RESUMEN
OBJECTIVES: Previous studies of the relationships between female reproductive factors and osteoarthritis (OA) have shown conflicting results. In this study, we aimed to explore the relationships between reproductive factors and joint replacement arthroplasty of the knee (TKRA) and hip (THRA) in a large nationwide population-based cohort of postmenopausal Korean women. METHODS: We included 1,134,680 subjects who participated in national health examinations in 2009 in the study. The study outcomes were incident THRA or TKRA due to severe hip or knee OA. The relationships between reproductive factors and THRA or TKRA were evaluated using a multivariable-adjusted proportional hazards model. RESULTS: During a mean follow-up duration of 8.2 years, 1,610 incident THRA cases and 60,670 incident TKRA cases were observed. Later age at menarche, longer breastfeeding, HRT and OC use were associated with increased risk of TKRA for severe knee OA, while later age at menopause and longer reproductive span were associated with decreased risk. With regard to THRA for severe hip OA, later menarche, longer breastfeeding, HRT more than 5 years, and OC use more than 1 year were associated with higher risk. The associations between reproductive factors and severe OA were more pronounced in underweight and younger subjects. CONCLUSION: We found that shorter estrogen exposure was associated with higher risk of TKRA due to severe knee OA, and such associations were more pronounced in underweight and younger subjects. The association between shorter estrogen exposure and THRA was not robust.
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Artroplastia de Reemplazo de Cadera/estadística & datos numéricos , Artroplastia de Reemplazo de Rodilla/estadística & datos numéricos , Osteoartritis de la Cadera/cirugía , Osteoartritis de la Rodilla/cirugía , Historia Reproductiva , Anciano , Estudios de Cohortes , Femenino , Humanos , Persona de Mediana Edad , Posmenopausia , República de Corea , Medición de RiesgoRESUMEN
AIMS: As cell-adapted foot-and-mouth disease virus (FMDV) with H56R mutation in VP3 has reduced thermostability, this study aimed to investigate the effect of thermostabilizers on cell-adapted FMDV for vaccine production. METHODS AND RESULTS: We examined the effect of 3% sucrose, 10% (or 25%) glycerol or 10% FBS on cell-adapted FMDV O/SKR/JC/2014, containing H56R mutation in VP3, as vaccine seed virus at -80, 4, 25 or 37°C for 2, 4 or 7 days. The stabilizing effect of 3% sucrose on O/SKR/JC/2014 was observed at 25, 37°C, and after repeated freeze-thaw cycles. Additionally, we tested the effect of 3% sucrose on the growth of FMDV or cells and did not observe any decrease in either viral growth or cell viability. CONCLUSIONS: Our study showed the protective effect of 3% sucrose on FMDV infectivity at various temperatures; this virus stock in 3% sucrose could be used for infecting cells without the removal of sucrose. SIGNIFICANCE AND IMPACT OF THE STUDY: We suggest that 3% sucrose-containing medium could be beneficial for the stable storage and transport of cell-adapted FMDV.
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Virus de la Fiebre Aftosa/crecimiento & desarrollo , Sacarosa/análisis , Excipientes de Vacunas/análisis , Vacunas Virales/química , Animales , Proteínas de la Cápside/genética , Supervivencia Celular/efectos de los fármacos , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/efectos de los fármacos , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/inmunología , Mutación , Sacarosa/farmacología , Temperatura , Excipientes de Vacunas/farmacología , Potencia de la VacunaRESUMEN
Environmental lung diseases are caused by exposure to adverse environmental conditions, such as atmospheric pressure changes or the ingestion or inhalation of toxic agents. The development of environmental lung diseases depends on the intensity and duration of exposure, the physiological and biological susceptibility of the host, and the toxic effects of the adverse environmental conditions encountered. A combination of clinical features, related exposure history, imaging findings, and a review of previous reports that support an association between exposure and the disease process is required for diagnosis.
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Contaminantes Atmosféricos/toxicidad , Exposición a Riesgos Ambientales/efectos adversos , Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/etiología , Presión Atmosférica , Susceptibilidad a Enfermedades , HumanosRESUMEN
OBJECTIVE: The ethnic and sex differences in the distributions of body mass index (BMI) and waist circumference (WC) among adults are largely unknown. Therefore, we aimed to investigate the percentiles of BMI and WC in groups divided according to age, sex, and ethnicity. PATIENTS AND METHODS: We conducted a population-based binational study of adults aged ≥20 years based on data from two sources: US National Health and Nutrition Examination Survey (2015 to 2020) and Korea National Health and Nutrition Examination Survey (2016 to 2019). RESULTS: Weight, height, and WC were measured in 13,144 American adults and 30,191 Korean adults. Overall, BMI increased at younger ages and decreased at older ages, which indicates a reversed U-shaped relationship, and differed in terms of age, sex, and ethnicity. Women in the other Hispanic, non-Hispanic white, non-Hispanic black, and "other ethnic groups" showed a common BMI peak at ages 50-54 years. The patterns of WC distribution were similar to those of BMI distribution. CONCLUSIONS: In this binational representative study, we found varied distributions of ethnic and sex differences in BMI and WC. Considering the differences in these distributions may help improve individual and personalized treatment strategies.
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Obesidad , Caracteres Sexuales , Adulto , Humanos , Femenino , Masculino , Estados Unidos/epidemiología , Índice de Masa Corporal , Circunferencia de la Cintura , Obesidad/epidemiología , Encuestas Nutricionales , República de CoreaRESUMEN
BACKGROUND AND OBJECTIVE: Recent studies reported that the lactone forms of 3-hydroxy- 3-methylglutaryl-coenzyme A reductase inhibitors, which are also known as statins, have a bone stimulatory effect. However, there are few reports on the effect of statins on periodontal ligament cells. This study examined the statin-induced osteoblastic differentiation of mouse periodontal ligament cells as well as its mechanism. MATERIAL AND METHODS: Mouse periodontal ligament cells were cultured with lovastatin or simvastatin, and their viability was measured. The levels of alkaline phosphatase (ALP), osteocalcin, bone sialoprotein and bone morphogenetic protein-2 mRNA expression were evaluated by RT-PCR. The osteoblastic differentiation was characterized by the ALP activity and Alizarin Red-S staining for calcium deposition. The activity of the osteocalcin gene (OG2) and synthetic osteoblast-specific elements (6× OSE) promoter with statins was also measured using a luciferase assay. For the signal mechanism of statins, the ERK1/2 MAPK activity was determined by western blot analysis. RESULTS: A statin treatment at concentrations < 1 µM did not affect the cell viability. Lovastatin or simvastatin at 0.1 µM increased the levels of ALP, osteocalcin, bone sialoprotein and bone morphogenetic protein-2 mRNA in mouse periodontal ligament cells. In addition, the ALP activity, mineralized nodule formation and OG2 and OSE promoter activity were higher in the lovastatin- or simvastatin-treated cells than the control cells. Western blot analysis confirmed that the statins stimulated the phosphorylation of ERK1/2. CONCLUSION: Lovastatin and simvastatin may stimulate the osteoblastic differentiation of periodontal ligament cells via the ERK1/2 pathway. This suggests that the statins may be useful for regenerating periodontal hard tissue.
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Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Osteoblastos/efectos de los fármacos , Ligamento Periodontal/citología , Fosfatasa Alcalina/análisis , Animales , Antraquinonas , Western Blotting , Proteína Morfogenética Ósea 2/análisis , Butadienos/farmacología , Calcificación Fisiológica/efectos de los fármacos , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colorantes , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sialoproteína de Unión a Integrina/análisis , Lovastatina/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Nitrilos/farmacología , Osteocalcina/análisis , Ligamento Periodontal/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Simvastatina/farmacología , Activación Transcripcional/efectos de los fármacosRESUMEN
Albumin microparticles containing Ofloxacin (Fluoroquinolone derivative commonly used in hospitals) were formulated by the spray dry method. By decreasing the pump feed rate or the total polymer concentration, a mixture of albumin/hypromellose acetate succinate (HPMCAS) microparticles and nanoparticles (MP/NP), containing Ofloxacin, were formulated. MP/NP were characterized, in vitro (particle size, zeta potential, and encapsulation efficiency). A comparison of the pharmacokinetics and biodistribution of aqueous Ofloxacin and Ofloxacin-loaded MP/NP, in Balb/c mice, revealed that peak concentrations were reduced in the serum, liver, spleen and brain, and a more sustained release was observed in serum and all of the organs tested for Ofloxacin MP/NP, compared to aqueous Ofloxacin. The MP/NP formulation allowed extended release by 24 h in the liver and more than 48 h in the brain. In serum, the elimination rate of Ofloxacin MP/NP is slower, the half life is longer, area under the plasma concentration time curve is decreased and volume of distribution is increased.
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Portadores de Fármacos/síntesis química , Nanopartículas/uso terapéutico , Ofloxacino/administración & dosificación , Ofloxacino/farmacocinética , Albúminas , Animales , Antibacterianos , Portadores de Fármacos/farmacocinética , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Tamaño de la Partícula , Farmacocinética , Distribución TisularRESUMEN
The federal government plans to rely on a policy of "consultation and concurrence" with state governments in developing nuclear waste repositories. The weaknesses of the concurrence approach are analyzed, and an alternative institutional framework for locating a waste repository is proposed: a siting jury that provides representation for state and local interests, while maintaining a high level of technical review. The proposal could be tested in the siting of away-from-reactor storage facilities for spent nuclear fuel.
RESUMEN
Experimental infection of susceptible cattle and pigs showed that the O/SKR/AS/2002 pig strain of foot-and-mouth disease virus (FMDV) causes an infection that is highly virulent and contagious in pigs but very limited in cattle. Pigs directly inoculated with, or exposed to swine infected with, strain O/SKR/AS/2002 showed typical clinical signs, including gross vesicular lesions in mouth and pedal sites. In addition, FMDV was isolated from, and FMDV genomic RNA was detected in, blood, serum, nasal swabs and oesophageal-pharyngeal (OP) fluid early in the course of infection. Antibodies against the non-structural protein (NSP) 3ABC were detected in both directly inoculated and contact pigs, indicating active virus replication. In contrast, the disease in cattle was atypical. After inoculation, lesions were confined to the infection site. A transient viraemia occurred 1 and 2 days after inoculation, and this was followed by the production of antibodies to NSP 3ABC, indicating subclinical infection. No clinical disease was seen, and no antibodies to NSP 3ABC were present in contact cattle. Additionally, no virus or viral nucleic acid was detected in blood, nasal swab and OP fluid samples from contact cattle. Thus, the virus appeared not to be transmitted from infected cattle to contact cattle. In its behaviour in pigs and cattle, strain O/SKR/AS/2002 resembled the porcinophilic FMDV strain of Cathay origin, O/TAW/97. However, the latter, unlike O/SKR/AS/2002, has reduced ability to grow in bovine-derived cells. The porcinophilic character of O/TAW/97 has been attributed to a deletion in the 3A coding region of the viral genome. However, O/SKR/AS/2002 has an intact 3A coding region.
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Enfermedades de los Bovinos/virología , Virus de la Fiebre Aftosa/patogenicidad , Fiebre Aftosa/patología , Enfermedades de los Porcinos/virología , Animales , Bovinos , Enfermedades de los Bovinos/patología , Modelos Animales de Enfermedad , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/aislamiento & purificación , Virus de la Fiebre Aftosa/fisiología , Miembro Posterior/patología , Antígenos O/clasificación , ARN Viral/genética , Análisis de Secuencia de ARN , Serotipificación , Porcinos , Enfermedades de los Porcinos/patología , Lengua/patologíaRESUMEN
In 2010 serotype O foot-and-mouth disease virus of the Mya98 lineage/SEA topotype spread into most East Asian countries. During 2010-2011 it was responsible for major outbreaks in the Republic of Korea where a monovalent O/Manisa vaccine (belonging to the ME-SA topotype) was applied to help control the outbreaks. Subsequently, all susceptible animals were vaccinated every 6â¯months with a vaccine containing the O/Manisa antigen. Despite vaccination, the disease re-occurred in 2014 and afterwards almost annually. This study focuses on the in vivo efficacy in pigs of a high quality monovalent commercial O1/Campos vaccine against heterologous challenge with a representative 2015 isolate from the Jincheon Province of the Republic of Korea. Initially, viral characterizations and r1 determinations were performed on six viruses recovered in that region during 2014-2015, centering on their relationship with the well characterized and widely available O1/Campos vaccine strain. Genetic and antigenic analysis indicated a close similarity among 2014-2015 Korean isolates and with the previous 2010 virus, with distinct differences with the O1/Campos strain. Virus neutralisation tests using O1/Campos cattle and pig post vaccination sera and recent Korean outbreak viruses predicted acceptable cross-protection after a single vaccination, as indicated by r1 values, and in pigs, by expectancy of protection. In agreement with the in vitro estimates, in vivo challenge with a selected field isolate indicated that O1/Campos primo vaccinated pigs were protected, resulting in a PD50 value of nearly 10. The results indicated that good quality oil vaccines containing the O1/Campos strain can successfully be used against isolates belonging to the O Mya98/SEA topotype.
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Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Inmunización , Enfermedades de los Porcinos/prevención & control , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Línea Celular , Protección Cruzada , Virus de la Fiebre Aftosa/clasificación , Virus de la Fiebre Aftosa/genética , Variación Genética , Filogenia , República de Corea , PorcinosRESUMEN
BACKGROUND: Human alpha(2)-antiplasmin (alpha(2)AP), the primary inhibitor of fibrinolysis, is secreted from the liver into plasma as a 464-residue protein with Met as the N-terminus. An R6W polymorphism has been suggested to affect fibrinolytic rate. Within circulating blood, antiplasmin-cleaving enzyme (APCE) cleaves Met-alpha(2)AP(R6) faster than Met-alpha(2)AP(W6) at the Pro12-Asn13 bond to yield Asn-alpha(2)AP. OBJECTIVES: To compare Met-alpha(2)AP(R6), Met-alpha(2)AP(W6) and Asn-alpha(2)AP for crosslinking with fibrin and the ability to protect fibrin from digestion by plasmin. METHODS AND RESULTS: Asn-alpha(2)AP utilizes Gln2 (Gln14 in Met-alpha(2)AP) to become crosslinked to fibrin approximately twelvefold faster than Met-alpha(2)AP(R6) or Met-alpha(2)AP(W6), and this enhances the resistance of fibrin to plasmin. All three forms of alpha(2)AP inhibit plasmin at identical rates. The N-terminal 12-residue peptide of Met-alpha(2)AP slows crosslinking of Met-alpha(2)AP(R6) or Met-alpha(2)AP(W6) by limiting access of factor XIIIa to Gln14 rather than shifting crosslinking to other Gln residues. Edman sequencing and mass analyses of tryptic peptides from each alpha(2)AP crosslinked with 5-(biotinamido)pentylamine showed Gln14 as the only major crosslinking site. Residues 5-8, GRQL in Met-alpha(2)AP(R6), and residues 1-8, MEPLGWQL in Met-alpha(2)AP(W6), slow fibrin crosslinking. CONCLUSION: Gln14 in both Met-alpha(2)AP(R6) and Met-alpha(2)AP(W6) is sheltered by the N-terminal 12-residue peptide, which, when cleaved, yields Asn-alpha(2)AP, which is rapidly crosslinked to fibrin and maximally protects it from plasmin. The R6 W polymorphism in Met-alpha(2)AP does not affect its crosslinking to fibrin, but it does slow cleavage by APCE and reduces the amount of Asn-alpha(2)AP available for rapid crosslinking to fibrin.
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Polimorfismo Genético , alfa 2-Antiplasmina/metabolismo , Aminas/química , Antígenos de Neoplasias/química , Biomarcadores de Tumor/química , Biotina/análogos & derivados , Biotina/química , Cromatografía de Afinidad , Reactivos de Enlaces Cruzados/química , Endopeptidasas , Factor XIII/metabolismo , Fibrina/química , Fibrinólisis , Gelatinasas , Humanos , Hígado/metabolismo , Proteínas de la Membrana , Modelos Biológicos , Péptidos/química , Estructura Terciaria de Proteína , Serina Endopeptidasas/químicaRESUMEN
BACKGROUND: The protein alpha1-antitrypsin is a prototype member of the serpin (serine protease inhibitor) family and is known to inhibit the activity of neutrophil elastase in the lower respiratory tract. Members of this family undergo a large structural rearrangement upon binding to a target protease, involving cleavage of the reactive-site loop. This loop is then inserted into the main body of the enzyme following the opening of a central beta sheet, leading to stabilization of the structure. Random mutageneses of alpha1-antitrypsin identified various mutations that stabilize the native structure and retard the insertion of the reactive-site loop. Structural studies of these mutations may reveal the mechanism of the conformational change. RESULTS: We have determined the three-dimensional structure of an uncleaved alpha1-antitrypsin with seven such stabilizing mutations (hepta alpha1-antitrypsin) at 2.7 A resolution. From the comparison of the structure with other serpin structures, we found that hepta alpha1-antitrypsin is stabilized due to the release of various strains that exist in native wild type alpha1-antitrypsin, including unfavorable hydrophobic interactions in the central hydrophobic core. The reactive-site loop of hepta alpha1-antitrypsin is an extended strand, different from that of the previously determined structure of another uncleaved alpha1-antitrypsin, and indicates the inherent flexibility of the loop. CONCLUSIONS: The present structural study suggests that the uncleaved alpha1-antitrypsin has many folding defects which can be improved by mutations. These folding defects seem to be utilized in a coordinated fashion in the regulation of the conformational switch of alpha1-antitrypsin. Some of the defects, represented by the Phe51 region and possibly the Met374 and the Thr59 regions, are part of the sheet-opening mechanism.
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Inhibidores de Serina Proteinasa/química , alfa 1-Antitripsina/química , Simulación por Computador , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Inhibidores de Serina Proteinasa/genética , alfa 1-Antitripsina/genéticaRESUMEN
Two human erythroleukemia cell lines, HEL and K562, express transglutaminase activity. The enzyme was identified as a tissue transglutaminase following chromatographic purification. All-trans-retinoic acid (10 microM) stimulated differentiation in HEL cells as judged by a 4-fold increase in hemoglobin content and a reduction in cell proliferation. The transglutaminase activity increased 9-fold. This increase in transglutaminase was the result of a pretranslational regulation of the gene as revealed by Northern blot analysis of mRNA. These changes were not a result of cell apoptosis, since parallel DNA degradation catalyzed by a Ca2(+)-dependent endonuclease could not be demonstrated. The K562 cells, in contrast, showed no transglutaminase induction following exposure to retinoic acid and displayed no changes in maturation markers or cell growth.
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Transglutaminasas/biosíntesis , Tretinoina/farmacología , División Celular/efectos de los fármacos , Línea Celular , Cicloheximida/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Cinética , Leucemia Eritroblástica Aguda , Peso Molecular , Transglutaminasas/genética , Transglutaminasas/aislamiento & purificaciónRESUMEN
1-(5-Aminopentyl)-3-phenylthiourea (PPTU), a recently developed inhibitor of the transglutaminase-catalyzed reaction (K.N. Lee, L. Fesus, S.T. Yancey, J.E. Girard, and S.I. Chung, (1985) J. Biol. Chem. 260, 14689-14694) was evaluated as a possible probe to examine the physiological role of transglutaminase (EC 2.3.2.13) in Chinese hamster ovary (CHO) cells. The [14C]PPTU in cell culture was readily taken up by CHO cells and was found to be covalently attached to high-molecular-weight proteins which are associated with the particulate fractions. Incubating cell homogenates, in the presence of Ca2+, incorporated the labeled PPTU exclusively into high-molecular-weight proteins that were also undergoing polymerization. PPTU at 0.1 mM, a concentration close to the Ki value reported for inhibition of tissue transglutaminase-catalyzed amine incorporation into the B chain of oxidized insulin, decreased high-molecular-weight protein polymerization, whereas PPTU at the same concentrations showed no effect on CHO cell proliferation or on in vitro calmodulin activation of cyclic nucleotide phosphodiesterase. These results suggest that transglutaminase may not be a constitutive enzyme in cell proliferation.
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Feniltiourea/análogos & derivados , Transglutaminasas/antagonistas & inhibidores , Animales , Cadaverina/análogos & derivados , Cadaverina/farmacología , División Celular/efectos de los fármacos , Línea Celular , Cricetinae , Cricetulus , Femenino , Cinética , Ovario , Feniltiourea/metabolismo , Feniltiourea/farmacología , Biosíntesis de Proteínas , Fracciones Subcelulares/metabolismoRESUMEN
The expression of hepatic stearoyl-CoA desaturase (SCD1) mRNA abundance and the fatty acid profile in male and female mice were compared. The expression of the SCD1 mRNA is significantly higher in female compared to male mice and that this difference is accompanied by higher levels of palmitoleic and oleic acid in the livers of female mice.
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Ácidos Grasos/análisis , Hígado/enzimología , Caracteres Sexuales , Estearoil-CoA Desaturasa/biosíntesis , Animales , Dieta , Ácidos Grasos Monoinsaturados/análisis , Femenino , Expresión Génica , Hígado/química , Masculino , Ratones , Ratones Endogámicos ICR , Ácido Oléico/análisis , ARN Mensajero/análisisRESUMEN
A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced. Amino acid sequence data identified human fructose-1,6-bisphosphate aldolase A, an intracellular protein, as a substrate for cellular transglutaminase.
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Fructosa-Bifosfato Aldolasa/metabolismo , Sondas Moleculares , Transglutaminasas/metabolismo , Aminas , Secuencia de Aminoácidos , Biotina/análogos & derivados , Western Blotting , Cromatografía de Afinidad , Neoplasias del Colon , Fructosa-Bifosfato Aldolasa/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9-11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.
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Butiratos/farmacología , Transglutaminasas/biosíntesis , Anticuerpos Monoclonales , Ácido Butírico , Cicloheximida/farmacología , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genética , Transglutaminasas/genéticaRESUMEN
Transglutaminases (EC 2.3.2.13) catalyze an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the epsilon-amino group of lysine residues in protein. Purified human erythrocyte transglutaminase was found to have another activity, i.e., GTP hydrolysis. Treatment of the enzyme with iodoacetamide, a cysteine-directed reagent, caused a 94% loss of TGase activity within 8 min, but no significant loss of GTPase activity. Cys-277, a known residue which is selectively modified by iodoacetamide, was replaced with Ser by site-directed mutagenesis to assess the role of the Cys-277 in the transglutaminase/GTPase activities. Wild-type cDNA, coding for human endothelial cell transglutaminase, and its C277S-mutated cDNA were cloned into a plasmid vector that contained a promoter from phage T7, and then expressed in Escherichia coli. The wild-type recombinant enzyme was indistinguishable from human erythrocyte transglutaminase in mobility on a SDS-polyacrylamide gel, immunoreactivity and catalytic activities for transglutaminase and GTPase. However, the recombinant enzyme was not blocked at the N-terminal alanine residue, as is the case in the naturally occurring erythrocyte enzyme. The C277S mutant enzyme showed no transglutaminase activity, but had Km and kcat values for GTPase activity that were comparable to those of wild-type recombinant and natural erythrocyte enzymes. These results demonstrate that Cys-277 is essential for transglutaminase activity, but not for GTPase activity, and that N-terminal blocking of tissue-type transglutaminase is not critical for either transglutaminase or GTPase activities.
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Cisteína , Eritrocitos/enzimología , GTP Fosfohidrolasas/genética , Transglutaminasas/genética , Sitios de Unión , Ditiotreitol , GTP Fosfohidrolasas/aislamiento & purificación , GTP Fosfohidrolasas/metabolismo , Humanos , Yodoacetamida , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/genética , Serina , Transglutaminasas/metabolismoRESUMEN
Chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), an orphan nuclear receptor belonging to the steroid-thyroid hormone receptor superfamily, plays an important role in cell fate determination of various tissues. However, the specific role of COUP-TFII in tooth development has not yet been elucidated. In the present study, we aimed to explore the role of COUP-TFII in dentin sialophosphoprotein (DSPP) expression and matrix mineralization in odontoblast-lineage cells. In primary human dental pulp cells (HDPCs) and murine dental papilla-derived cells (MDPC-23) cultured in a mineralizing medium, the expression of COUP-TFII was induced along with the increased odontoblast-specific dentin matrix protein-1 (DMP-1) and DSPP expression. Endogenous expression of COUP-TFII in maxillary second molar germs of rats showed an increasing tendency as development of the tooth progressed. Also, COUP-TFII protein was detected in greater quantity in the odontoblastic layer of second molar germs than in that of third molar germs of rats. Overexpression of COUP-TFII using an adenoviral system upregulated the expression of odontoblast-specific genes with increased alkaline phosphatase activity and matrix mineralization in odontoblast-lineage cells. In contrast, knockdown of COUP-TFII using small interfering RNA decreased the expression of odontoblast-specific genes, which reduced matrix mineralization. Mechanistic studies revealed that COUP-TFII increased DSPP transcription by direct binding on the DSPP promoter. In addition, COUP-TFII physically interacted with the homeodomain transcription factor Msx2 and antagonistically regulated the Msx2 effect on DSPP promoter activity. Taken together, these results suggest that COUP-TFII has a stimulatory role in DSPP expression and matrix mineralization in odontoblast-lineage cells.
Asunto(s)
Factor de Transcripción COUP II/metabolismo , Dentinogénesis , Proteínas de la Matriz Extracelular/metabolismo , Odontoblastos/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Proteínas de Homeodominio/metabolismo , Humanos , Inmunoprecipitación , Ratones , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Coloración y Etiquetado , TransfecciónRESUMEN
Three peptides corresponding to glycine-rich internal sequences of the guinea pig liver transglutaminase molecule were synthesized. These were peptide 1 (amino acid residues 520-544), peptide 2 (amino acid residues 345-367) and peptide 3 (amino acid residues 45-69). All of the synthetic peptides demonstrated significant binding ability for both ATP and GTP. Peptide 1 was the best protector of transglutaminase activity from both ATP and GTP inhibition, while peptides 2 and 3 protected the activity only from GTP inhibition. The data shown here lead us to propose putative binding site(s) for ATP and GTP guinea pig liver transglutaminase.