RESUMEN
OBJETIVO: El implante biodegradable de dexametasona ozurdex está aprobado por la Agencia Europea del Medicamento para el tratamiento de uveítis intermedia y posterior y el edema macular tras oclusión venosa retiniana y edema macular diabético. El propósito de este estudio es evaluar los efectos en la presión intraocular en una cohorte de pacientes de la práctica clínica en la vida real. MÉTODOS: Estudio retrospectivo de pacientes tratados con ozurdex en el Hospital Universitario Cruces en un periodo de seguimiento de 6 meses. Se registraron las siguientes variables: edad, sexo, diagnóstico e historia de glaucoma, presión intraocular, tratamiento antihipertensivo y grosor macular medidos antes de la inyección y después de ella tras 1, 2, 4 y 6 meses. Los test estadísticos utilizados fueron: prueba U de Mann-Whitney, test de Chi cuadrado (con corrección de Fisher en caso necesario) y test de Wilcoxon. El nivel de significación estadística se estableció en p < 0,05. RESULTADOS: Se evaluaron los efectos de 75 inyecciones en 67 pacientes (35 mujeres: 52%; edad media: 62 años). La presión intraocular media previa a la inyección fue 15,9 mmHg y 1, 2, 4 y 6 meses tras la inyección: 18,80 (p = 0,627); 18,84 (p = 0,494); 17,02 (p = 0, 796) y 15,5 (p = 0,829). No se observaron diferencias estadísticamente significativas en las medidas de presión intraocular entre pacientes con historia previa de glaucoma y aquellos sin diagnóstico previo. CONCLUSIONES: Ozurdex muestra un perfil de seguridad excelente en términos de presión intraocular en la práctica clínica. Incluso los pacientes con antecedente de glaucoma muestran tal perfil. Ozurdex es una buena opción terapéutica para enfermedades retinianas en estos pacientes
OBJECTIVE: The dexamethasone biodegradable implant, ozurdex, is approved by the US Food and Drug Administration and by the European Medical Agency for the treatment of intermediate and posterior uveitis, and for the treatment of macular edema following retinal vein occlusion and diabetic macular edema. The aim of this study was to assess the effects of intraocular pressure in a cohort of patients from a real-life clinical practice. METHODS: Retrospective review was performed on the clinical records of patients treated with Ozurdex in Hospital Universitario Cruces in a 6 month period. The following variables were recorded: age, gender, diagnosis and history of glaucoma; intraocular pressure, antihypertensive treatment and macular thickness were recorded before the injection, and in the 1st, 2nd, 4th, and 6th months after the injection. Statistical tests: Mann-Whitney U test, Chi square test (with Fisher correction when needed) and Wilcoxon test. The level of statistical significance was set at P<.05. RESULTS: The effects of 75 injections given to 67 patients (35 women: 52%; mean age: 62) were evaluated. Mean intraocular pressure before the injection was 15.9 mmHg and at 1st, 2nd, 4th and 6th months after de injection it was 18.80 (P=.627), 18.84 (P=.494), 17.02 (P=.796) and 15.5 (P=.829), respectively. No statistically significant differences were observed in intraocular pressure measurements at the mentioned follow-up visits between patients with and without a history of glaucoma. CONCLUSIONS: In real-life clinical practice, ozurdex shows an excellent safety profile in terms of intraocular hypertension. Patients with a history of glaucoma may also show this profile, with ozurdex being a good option to treat retinal diseases in these patients
Asunto(s)
Femenino , Humanos , Masculino , Presión Intraocular/genética , Prácticas Clínicas , Prácticas Clínicas/métodos , Glaucoma/diagnóstico , Uveítis/metabolismo , Edema Macular/metabolismo , Edema Macular/patología , Enfermedades de la Retina/diagnóstico , Presión Intraocular/fisiología , Prácticas Clínicas/normas , Prácticas Clínicas/tendencias , Glaucoma/patología , Uveítis/patología , Edema Macular/complicaciones , Edema Macular/diagnóstico , Enfermedades de la Retina/metabolismo , Estudios RetrospectivosRESUMEN
OBJECTIVE: The dexamethasone biodegradable implant, ozurdex, is approved by the US Food and Drug Administration and by the European Medical Agency for the treatment of intermediate and posterior uveitis, and for the treatment of macular edema following retinal vein occlusion and diabetic macular edema. The aim of this study was to assess the effects of intraocular pressure in a cohort of patients from a real-life clinical practice. METHODS: Retrospective review was performed on the clinical records of patients treated with Ozurdex in Hospital Universitario Cruces in a 6 month period. The following variables were recorded: age, gender, diagnosis and history of glaucoma; intraocular pressure, antihypertensive treatment and macular thickness were recorded before the injection, and in the 1st, 2nd, 4th, and 6th months after the injection. STATISTICAL TESTS: Mann-Whitney U test, Chi square test (with Fisher correction when needed) and Wilcoxon test. The level of statistical significance was set at P<.05. RESULTS: The effects of 75 injections given to 67 patients (35 women: 52%; mean age: 62) were evaluated. Mean intraocular pressure before the injection was 15.9 mmHg and at 1st, 2nd, 4th and 6th months after de injection it was 18.80 (P=.627), 18.84 (P=.494), 17.02 (P=.796) and 15.5 (P=.829), respectively. No statistically significant differences were observed in intraocular pressure measurements at the mentioned follow-up visits between patients with and without a history of glaucoma. CONCLUSIONS: In real-life clinical practice, ozurdex shows an excellent safety profile in terms of intraocular hypertension. Patients with a history of glaucoma may also show this profile, with ozurdex being a good option to treat retinal diseases in these patients.
Asunto(s)
Dexametasona/farmacología , Presión Intraocular/efectos de los fármacos , Hipertensión Ocular/inducido químicamente , Anciano , Antihipertensivos/uso terapéutico , Dexametasona/administración & dosificación , Dexametasona/efectos adversos , Dexametasona/uso terapéutico , Retinopatía Diabética/tratamiento farmacológico , Implantes de Medicamentos , Femenino , Glaucoma/complicaciones , Humanos , Inyecciones Intravítreas , Edema Macular/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Hipertensión Ocular/epidemiología , Oclusión de la Vena Retiniana/tratamiento farmacológico , Estudios Retrospectivos , Tonometría OcularRESUMEN
BACKGROUND: To describe the socioeconomic profiles of geographical areas on Montreal Island in which human immunodeficiency virus (HIV) seropositive women delivering live births between 1989 and 1993 reside. METHODS: Leftover dried blood spot filter paper specimens collected from newborns were irretrievably unlinked from identifying information prior to testing. Seroprevalence estimates were calculated based on Western blot confirmed positive samples. Using data from the Canadian census, Revenue Canada, and provincial birth records, the socioeconomic characteristics of postal zones in which seropositive mothers reside were described. RESULTS: Montreal Island had an overall 5-year HIV seroprevalence rate estimate of 16.6 (95% CI: 14.1-19.3) per 10000 childbearing women. Areas in which at least one seropositive woman gave birth had lower mean infant birthweights and higher percentages of single mothers and single-parent families. The HIV-positive neonatal blood specimens were more likely to originate from areas where a higher proportion of residents reported less education, greater unemployment, and lower income. CONCLUSIONS: Higher HIV infection rates were found among childbearing women from lower socioeconomic areas of Montreal. Increased understanding of the relationship between socioeconomic status and HIV acquisition and transmission is required to inform the development of targeted HIV prevention programmes.
Asunto(s)
Infecciones por VIH/epidemiología , Complicaciones Infecciosas del Embarazo/epidemiología , Adulto , Escolaridad , Empleo , Etnicidad , Femenino , Seropositividad para VIH/epidemiología , Humanos , Renta , Masculino , Embarazo , Prevalencia , Quebec/epidemiología , Factores SocioeconómicosAsunto(s)
Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Adolescente , Adulto , Secuencia de Bases , ADN Viral/análisis , Brotes de Enfermedades , Femenino , VIH-1/clasificación , Humanos , Masculino , Epidemiología Molecular , Datos de Secuencia Molecular , Terranova y Labrador/epidemiología , Filogenia , Reacción en Cadena de la Polimerasa , Asunción de Riesgos , Análisis de Secuencia de ADNRESUMEN
Metastatic Lewis lung carcinoma (LLC-LN7) cells have increased protein kinase A (PKA) activity and are more invasive in vitro than are non-metastatic (LLC-C8) cells. To determine whether PKA mediates the in vitro invasiveness and in vivo metastatic capabilities of these tumor cells, the LLC variants were stably transfected to over-express the C alpha subunit of PKA, and thus to have increased PKA activity, or to express a mutant cAMP-resistant PKA R1 alpha subunit which blocks PKA activation. Wild-type LLC-LN7 tumor cells were invasive in vitro and in vivo, recurred after tumor excision and metastasized to the lungs. However, they lost these properties after transfection to express the mutant R1 alpha that blocks PKA activation. The non-invasive, non-recurring and non-metastatic LLC-C8 cells gained the capacity to invade, to recur following tumor excision and to metastasize when transfected to express the PKA C alpha subunit.
Asunto(s)
Carcinoma Pulmonar de Lewis/enzimología , Carcinoma Pulmonar de Lewis/patología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Animales , Membrana Basal/enzimología , Membrana Basal/patología , Carcinoma Pulmonar de Lewis/secundario , Movimiento Celular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Neoplasias Pulmonares/secundario , Sustancias Macromoleculares , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Recurrencia Local de Neoplasia/enzimología , Recurrencia Local de Neoplasia/patología , TransfecciónRESUMEN
We have previously noted an association between proviral load and the severity of immune disease in individuals with a wide range of CD4 cell counts. Using the quantitative DNA polymerase chain reaction technology developed in our laboratory, we sought to extend these observations, with a view to establishing guidelines for the use of proviral load in a clinical context. We studied 199 patients with a range of CD4 cell counts attending an urban tertiary care center. Proviral load/10(6) peripheral blood mononuclear cells (PBMCs) was measured using a microtiter plate assay designed specifically for this purpose. Human immunodeficiency virus proviral DNA was detected in 193 of 199 clinical samples. Levels of proviral load were tabulated for patients and evaluated in seven categories defined by CD4 cell counts. Although a wide range of proviral loads was observed in each category of patients, there was a trend toward increasing proviral load with decreasing CD4 cell count. Statistically significant relationships were observed between proviral load and the CD4 cell count and the CD4 cell percentage (Spearman's correlation coefficient -0.19, p = 0.01 for both absolute CD4 and CD4 percentage). These relationships were quite weak and could not be taken to explain disease progression in isolation. If we defined a cutoff between low and high proviral loads at 100 copies/10(6) PBMCs, we noted that 52% (24 of 46) of patients with CD4 cell counts > 400/microliters had lower loads, as compared with 16% (24 of 143) of those with more advanced disease (p < 0.01). There is a weak, but statistically significant association between proviral load and CD4 cell depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
ADN Viral/sangre , Infecciones por VIH/virología , VIH/genética , Leucocitos Mononucleares/virología , Provirus/genética , Recuento de Linfocito CD4 , Progresión de la Enfermedad , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
The optimal temperature of blood cardioplegia remains controversial. Interstitial myocardial pH was monitored online with a probe that was inserted in the anterior wall of the left ventricle. Venous pH, lactate production, and creatine kinase and troponin T release were measured in coronary sinus blood obtained in 14 dogs after ischemic arrest periods of 5, 10, 20, and 40 minutes with warm (n = 7; mean myocardial temperature, 35 degrees +/- 2 degrees C) and cold (n = 7; mean myocardial temperature, 12 degrees +/- 1 degree C) blood cardioplegic protection. Blood cardioplegic solution was delivered at a rate of 100 mL/min during the 10 minutes between each ischemic arrest. The interstitial myocardial pH decreased significantly (p < 0.05) from 7.1 +/- 0.3 to 6.53 +/- 0.3 after ischemia in animals perfused with warm blood cardioplegia and from 7.04 +/- 0.3 to 6.64 +/- 0.1 in those receiving cold blood cardioplegic protection; however, the difference between the groups was not significant (p > 0.05). Lactate production and creatine kinase and troponin T release increased significantly after ischemia, but there was no difference in the changes between the warm and cold blood cardioplegia groups. In conclusion, ischemia caused significant changes in all variables measured, and these changes were directly proportional to the duration of ischemia. However, there was no significant difference (p > 0.05) in the myocardial metabolic changes between the warm and cold blood cardioplegia groups in terms of the duration of ischemic arrest studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Creatina Quinasa/sangre , Paro Cardíaco Inducido/métodos , Hipertermia Inducida , Hipotermia Inducida , Lactatos/sangre , Aturdimiento Miocárdico/metabolismo , Miocardio/metabolismo , Troponina/sangre , Análisis de Varianza , Animales , Biomarcadores/sangre , Perros , Concentración de Iones de Hidrógeno , Ácido Láctico , Aturdimiento Miocárdico/etiología , Factores de Tiempo , Troponina T , VenasRESUMEN
Blood sampling on filter paper has many advantages for the detection of perinatal human immunodeficiency virus (HIV) infection by the polymerase chain reaction (PCR). However, if the method is to be widely used, an assessment of its performance under field conditions is required. To simulate conditions in the field, 50-microliters aliquots of whole blood containing low levels of HIV proviral DNA (4 to 1,024 copies per 100,000 nucleated cells) were spotted onto filter paper; dried; and subjected to heat, humidity, and prolonged storage at room temperature. After exposure, the DNA was recovered and amplified with primers to human leukocyte antigen DQ alpha- and HIV-specific sequences. Treatment at 37 degrees C and 60% humidity for 7 days, storage for 12 weeks at 22 degrees C, and freeze-thawing twice had no adverse effect on PCR reactivity when compared with the results obtained with reference spots stored at -20 degrees C. The lower limits of HIV detection in all tests ranged from 4 to 16 HIV copies per 100,000 cells. Fixation in 70% ethanol improved the amplification of low levels of HIV DNA and reduced biohazard risks. These findings suggest that dried blood spots will provide a powerful new resource for testing for HIV by PCR, especially in remote areas where refrigeration and immediate sample processing are unavailable.
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Recolección de Muestras de Sangre/métodos , ADN Viral/sangre , VIH-1/aislamiento & purificación , ADN Viral/genética , Estudios de Evaluación como Asunto , Femenino , Infecciones por VIH/diagnóstico , Humanos , Recién Nacido , Papel , Reacción en Cadena de la Polimerasa , EmbarazoRESUMEN
Variable-number-tandem-repeats (VNTRs) are highly polymorphic and provide informative genetic markers for distinguishing between individuals. We have used PCR amplification of VNTR locus pMCT118 to identify mislabelled specimens submitted for HIV PCR testing. The method is rapid, can be applied to large numbers of samples and eliminates the need for radioactive probes. DNA samples (10 ng) are amplified for 25 cycles using fluorescence-labelled oligonucleotide primers (blue dye). An aliquot of the PCR product is then combined with an internal lane size standard (labelled with a red dye), electrophoresed through a 2% agarose gel on an automated fluorescence DNA fragment analyser and the size and quantity of the fragments determined automatically relative to the internal standard. Fifteen alleles, ranging in size from 398 tp 709 bp were readily identified in a random sampling of DNA from 63 unrelated HIV-infected patients. Fragment size was reproducible and corresponded to alleles containing from 16 to 35 repeats of a 16 bp unit. VNTR genotyping will prove useful for resolving discordant results due to specimen mix-up and ensuring that the correct samples have been analyzed.
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Dermatoglifia del ADN , Infecciones por VIH/diagnóstico , VIH/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alelos , Secuencia de Bases , Estudios Transversales , Reacciones Falso Positivas , Humanos , Masculino , Datos de Secuencia MolecularRESUMEN
OLIGSCAN (oligonucleotide scanner) is a computer program for IBM-PC-compatible computers that allows the user to scan up to 200 DNA sequences for homology to oligonucleotide sequences of interest. Once a core sequence of longer than the user-defined minimum length is found, the remainder of the oligonucleotide is compared to the corresponding positions of the larger sequence to identify matches or mismatches flanking the core region. This algorithm results in identification of the longest possible homologous regions first. The program was originally designed to assist in the identification of potential annealing sites for polymerase chain reaction (PCR) primers in the genomic DNA of related strains of viruses. However, it may also be used for more general pattern-identification purposes, including scanning for various sequence motifs of functional importance. We present the analysis of homology to an oligonucleotide primer in 16 complete genomic sequences of the human and simian immunodeficiency viruses.
Asunto(s)
ADN , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido Nucleico , Programas Informáticos , Algoritmos , Animales , Secuencia de Bases , VIH-1/genética , VIH-2/genética , Humanos , Datos de Secuencia Molecular , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
The nucleotide string TAAAACGAAAGT is the longest perfect homology shared by all sequenced human papillomavirus genomes. This nucleotide string, which was also found to be highly specific for human papillomavirus genomes, shares the same genomic position in all viral types (5' end of the E1 open reading frame) and putatively codes in every case for the same amino acids. One possible evolutionary model was used to estimate the probability of random occurrence of the nucleotide string in 10 human papillomavirus genomes. It assumed that the universal string had been subjected to the same mutation rate as the entire E1 open reading frame. The estimated probability was found to be very low, suggesting that the conservation of the string could not have resulted from random divergence and that its conservation among human papillomaviruses is likely to reflect the occurrence of biological constraints. It is speculated that this nucleotide string may be required to code for amino acids indispensable for the nuclear localization of E1-coded peptides or to bind cellular factors affecting viral replicative functions. Definitive evidence is expected to come from oligonucleotide-protein binding experiments and from site-directed mutagenesis of cloned HPV genomes. This motif, universal among human papillomaviruses, is being successfully used in the design of consensus primers from the early region.
Asunto(s)
Genoma Viral , Papillomaviridae/genética , Homología de Secuencia de Ácido Nucleico , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Consenso , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/genética , Sistemas de Lectura Abierta/genética , Papillomaviridae/clasificación , FilogeniaRESUMEN
We evaluated and compared the in vitro characteristics of direct current ablation using high energy ablation (Hewlett-Packard defibrillator) and a new form of low energy ablation (low energy ablation power supply, Cardiac Recorders, UK). Two new catheters with a large distal electrode have been recently introduced for catheter ablation: a low energy 7F bipolar catheter (Bard) with a contoured distal electrode, and a 7F deflectable catheter with a 4-mm tip (Mansfield). In vitro studies were carried out in a large tank filled with physiological saline while recording voltage, current, and pressure. High speed cinematography at 32,000 frames per second (Cordin, Utah) was done to assess the dynamic behavior of the vapor globe with both systems of energy delivery. We evaluated shocks of 50, 100, 150, 200, and 300 joules with the conventional system, and shocks of 10, 15, 20, 30, and 40 joules with the new system, and also compared the effects of varying catheter design with both systems of energy delivery. The conventional system using high energy showed significant arcing and increases in pressure. Low energy direct current ablation produces nonarcing shocks with 20 joules or less, and significantly less vapor globe and gas formation during arcing shocks, with a shorter duration of increase in pressure. This new system using low energy direct current may reduce the risk and complications reported with high energy ablations.
Asunto(s)
Cardiología/instrumentación , Electrocoagulación , Cateterismo , Electricidad , Electrocoagulación/instrumentación , Películas CinematográficasRESUMEN
DC shocks within the coronary sinus have been abandoned because of the risk of cardiac rupture and tamponade. Catheter ablation using DC energy to electrodes straddling the ostium of the coronary sinus, when used clinically, has been reported to result in cardiac tamponade in as many as 16% of patients. A new system of energy delivery maximizes voltage while decreasing the undesirable effects caused by barotrauma. This system includes 1) a low-energy ablation power supply with a brief time-constant capacitive discharge that delivers up to 40 J and 3,000 V and 2) a low-energy ablation catheter with a contoured distal electrode. We performed in vitro and in vivo studies of this new system and compared arcing shocks with nonarcing shocks. Ablations were performed using unipolar distal shocks (D) and unipolar shocks to both electrodes made electrically common (P-D). In vitro studies were done in a large tank filled with physiological saline while recording voltage, current, and pressure. High-speed cinematography (32,000 frames/sec) of shocks of 10-40 J permitted detailed analysis of the vapor globe. Anodal shocks of less than 20 J showed no arcing or only minimal vapor globe formation. For D and P-D anodal shocks of 40 J, the diameters of the vapor globe were 31 and 22 mm, respectively, corresponding to pressure recordings of 11 and 4.9 atm. The pressure rise lasted less than 50 mu sec. In vivo studies involved 18 dogs that received nonarcing shocks (one to six shocks of 15 J) and 18 dogs that received arcing shocks (one to three shocks of 40 J). Each group was divided between D and P-D shocks; catheter ablation was performed at a mean +/- SEM distance of 2.94 +/- 0.92 cm within the coronary sinus. All dogs tolerated the procedure without cardiac rupture or tamponade. When killed 2-4 days later, the dogs had edema and hyperemia or hemorrhage in the area of the coronary sinus. We compared the effects of multiple (three to six) nonarcing shocks with the effects of one to three arcing shocks. Disruption or rupture of the coronary sinus within the epicardial fat space occurred in two of 12 dogs (17%) with multiple nonarcing shocks but in 13 of 18 dogs (72%) with arcing shocks (p less than 0.003). Occlusion of the coronary sinus occurred in two of 12 dogs (17%) with multiple nonarcing shocks and in nine of 18 dogs (50%) with arcing shocks (p less than 0.06).(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Vasos Coronarios/cirugía , Electrocoagulación/métodos , Sistema de Conducción Cardíaco/cirugía , Animales , Arritmias Cardíacas/cirugía , Taponamiento Cardíaco/prevención & control , Perros , Femenino , Rotura Cardíaca/prevención & control , Masculino , Modelos Cardiovasculares , Modelos Estructurales , Síndrome de Wolff-Parkinson-White/cirugíaRESUMEN
A series of rapidly dividing epithelial (RDE) cell lines have been isolated from primary cultures of rat ventral prostate (RVP) epithelial cells. Unlike androgen-dependent secretory epithelial cells, the RDE cells in culture do not express the androgen-dependent secretory proteins, nor do they express the androgen-repressed cell death sequences (TRPM-2) found in the epithelial cells during prostatic regression. Screening of a cDNA clone library established from RDE cell mRNA has yielded a number of RDE cell-specific sequences. One of these, RDE-.25 is a 250-base mRNA. The sequence of RDE-.25 shows considerable homology with the rat growth hormone gene and two murine oncogene sequences. We believe that the absence of androgen-repressed cell death sequence expression confers androgen independence for survival and growth, while the expression of RDE-.25 may represent an autocrine growth stimulus which greatly increases the rate of cell division in these cells.
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Andrógenos/farmacología , Próstata/efectos de los fármacos , ARN Mensajero/genética , Animales , Secuencia de Bases , Northern Blotting , División Celular/efectos de los fármacos , Línea Celular , ADN/análisis , Células Epiteliales , Masculino , Ratones , Datos de Secuencia Molecular , Próstata/citología , RatasRESUMEN
Curare action on nicotinic acetylcholine receptors has a number of facets, of which the best known is competitive antagonism. Here we describe the weak agonist action of 10(-5) M curare on the murine skeletal muscle cell line, G8. Although curare induces no depolarization in G8 cells, single-channel recordings reveal short-lived curare-induced currents. A feature of these brief events is the multiplicity of conductance levels (of the four levels with conductances of 48, 37, 14, and 6 pS, none had a lifetime greater than 1.5 ms). Most well-resolved events (about 17% of which are to a subconductance) last less than 0.5 ms, with activation occurring predominantly as isolated events rather than in bursts. Agonism is not, however, a high probability action for curare: calculations based on the frequency of events at half-saturating conditions suggest that curare-induced channel openings occur during less than 1% of acetylcholine receptor-curare binding episodes. The outcome is (a) an agonist action too feeble to perturb the membrane voltage and (b) a powerful competitive antagonist action.
Asunto(s)
Curare/farmacología , Músculos/metabolismo , Receptores Colinérgicos/metabolismo , Animales , Línea Celular , Electrofisiología , Embrión de Mamíferos/metabolismo , Femenino , Cinética , Membranas/fisiología , Ratones , EmbarazoRESUMEN
Several techniques for the separation of rat ventral prostate cells, using density gradient centrifugation or mechanical means have been published, yielding fibroblast and epithelial cell populations of varying purities and viability. These techniques are often tedious, yield relatively limited numbers of cells, demand considerable technical expertise, and result in the isolation of cells of limited viability. Two techniques for the isolation and establishment of epithelial and fibroblast cell cultures from mature rat ventral prostate are described here. Collagenase/trypsin digestion of the tissue yields a single-cell suspension of both cell types that are separated on Percoll isopycnic centrifugation gradients. A continuous gradient system allows for the separation of a greater number of cells with very high degrees of purity. A second technique based on a step-gradient system produces reproducible subfractionation of the epithelial cell component of the prostate within a considerable shorter period of time. An improved medium for plating epithelial and fibroblast cells has also been developed. The separated cells are plated on collagen and/or fibronectin-coated dishes in a serum-free plating medium that is later replaced with a serum containing growth medium. The plating medium greatly increases the plating efficiency of the isolated cell types, particularly the epithelial cells.
Asunto(s)
Separación Celular/métodos , Fibroblastos , Próstata/citología , Animales , Células Cultivadas , Centrifugación por Gradiente de Densidad , Colágeno , Sondas de ADN , Células Epiteliales , Fibronectinas , Masculino , Povidona , Ratas , Dióxido de SilicioRESUMEN
Androgen-independent cell lines have been clonally selected from primary cultures of androgen-dependent epithelial cells from the rat ventral prostate. These rapidly dividing epithelial-like cells (RDE) have altered morphology and adherence characteristics. Unlike normal prostate epithelial cells, the RDE cell lines do not require androgens for cell division or cell survival. In the presence of physiological concentrations of testosterone, the isoelectric focusing patterns of prostatic acid phosphatases are abnormal in these RDE cells, and the prostate steroid-binding protein genes are not expressed. The loss of androgen dependence is not due to the inability of RDE cells to metabolize testosterone to 5 alpha-dihydrotestosterone, the active androgen, since the RDE cell lines metabolize testosterone in a manner similar to normal androgen-dependent epithelial cells. When RDE cells are grown on collagen matrices, the cells assume ductlike structures, similar to prostatic acini, although PSBP gene expression is not induced. When seeded into soft agar these cell lines form distinct foci, suggesting that they are potentially tumorigenic.
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Andrógenos/farmacología , Próstata/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Proteína de Unión a Andrógenos/genética , Animales , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dihidrotestosterona/metabolismo , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Cinética , Masculino , Próstata/citología , Próstata/metabolismo , Prostateína , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Secretoglobinas , Testosterona/metabolismo , Testosterona/farmacología , UteroglobinaRESUMEN
The involution of the prostate that occurs after castration is thought to be an active process, requiring protein synthesis. A number of "castration-induced" proteins which might be involved in this process have been identified. We recently described a group of "testosterone-repressed" mRNA sequences in the prostate which could code for these proteins. Because of their potential importance in the autophagic response we have cloned these sequences, and we report here the characterization of the most abundant of these sequences (TRPM-2), and the kinetics of the induction of this gene in the prostate after castration. TRPM-2 is induced to a maximum level of approximately 1440 ppm of total RNA six days after castration, by which time the androgen dependent, prostate steroid binding protein (PSBP) mRNA sequences have diminished to undetectable levels. The translation product of TRPM-2 is a protein of approximately 46,000 daltons, with a pI of 5.9-6.3. Since this gene is expressed in other involuting tissues, it may play an important role in the process of tissue regression.