RESUMEN
BACKGROUND AND PURPOSE: Diagnostic errors in radiology are classified as perception or interpretation errors. This study determined whether specific conditions differed when perception or interpretation errors occurred during neuroradiology image interpretation. MATERIALS AND METHODS: In a sample of 254 clinical error cases in diagnostic neuroradiology, we classified errors as perception or interpretation errors, then characterized imaging technique, interpreting radiologist's experience, anatomic location of the abnormality, disease etiology, time of day, and day of the week. Interpretation and perception errors were compared with hours worked per shift, cases read per shift, average cases read per shift hour, and the order of case during the shift when the error occurred. RESULTS: Perception and interpretation errors were 74.8% (n = 190) and 25.2% (n = 64) of errors, respectively. Logistic regression analyses showed that the odds of an interpretation error were 2 times greater (OR, 2.09; 95% CI, 1.05-4.15; P = .04) for neuroradiology attending physicians with ≤5 years of experience. Interpretation errors were more likely with MR imaging compared with CT (OR, 2.10; 95% CI, 1.09-4.01; P = .03). Infectious/inflammatory/autoimmune diseases were more frequently associated with interpretation errors (P = .04). Perception errors were associated with faster reading rates (6.01 versus 5.03 cases read per hour; P = .004) and occurred later during the shift (24th-versus-18th case; P = .04). CONCLUSIONS: Among diagnostic neuroradiology error cases, interpretation-versus-perception errors are affected by the neuroradiologist's experience, technique, and the volume and rate of cases read. Recognition of these risk factors may help guide programs for error reduction in clinical neuroradiology services.
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Competencia Clínica , Errores Diagnósticos , Radiología , Femenino , Humanos , Radiólogos , Factores de RiesgoRESUMEN
OBJECTIVE: To examine the effects of direct oral anticoagulants (DOACs) on bleeding complications following dental surgeries. SUBJECTS AND METHODS: This 6-year retrospective study collected data from records of patients undergoing oral surgical procedures within a university setting. An electronic health record database was searched using current procedural terminology codes for oral surgical procedures. Information regarding patient, procedural factors, and postoperative complications were extracted. Data were analyzed by Fisher's exact test. RESULTS: Of patients who had a procedural code associated with oral surgery, only 0.11% (12/11,320) took a DOAC. Twelve patients (10 males, age ranging from 44 to 90 years) underwent 17 surgeries by nine different practitioners involving 98 extractions, 14 alveoloplasties, two tuberosity reductions, and two tori removals. In nine cases, the DOAC was discontinued a mean of 52.5 hrs prior to surgery (range 12-120 hrs). Bleeding complications were not reported for patients whose drug was discontinued or continued. Documentation of drug continuation/discontinuation was poor. CONCLUSIONS: Bleeding was not observed with direct oral anticoagulation use in this oral surgery cohort. Drug discontinuation/continuation was not a factor in bleeding outcomes, and direct oral anticoagulation interruption was variable and poorly documented.
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Anticoagulantes/administración & dosificación , Procedimientos Quirúrgicos Orales/efectos adversos , Hemorragia Posoperatoria/etiología , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Anticoagulantes/efectos adversos , Documentación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Privación de TratamientoRESUMEN
High-throughput screening for the induction of a luciferase reporter gene in a thrombopoietin (TPO)-responsive cell line resulted in the identification of 4-diazo-3-hydroxy-1-naphthalenesulfonic acids as TPO mimics. Modification of the core structure and adjustment of unwanted functionality resulted in the development of (5-oxo-1,5-dihydropyrazol-4-ylidene)hydrazines which exhibited efficacies equivalent to those of TPO in several cell-based assays designed to measure thrombopoietic activity. Furthermore, these compounds elicited biochemical responses in TPO-receptor-expressing cells similar to those in TPO itself, including kinase activation and protein phosphorylation. Potencies for the best compounds were high for such low molecular weight compounds (MW < 500) with EC(50) values in the region of 1-20 nM.
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Compuestos Azo/síntesis química , Hidrazinas/síntesis química , Megacariocitos/efectos de los fármacos , Naftalenosulfonatos/síntesis química , Proteínas de Neoplasias , Pirazoles/síntesis química , Receptores de Citocinas , Trombopoyetina/química , Animales , Compuestos Azo/química , Compuestos Azo/farmacología , División Celular , Línea Celular , Evaluación Preclínica de Medicamentos , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Genes Reporteros , Hidrazinas/química , Hidrazinas/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Imitación Molecular , Peso Molecular , Naftalenosulfonatos/química , Naftalenosulfonatos/farmacología , Fosforilación , Fosfotransferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pirazoles/química , Pirazoles/farmacología , Receptores de Trombopoyetina , Relación Estructura-Actividad , Trombopoyetina/metabolismoRESUMEN
The successful cloning and subsequent clinical application of recombinant cytokines and/or growth factors has generated a number of important therapeutics. In contrast to the G-protein-coupled receptors, identification of small-molecule agonists of the cytokine and/or growth factor receptor family has proved difficult. The first small peptides and non-peptidic small-molecule agonists for several receptors have recently been reported. The initial identification and/or crucial characterization of these molecules as true mimics was dependent on the use of cell-based functional assays. This article will review recent cell-based assay technologies that are suitable for HTS and that are being applied to the discovery of novel cytokine and growth factor mimics.
RESUMEN
A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.
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Bencimidazoles/farmacología , Guanidinas/farmacología , Proteínas de la Leche , Proteínas Proto-Oncogénicas , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Animales , Bencimidazoles/química , Bencimidazoles/metabolismo , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Proteínas de Unión al ADN/metabolismo , Dimerización , Femenino , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/citología , Guanidinas/química , Guanidinas/metabolismo , Humanos , Janus Quinasa 1 , Janus Quinasa 2 , Recuento de Leucocitos , Leucopoyesis , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Fosforilación , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Especificidad de la Especie , Transactivadores/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
This paper summarizes a two-phase project that reviewed the Commercial Vehicle Safety Alliance's out-of-service criteria for vehicles. The first phase examined relevant background information and conducted a questionnaire survey of CVSA inspectors and industry representatives. The second phase of the project involved extensive collection and evaluation of accident data. The results of both phases show a high level of support, in terms of contribution to vehicle accidents, for four of the vehicle criteria (regarding brakes, load securement, tires, and wheels and rims). There was some support for coupling devices, fuel systems, lighting devices, steering and suspension. The support for the remaining vehicle criteria (exhaust systems, frames, van and open top trailer bodies, and windshield wipers) was little to none. Further research continuing the study of accident data is recommended to confirm these findings. However, the data must be collected in a consistent and detailed manner if accurate information on the relationship of accidents and vehicle criteria is to be established.
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Vehículos a Motor , Seguridad , Diseño de Equipo , Humanos , OregonRESUMEN
STUDY OBJECTIVE: In transthoracic defibrillation, the American Heart Association (AHA) recommends wide separation of electrodes and avoidance of gel smearing between electrodes. Few data support this recommendation. Our objective was to determine the importance of electrode placement and gel-application technique on transcardiac defibrillation current and the effect of changes caused by postexercise vasodilation and sweating. METHODS: Our subjects were 10 normal adults, 5 men and 5 women, who ranged in age from 22 to 48 years. We determined interelectrode impedance (Z) using a validated test-pulse method that does not require shock delivery. Electrode placement/gel-application techniques were varied among four types: (1) AHA-recommended technique (apex-to-anterior electrode placement, no smearing of gel between electrodes); (2) parasternal-to-anterior placement, electrodes within 2 cm of each other, no smearing of gel between electrodes; (3) parasternal-to-anterior placement, electrodes within 2 cm of each other with smearing of gel between electrodes (worst-case scenario); and (4) apex-to-anterior placement, smearing of gel between electrodes. To assess the effect of cutaneous vasodilation and sweating on interelectrode impedance, we repeated these measurements after the subjects performed 12 to 18 minutes of treadmill exercise. The ratio of predicted transcardiac current of the AHA technique to that of the nonstandard technique was estimated with this formula: square root of Z, non-standard technique divided by square root of Z, AHA technique. RESULTS: Resting interelectrode impedance declined 38% from 58 +/- 10.3 omega (AHA-recommended technique) to 36 +/- 7.6 omega (electrode paddles adjacent, gel smeared between) (P < .01). Predicted transcardiac current ratio was reduced to .78 +/- .09 (P < .01), a 22% reduction. We noted no change in the results after exercise. CONCLUSION: Adjacent placement of electrodes and smearing of gel between electrodes creates a low-impedance pathway along the chest wall, which shunts current away from the heart. Thus improper application of electrodes and gel substantially degrades transcardiac current and may result in failed defibrillation. Sweating and vasodilation did not cause a similar problem.
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Cardioversión Eléctrica/instrumentación , Impedancia Eléctrica , Electrodos/normas , Adulto , Sesgo , Ejercicio Físico/fisiología , Prueba de Esfuerzo , Femenino , Geles , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sudoración/fisiología , Vasodilatación/fisiologíaRESUMEN
The case is reported of an infant with hyperammonaemia secondary to severe distal renal tubular acidosis. A clinical association between increased concentrations of ammonia in serum and renal tubular acidosis has not previously been described. In response to acidosis the infant's kidneys presumably increased ammonia synthesis but did not excrete ammonia, resulting in hyperammonaemia. The patient showed poor feeding, frequent vomiting, and failure to thrive, but did not have an inborn error of metabolism. This case report should alert doctors to consider renal tubular acidosis in the differential diagnosis of severely ill infants with metabolic acidosis and hyperammonaemia.
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Acidosis Tubular Renal/sangre , Amoníaco/sangre , Acidosis Tubular Renal/complicaciones , Insuficiencia de Crecimiento/etiología , Femenino , Estudios de Seguimiento , Humanos , Lactante , Nefrocalcinosis/diagnóstico por imagen , Nefrocalcinosis/etiología , UltrasonografíaRESUMEN
The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter.
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Proteínas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazoles/farmacología , Tiazolidinedionas , Factores de Transcripción/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/anatomía & histología , Tejido Adiposo/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN/química , Activación Enzimática , Expresión Génica/efectos de los fármacos , Humanos , Leptina , Hígado/anatomía & histología , Datos de Secuencia Molecular , Tamaño de los Órganos/efectos de los fármacos , Pioglitazona , Regiones Promotoras Genéticas , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/agonistas , Rosiglitazona , Factores de Transcripción/agonistasRESUMEN
It has frequently been proposed that while the constitutive secretory pathway is present in all cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine, or exocrine types. In this study we provide evidence that suggests that this distinction is not as restrictive as proposed. We have identified a population of post-Golgi storage vesicles in several constitutive secretory cells using [35S]SO4-labeled glycosaminoglycan (GAG) chains as a marker. A fraction of this pool of vesicles can undergo exocytosis in response to stimuli such as cytoplasmic Ca2+ and phorbol esters. The effect of Ca2+ was demonstrated both in intact cells in the presence of the ionophore A23187 and in streptolysin-O-permeabilized semi-intact cells. N-ethylmaleiimide, under conditions known to block regulated and constitutive secretion, inhibited the stimulated secretion from these cells, suggesting that the observed release of labeled GAG chains was not due to a leakage artefact. Subcellular fractionation revealed that the stored GAG chains were in low-density membrane granules (d approximately 1.12 g/ml), whose size was greater than that of synaptic-like vesicles found in PC12 cells. In addition, in CHO cells that express epitope-tagged rab 3D, the labeled GAG chains were found to cofractionate with the exogenous rab protein. When expressed in the regulated cell line AtT-20, this tagged rab protein was found to colocalize with ACTH-containing dense-core granules by indirect immunofluorescence. Taken together, these results provide evidence for the presence of a cryptic regulated secretory pathway in "constitutive" cells and suggest that the regulated secretory pathway is more widespread amongst different cell types than previously believed.
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Gránulos Citoplasmáticos/metabolismo , Exocitosis/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células CHO , Calcio/fisiología , Línea Celular , Cricetinae , Gránulos Citoplasmáticos/química , Etilmaleimida/farmacología , Femenino , Proteínas de Unión al GTP/análisis , Proteínas de Unión al GTP/genética , Expresión Génica , Glicosaminoglicanos/análisis , Glicosaminoglicanos/metabolismo , Glicósidos/metabolismo , Aparato de Golgi/fisiología , Células L , Ratones , Células PC12 , ARN Mensajero/análisis , Ratas , Proteínas de Unión al GTP rab3RESUMEN
The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.
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Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , Proteínas/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Células Cultivadas , Secuencia de Consenso , Cartilla de ADN , Exones , Humanos , Intrones , Leptina , Datos de Secuencia Molecular , Obesidad/genética , Oligodesoxirribonucleótidos , Epiplón , Fenotipo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , Roedores , Transcripción GenéticaRESUMEN
Paris suggests that some cultures provide protective factors that can suppress the emergence of borderline personality disorder (BPD). Yet all cultures contain some individuals who perceive themselves as unable to meet what is expected of them, and the resultant distress is expressed through a variety of "ethnic" disorders such as susto or nervios. When viewed in this context, BPD is similar to these disorders, notably in the perceived sense of social failure, marginality and powerlessness.
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Trastorno de Personalidad Limítrofe/psicología , Comparación Transcultural , Países en Desarrollo , Trastorno de Personalidad Limítrofe/diagnóstico , Emigración e Inmigración , Humanos , Controles Informales de la Sociedad , Valores SocialesRESUMEN
Recent studies on developmentally regulated hemolymph proteins in insects have shown that two proteins, a lipoprotein and a member of a hexamerin gene family, bind riboflavin. The biosynthesis, developmental regulation, and properties of these proteins are described and compared with the riboflavin-binding proteins and flavin distributions in vertebrates. The importance of riboflavin-binding proteins in insect development is discussed in relation to existing information and avenues for future research are presented.
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Proteínas Portadoras/metabolismo , Flavinas/metabolismo , Insectos/metabolismo , Proteínas de Transporte de Membrana , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Insectos/genética , Datos de Secuencia Molecular , Riboflavina/metabolismo , VertebradosRESUMEN
Bacteria which are infectious by inoculation in lepidoptera have been isolated and characterized from semispecies comprising the Drosophila paulistorum complex. These microorganisms are pathogenic toward lepidopteran hosts such as Heliothis virescens when introduced by injection of Drosophila tissue extracts and have been given the trivial name DpLE (D. paulistorum lepidopteran entomopathogen). The DpLE from two of the semispecies, Transitional and Andean, were determined to be related to Proteus vulgaris based upon nucleotide sequence comparisons of 16S rDNA genes. Infectivity and 16S rDNA-based PCR assays showed the bacterium to be localized in a number of drosophilid tissues except adult heads and thoraces. Based upon similar experiments, the DpLE in transinfected Heliothis larvae were found in all tissues assayed prior to the onset of mortality. Stocks of Drosophila which had spontaneously lost DpLE continued to produce sterile sons when crossed with incompatible semispecies' females, confirming that the bacilliform DpLE is not the causative agent of the Drosophila paulistorum intersemispecific hybrid male sterility. Acquisition of the sequences of the 16S rDNA molecules of DpLE from all six semispecies permitted the construction of a phylogenetic tree in which the groupings were found not to be congruent with the phylogenies of their insect hosts.
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Drosophila/microbiología , Enterobacteriaceae/aislamiento & purificación , Mariposas Nocturnas/microbiología , Animales , Secuencia de Bases , ADN Bacteriano/genética , ADN Ribosómico/genética , Enterobacteriaceae/clasificación , Enterobacteriaceae/patogenicidad , Enterobacteriaceae/ultraestructura , Femenino , Fertilidad , Masculino , Datos de Secuencia Molecular , FilogeniaRESUMEN
An N-terminal amino acid sequence of a 16.9 kDa hemolymph polypeptide, "Vesicle Promoting Factor" (VPF) from Trichoplusia ni, revealed a high sequence homology (70%) with Manduca sexta apolipophorin-III. A polyclonal antibody developed against VPF, however, was not immunoreactive with either purified M. sexta or T. ni apolipophorin-III. Immunoblots of tissue homogenates of T. ni indicated that VPF was present in imaginal wing discs, central nervous system (CNS), silk glands, midgut and hemocytes from fifth instar larvae, and also in the IAL-TND1 cell line which can grow as either fluid-filled multicellular vesicles or multicellular aggregates. VPF was also detected immunologically in the hemolymph of adults of T. ni, and in hemolymph of adults and larvae of Galleria mellonella and Heliothis virescens. Testes, midgut, hemocytes, and wing discs, but not Malpighian tubules, of T. ni released VPF into tissue culture medium during a 3 h incubation period.
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Mariposas Nocturnas/química , Péptidos/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Apolipoproteínas/aislamiento & purificación , Western Blotting , Línea Celular , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Especificidad de la EspecieRESUMEN
OBJECTIVES: Patients with a diagnosis of borderline personality disorder were studied to learn how they experienced the disorder and its treatment. METHODS: Life history narratives were obtained from ten patients with borderline personality disorder in a series of 90-minute interviews held over the course of a year. The interviews had minimal structure; patients were simply asked to talk about themselves. RESULTS: The narratives revealed striking similarities in the patients' experience with borderline personality disorder. Reports of their experience differed markedly from clinical descriptions of the disorder. Common themes of estrangement, inadequacy, and despair were identified, as well as common coping strategies, primarily dissociation and avoidance of self-disclosure. CONCLUSIONS: Patients' experiences with borderline personality disorder were highly consistent but differed markedly from clinical descriptions. The patient narratives provided information that could lead to more effective treatment of the disorder.
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Trastorno de Personalidad Limítrofe/psicología , Adaptación Psicológica , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Autoimagen , Autorrevelación , Conducta SocialRESUMEN
We measured stimulant-induced changes of exocytosis that are associated with increases in Cl secretion (i.e., short circuit current, ISC, in microA/cm2) and apical (ap) Cl permeability (PCl) and basolateral (bl) K permeability (PK) (both in cm/s) in T84 monolayers. PCl and PK were measured by permeabilizing the bl or ap membrane with nystatin. PCl was also measured with a fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). A noninvasive and sensitive method (release of 35SO4-labeled glycosaminoglycan [GAG], a fluid-phase marker of Golgi-derived vesicles) was used to measure exocytosis at both ap and bl membranes. At rest, ISC = 3.6, PK = 0.8 x 10(-6), PCl = 2.1 x 10(-6) with SPQ and 2.4 x 10(-6) electrically, and there was constitutive GAG secretion (i.e., exocytosis) to both ap and bl sides (bl > 2 x ap). Carbachol (C) increased: ISC (delta = 18.6), PK (6.5x), PCl (1.8-2.9x), and exocytosis to both ap (2.2-3.5x) and bl (2.0-3.0x) membranes. Forskolin (F) increased ISC (delta = 29), PCl (5.5-11x) and ap exocytosis (1.5-2x), but had no effect on PK or bl exocytosis. Synergistic effects on ISC occurred when C was added to F-treated cells but not vice versa, even though the characteristic effects of F+C on PCl, PK, and/or GAG secretion were identical to those exhibited when stimulants were added individually. Cl secretion results from coordinated activation of channels at ap and bl membranes, and exocytosis may play a role in these events.
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Permeabilidad de la Membrana Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Cloruros/farmacocinética , Exocitosis , Potasio/farmacocinética , Carbacol/farmacología , Colforsina/farmacología , Glicosaminoglicanos/metabolismo , Aparato de Golgi/metabolismo , Humanos , Potenciales de la Membrana , Nistatina/farmacología , Células Tumorales CultivadasRESUMEN
The 85K storage protein that accumulates in the hemolymph of Galleria mellonella during the final larval instar was isolated and purified from newly molted pupae. The separation of fresh hemolymph proteins from larvae or pupae by different chromatographic and electrophoretic procedures indicated the native protein had a M(r) of 170,000 and consisted of two identical 85K subunits. Crosslinking experiments using fresh hemolymph followed by Western blotting also indicated a dimeric structure for the native protein. Analyses of the dimer purified from pupal hemolymph indicated that 85K was a glycoprotein, containing approximately 6.5% neutral sugar and about 1.9% amino sugar. Like other insect flavin-binding proteins, 85K has a relatively high histidine content but an uncharacteristically high arginine content. The purified 85K dimer did not bind riboflavin, suggesting that the integrity of the molecule had been altered during purification. However, 85K purified in low yield by Affi-Gel Blue chromatography, did bind riboflavin, indicating that under certain, undefined conditions the functional integrity of the protein could be retained during purification.
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Proteínas Portadoras/aislamiento & purificación , Hemolinfa/química , Proteínas de Transporte de Membrana , Mariposas Nocturnas/química , Riboflavina/metabolismo , Secuencia de Aminoácidos , Animales , Carbohidratos/análisis , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Larva/química , Datos de Secuencia Molecular , Peso Molecular , Pupa/químicaRESUMEN
Recent studies using the fungal metabolite brefeldin A (BFA) have provided important insights into the dynamics and the organization of the ER/Golgi membrane system. Here we examined the effect of BFA on the functional integrity of the distal part of the secretory pathway, i.e., transport between trans-Golgi cisternae and the cell surface. To assay export via the constitutive pathway, we followed the movement of vesicular stomatitis virus (VSV) G glycoprotein that had been accumulated in the trans-Golgi network (TGN) by incubation of infected BHK-21 cells at 20 degrees C. Addition of BFA rapidly and reversibly inhibited cell surface transport of G protein. The block to secretion was not due to redistribution of externalized G protein to internal pools. It was also not due to collapse of TGN to the ER, since VSV G protein blocked in treated cells resided in compartments that were distinct from the ER/Golgi system. Similar effects were found with a bulk-flow marker: BFA blocked constitutive secretion of glycosaminoglycan chains that had been synthesized and sulfated in the trans-Golgi cisternae. To examine export via the regulated secretory pathway, we assayed secretion of [35S]SO4 labeled secretogranin II from PC12 cells, a marker that has been used to study secretory granule budding from the TGN (Tooze, S. A., U. Weiss, and W. B. Huttner. 1990. Nature [Lond.]. 347:207-208). BFA potently inhibited secretion of sulfated secretogranin II induced by K+ depolarization. Inhibition was at the level of granule formation, since BFA had no effect on regulated secretion from preformed granules. Taken together, the results suggest that BFA blocks export via both the constitutive and the regulated pathways. In contrast, endocytosis and recycling of VSV G protein were not blocked by BFA, consistent with previous studies that endocytosis is unaffected (Misumi, Y., Y. Misumi, K. Miki, A Takatsuki, G. Tamura, and Y. Ikehara. 1986. J. Biol. Chem. 261:11398-11403). These and earlier results suggest that the exo/endocytic pathway of mammalian cells consist of two similar but distinct endomembrane systems: an ER/Golgi system and a post-Golgi system. BFA prevents forward transport without affecting return traffic in both systems.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/metabolismo , Ciclopentanos/farmacología , Glicosaminoglicanos/biosíntesis , Aparato de Golgi/metabolismo , Glicoproteínas de Membrana , Transferrina/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Autorradiografía , Brefeldino A , Fraccionamiento Celular/métodos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Centrifugación por Gradiente de Densidad , Cricetinae , Glicoproteínas/metabolismo , Glicosaminoglicanos/aislamiento & purificación , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Cinética , Sulfatos/metabolismo , Radioisótopos de AzufreRESUMEN
Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.