cell lines HAK-1A and HAK-1B were used in this study; the latter was also used for xenograft experiments in nude mice to assess in vivo efficacy of combination treatment. CD44v9 positivity was significantly higher in HAIC-treated tissues (5/7) than in non-pretreated tissues (2/30), suggesting the involvement of CD44v9 in the resistance to HAIC. xCT was significantly expressed in poorly differentiated HCC tissues. Combination treatment effectively killed the CD44v9-harboring HAK-1B cells through ROS-mediated apoptosis and significantly decreased xenografted tumor growth. In conclusion, the xCT inhibitor SASP augmented ROS-mediated apoptosis in CDDP-treated HCC cells, in which the CD44v9-xCT system functioned. As CD44v9 is typically expressed in HAIC-resistant HCC cells, combination treatment with SASP with CDDP may overcome such drug resistance.
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Sistema de Transporte de Aminoácidos y+/fisiología , Carcinoma Hepatocelular/tratamiento farmacológico , Receptores de Hialuranos/fisiología , Neoplasias Hepáticas/tratamiento farmacológico , Sulfasalazina/farmacología , Anciano , Anciano de 80 o más Años , Sistema de Transporte de Aminoácidos y+/análisis , Animales , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/química , Cisplatino/farmacología , Resistencia a Antineoplásicos , Femenino , Células Hep G2 , Humanos , Receptores de Hialuranos/análisis , Neoplasias Hepáticas/química , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
AIM: To elucidate the role of STAT3 in hepatocarcinogenesis and biliary ductular proliferation following chronic liver injury. METHODS: We investigated thioacetamide (TAA)-induced liver injury, compensatory hepatocyte proliferation, and hepatocellular carcinoma (HCC) development in hepatic STAT3-deficient mice. In addition, we evaluated TAA-induced biliary ductular proliferation and analyzed the activation of sex determining region Y-box9 (SOX9) and Yes-associated protein (YAP), which regulate the transdifferentiation of hepatocytes to cholangiocytes. RESULTS: Both compensatory hepatocyte proliferation and HCC formation were significantly decreased in hepatic STAT3-deficient mice as compared with control mice. STAT3 deficiency resulted in augmentation of hepatic necrosis and fibrosis. On the other hand, biliary ductular proliferation increased in hepatic STAT3-deficient livers as compared with control livers. SOX9 and YAP were upregulated in hepatic STAT3-deficient hepatocytes. CONCLUSION: STAT3 may regulate hepatocyte proliferation as well as transdifferentiation into cholangiocytes and serve as a therapeutic target for HCC inhibition and biliary regeneration.
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Sistema Biliar/fisiología , Carcinogénesis/patología , Carcinoma Hepatocelular/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas/patología , Regeneración , Factor de Transcripción STAT3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sistema Biliar/citología , Carcinoma Hepatocelular/inducido químicamente , Proteínas de Ciclo Celular , Proliferación Celular , Transdiferenciación Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/fisiología , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas Experimentales/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfoproteínas/metabolismo , Fosforilación , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción STAT3/genética , Tioacetamida/toxicidad , Regulación hacia Arriba , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.
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Tejido Adiposo/patología , Proteínas del Ojo/farmacología , Hígado Graso/metabolismo , Hígado Graso/prevención & control , Factores de Crecimiento Nervioso/farmacología , Inhibidores de Proteasas/farmacología , ARN Mensajero/metabolismo , Serpinas/farmacología , Adenoviridae/genética , Alanina Transaminasa/sangre , Animales , Deficiencia de Colina/complicaciones , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Dieta , Modelos Animales de Enfermedad , Regulación hacia Abajo , Proteínas del Ojo/genética , Hígado Graso/genética , Hígado Graso/patología , Inyecciones Intramusculares , Interleucina-1beta/genética , Interleucina-6/genética , Macrófagos del Hígado , Cirrosis Hepática/prevención & control , Masculino , Metionina/administración & dosificación , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Factores de Crecimiento Nervioso/genética , Estrés Oxidativo , PPAR gamma/genética , Serpinas/genética , Factor de Necrosis Tumoral alfa/genética , Regulación hacia ArribaRESUMEN
Doublecortin-like kinase 1 (DCLK1), a marker for intestinal and pancreatic cancer stem cells, is highly expressed in neuroblastomas. This study was conducted to assess DCLK1 expression levels in pancreatic neuroendocrine tumor (PNET) tissues and to explore the roles of this molecule in clinical tissue from multiple PNET patients, cells (BON1, QGP1, and CM) and tumor xenografts. Immunohistochemically, all PNET tissues highly and diffusely expressed DCLK1 as a full-length isoform, identical to that detected in primary liver NETs. A DCLK1-overexpressing PNET cell line (QGP1-DCLK1) exhibited epithelial-mesenchymal transition (EMT)-related gene signatures, and robust upregulation of Slug (SNAI2), N-Cadherin (CDH2), and Vimentin (VIM) was validated by real-time PCR and immunoblotting. QGP1-DCLK1 cells had increased cell migration in a wound-healing assay and formed significantly larger xenograft tumors in nude mice. The factors involved in the formation of the fast-growing tumors included p-FAK (on Tyr925), p-ERK1/2, p-AKT, Paxillin, and Cyclin D1, which upon knockdown or pharmacologic inhibition of DCLK1 abolished the expression of these molecules. In conclusion, robust and ubiquitous expression of DCLK1 was first demonstrated here in human PNET tissue specimens and cells. DCLK1 characterized the PNET cell behavior, inducing p-FAK/SLUG-mediated EMT. These findings suggest the possibility of developing novel therapeutic strategies against PNETs by targeting DCLK1.Implications: Evidence here reveals that human PNETs diffusely and robustly express the cancer stem cell marker DCLK1, which drives SLUG-mediated EMT, and suggests that NETs share biological features for druggable targets with other tumors, including neuroblastoma that also highly expresses DCLK1. Mol Cancer Res; 15(6); 744-52. ©2017 AACR.
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Transición Epitelial-Mesenquimal/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Quinasas Similares a Doblecortina , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Tirosina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND & AIM: Aflibercept known as ziv-aflibercept in the United States is a soluble decoy receptor of both vascular endothelial growth factor (VEGF) receptor-1 and -2 known to inhibit the binding of VEGF and placental growth factor (PlGF) to VEGF receptor-1 and -2. Here, we analyzed the mechanisms of the antitumor effects of aflibercept in mouse hepatoma models. METHODS: In in vitro studies, we determined the effects of aflibercept on human umbilical vein cell (HUVEC) proliferation and bone marrow (BM) cell differentiation to endothelial progenitor cells (EPCs). In in vivo experiments, aflibercept was injected intraperitoneally in hepatoma cell tumor-bearing mice, and its inhibitory effects on tumor growth and BM cell migration to tumor tissues were evaluated. RESULTS: Aflibercept suppressed phosphorylation of VEGF receptor-1 and -2 in HUVEC and dose-dependently inhibited VEGF-induced HUVEC proliferation. It suppressed the differentiation of BM cells to EPCs and migration of BM cells to tumor tissues. It also suppressed tumor growth and prolonged survival time of tumor-bearing mice without side effects. In tumor tissues, aflibercept upregulated the expression of hypoxia inducible factor1-α, VEGF, PlGF, fibroblast growth factor-2, platelet derived growth factor-BB, and transforming growth factor-α and reduced microvascular density. It also reduced sinusoidal density in noncancerous liver tissues. CONCLUSIONS: Our results demonstrated potent antitumor activity for aflibercept in a mouse model of hepatocellular carcinoma. These effects were mediated through inhibition of neovascularization, caused by inhibition of endothelial cell proliferation, EPC differentiation, and BM cell migration to tumor tissues.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Células de la Médula Ósea/citología , Carcinoma Hepatocelular/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Receptores de Factores de Crecimiento Endotelial VascularRESUMEN
During axon guidance, growth cones navigate toward attractive cues by inserting new membrane on the cue side. This process depends on Ca(2+) release from endoplasmic reticulum (ER) Ca(2+) channels, but the Ca(2+) sensor and effector governing this asymmetric vesicle export remain unknown. We identified a protein complex that controls asymmetric ER Ca(2+)-dependent membrane vesicle export. The Ca(2+)-dependent motor protein myosin Va (MyoVa) tethers membrane vesicles to the ER via a common binding site on the two major ER Ca(2+) channels, inositol 1,4,5-trisphosphate and ryanodine receptors. In response to attractive cues, micromolar Ca(2+) from ER channels triggers MyoVa-channel dissociation and the movement of freed vesicles to the cue side, enabling growth cone turning. MyoVa-Ca(2+) channel interactions are required for proper long-range axon growth in developing spinal cord in vivo. These findings reveal a peri-ER membrane export pathway for Ca(2+)-dependent attraction in axon guidance.
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Orientación del Axón , Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Miosina Tipo V/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Calcio , Canales de Calcio/química , Señalización del Calcio , Exocitosis , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Unión Proteica , Médula Espinal/metabolismo , Vesículas Transportadoras , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34(+) cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34(+) cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34(+) cells were injected via the spleen. After 7 days, CD34(+) cells were effectively expanded in a serum-free culture medium. Expanded CD34(+) cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34(+) cells increased compared with nonexpanded CD34(+) cells. Expanded CD34(+) cell transplantation reduced liver fibrosis, with a decrease of αSMA(+) cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34(+) cells over non-expanded CD34(+) cells. The inhibition of integrin αvß3 and αvß5 disturbed the engraftment of transplanted CD34(+) cells and aggravated liver fibrosis. These findings suggest that expanded CD34(+) cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model.
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Transglutaminase is a Ca2+-dependent enzyme catalyzing protein cross-linking reactions. We investigated the contribution of this enzyme to the resealing of the injured plasma membrane in animal cells, using a lung cancer-derived cell line, A549. After mechanical injury by razor-scratching, the level of membrane resealing was estimated by differential incorporation of dextrans labeled with two distinct fluorescent dyes. The recovery level was decreased in the presence of excess primary amine, as a competitive inhibitor of transglutaminase. We established a cell line that stably expresses shRNA (short hairpin RNA) to specifically inhibit the expression of TGase 2 (tissue-type isozyme of transglutaminase) and confirmed the suppressed resealing level in the cell. Furthermore, additional expression of TGase 2 rescued the ability for membrane resealing. These results show that, after mechanical damage, this enzyme appeared to contribute to membrane resealing.
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Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
Transglutaminases are Ca(2+)-dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca(2+)-binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury.
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Physarum polycephalum/metabolismo , Transglutaminasas/metabolismo , Actinas/aislamiento & purificación , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biotina/análogos & derivados , Biotina/metabolismo , Cadaverina/análogos & derivados , Cadaverina/metabolismo , Proteínas de Unión al Calcio/aislamiento & purificación , Proteínas de Unión al Calcio/metabolismo , Translocasas Mitocondriales de ADP y ATP/aislamiento & purificación , Translocasas Mitocondriales de ADP y ATP/metabolismo , Datos de Secuencia Molecular , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Estrés MecánicoRESUMEN
Monoclonal antibodies (Mabs) are important biomolecules in immunology and have widespread applications in prognosis, diagnosis, and therapeutics. Here, we describe a novel approach called single-cell RT-PCR-linked in vitro expression (SICREX), which enables the high-throughput generation and screening of Mabs. This approach entails the isolation of B cells from immunized mouse spleen or human peripheral blood using magnetic microbeads conjugated with a B-cell-selective marker, anti-CD19. The light chain (Lc) and Fd portion of heavy-chain (Hc) genes of each cell are separately amplified by RT-PCR and then combined with the sequences of a T7 promoter, a ribosome binding site (rbs), and a T7 terminator by an overlapping PCR technique. The paired full-length DNA fragments of Lc and Hc genes from single B cells are simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by an enzyme-linked immunosorbent assay to find positive fragments possessing the affinity for the antigen. From spleen cells of an immunized mouse with calcium binding protein 40, a Fab fragment with K(d) of 1.6 (+/- 0.3) x 10(-8) against the antigen was obtained. From human peripheral blood, Fab fragments against a blood group B-BSA were obtained in a similar manner. The SICREX approach is simple, rapid and versatile, allowing the high-throughput generation of naturally paired Lc and Hc with antigen-binding activity from various animal sources.
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Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Linfocitos B/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/citología , Secuencia de Bases , Separación Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ingeniería de Proteínas/métodos , Sensibilidad y Especificidad , Bazo/citologíaRESUMEN
Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.
Asunto(s)
Factor XIIIa/genética , Factor XIIIa/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Pruebas Genéticas/métodos , Biblioteca de Péptidos , Transglutaminasas/genética , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Caseínas/metabolismo , Clonación Molecular , Glutamina/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Cobayas , Humanos , Datos de Secuencia Molecular , Proteína Glutamina Gamma Glutamiltransferasa 2 , Especificidad por SustratoRESUMEN
Transglutaminase (TGase) is an enzyme that modifies proteins by crosslinking or polyamination. Physarum polycephalum, an acellular slime mold, is the evolutionally lowest organism that has a mammalian-type transglutaminase. We have cloned a cDNA for Physarum polycephalum TGase (PpTGB), homologous to a previously identified TGase (PpTGA), whose sequence is similar to that of mammalian TGases. PpTGB encodes a primary sequence identical to that of PpTGA except for 11 amino acid residues at the N-terminus. Reverse transcription-PCR and Western blotting analyses showed that both PpTGA and PpTGB are expressed in microplasmodia and macroplasmodia during their life cycle, except for in sporangia. For biochemical characterization, we carried out the ectopical expressions of PpTGA and PpTGB in Dictyostelium discoideum. Subcellular fractionation of these Dictyostelium cells showed that the expressed PpTGA, but not PpTGB, localizes to the membrane fraction. Furthermore, in Physarum, subcellular fractionation and immunostaining indicated specific localization at the plasma membrane in macroplasmodia, while the localization was entirely cytoplasmic in microplasmodia.
Asunto(s)
Physarum polycephalum/enzimología , Transglutaminasas/química , Transglutaminasas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad de la EspecieRESUMEN
Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of approximately 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2).