RESUMEN
The basolateral Na+/K+/2Cl(-) cotransporter (NKCC1) has been shown to be an independent regulatory site for electrogenic Cl(-) secretion. The proinflammatory phorbol ester, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), inhibits basal and cyclic adenosine monophosphate (cAMP)-stimulated NKCC1 activity in T84 intestinal epithelial cells and decreases the steady state levels of NKCC1 mRNA in a time- and dose-dependent manner. The levels of NKCC1 protein also fall in accordance with the NKCC1 mRNA transcript and these levels are unaffected by 4alpha-phorbol, which does not activate PKC. Inhibition of maximal (cAMP-stimulated) NKCC1 functional activity by PMA was first detected by 1 h, whereas decreases in the steady state levels of NKCC1 mRNA were not detectable until 4 h. NKCC1 mRNA expression recovers toward control levels with extended treatment of cells with PMA suggesting that the PMA effects on NKCC1 expression are mediated through activation of PKC. Although NKCC1 mRNA and protein levels return to control values after extended PMA exposure, NKCC1 functional activity does not recover. Immunofluorescence imaging suggest that the absence of functional recovery is due to failure of newly synthesized NKKC1 protein to reach the cell surface. We conclude that NKCC1 has the capacity to be regulated at the level of de novo expression by PKC, although decreased NKCC1 expression alone cannot account for either early or late loss of NKCC1 function.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteína Quinasa C/metabolismo , División Celular , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Simportadores de Cloruro de Sodio-Potasio , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND & AIMS: Phenylimidazothiazoles have recently been shown to activate wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in transfected cells and were proposed as therapy for cystic fibrosis. The aim of this study was to investigate the effects of phenylimidazothiazoles on regulated transepithelial Cl- transport in intact epithelia. METHODS: T84 intestinal epithelial cells grown on permeable supports and stripped human colonic mucosal sheets were studied by conventional current-voltage clamping. Selective permeabilization of apical or basolateral membranes with the monovalent ionophore nystatin was used to isolate basolateral K+ and apical Cl- channel activity, respectively. 86Rb+ uptake was assessed for Na/K/2Cl cotransporter and Na+,K(+)-adenosine triphosphatase activity. RESULTS: In T84 monolayers and human colon, levamisole and its brominated derivative bromotetramisole failed to activate transepithelial secretion. In fact, these compounds dose-dependently inhibited secretory responses to the cyclic adenosine monophosphate agonist forskolin and the Ca2+ agonist carbachol. In permeabilized T84 monolayers, phenylimidazothiazoles weakly activated apical Cl- currents (consistent with their reported action on CFTR) and did not affect bumetanide-sensitive or bumetanide-insensitive 86+Rb+ uptake. Instead, they profoundly inhibited the basolateral Ba(2+)-sensitive and Ba(2+)-insensitive K+ currents. CONCLUSIONS: Phenylimidazothiazoles block K+ channels required for Cl(-)-secretory responses elicited by diverse pathways in model epithelia and native colon, an effect that outweighs their ability to activate apical Cl- channels.
Asunto(s)
Cloruros/metabolismo , Mucosa Intestinal/metabolismo , Levamisol/farmacología , Bloqueadores de los Canales de Potasio , Calcio/fisiología , Proteínas Portadoras/metabolismo , Línea Celular , Membrana Celular/metabolismo , Cloruros/fisiología , Colforsina/farmacología , Colon/efectos de los fármacos , Colon/fisiología , Conductividad Eléctrica , Humanos , Técnicas In Vitro , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Canales de Potasio/fisiología , Transducción de Señal/fisiología , Simportadores de Cloruro de Sodio-Potasio , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Tetramisol/análogos & derivados , Tetramisol/farmacologíaRESUMEN
CP1 is a yeast protein which binds to the highly conserved DNA element I (CDEI) of yeast centromeres. We have purified CP1 to near homogeneity; it is comprised of a single polypeptide of molecular weight 58,400. When bound to yeast CEN3 DNA, CP1 protects a 12-15-base pair region centered over CDEI. Methylation interference experiments show that methylations of residues located outside of the 8-base pair CDEI sequence have no detectable effect on CP1 binding, suggesting that the DNA sequences important for CP1 recognition are confined to the CDEI octanucleotide. The equilibrium constant for CP1 binding to CEN3 DNA is relatively low, 3 x 10(8) M-1. Using a novel method to determine relative DNA binding constants, we analyzed the effect of CDEI mutations on CP1 binding. A C to T point mutation at position 5 (CO1) reduces the equilibrium constant about 35-fold, while the insertion of an additional T at this position (CAT) reduces the equilibrium constant 1,400-fold. The effect of these mutations on mitotic centromere function in vivo was assessed using a plasmid stability assay. While the CO1 mutation had a slight effect, the CAT mutation significantly impaired function, implying that CP1 binding is required for the optimal mitotic function of yeast centromeres.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Centrómero/metabolismo , Cromosomas/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/metabolismo , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Plásmidos , Unión Proteica , Mapeo Restrictivo , Saccharomyces cerevisiae/genéticaRESUMEN
In response to the need for development of measures to effectively control human dental caries, a postdocroral research training program in cariology has been established at the University of Minnesota School of Dentistry. This article discusses the concept of cariology, the background leading to the development of the program, and the goals and structure of the program. The recruiting and training of individuals to perform and direct multidisciplinary basic and applied research in cariology are explained. A didactic and clinical program is presented which gives a comprehensive introduction to cariology to trainees with a Ph.D degree and little previous knowledge of the multifactorial nature of dental caries.
Asunto(s)
Caries Dental , Educación de Posgrado en Odontología , Programas Nacionales de Salud , Facultades de Odontología , Curriculum , Minnesota , National Institutes of Health (U.S.) , Organización y Administración , Investigación , Estados Unidos , UniversidadesAsunto(s)
Anticuerpos Antivirales/análisis , Orthomyxoviridae/inmunología , África Oriental , Animales , China , Pruebas de Fijación del Complemento , Brotes de Enfermedades/veterinaria , Reservorios de Enfermedades , Haplorrinos , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas Virales , Macaca , Enfermedades de los Monos/epidemiología , Neuraminidasa , Infecciones por Orthomyxoviridae/veterinaria , Serotipificación , Factores de TiempoAsunto(s)
Sueros Inmunes , Pruebas de Neutralización , Virus Vaccinia/inmunología , Animales , Antígenos Virales , Proteínas del Sistema Complemento , Estudios de Evaluación como Asunto , Cobayas/inmunología , Pruebas de Inhibición de Hemaglutinación , Calor , Humanos , Hidróxidos , Macaca/inmunología , Compuestos de Amonio Cuaternario , Conejos/inmunología , Serpientes , Factores de Tiempo , Ponzoñas , ZimosanAsunto(s)
Formación de Anticuerpos , Vacuna contra Viruela , Vacunación , Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla , Adulto , Animales , Embrión de Pollo/inmunología , Pruebas de Fijación del Complemento , Pruebas de Inhibición de Hemaglutinación , Humanos , Esquemas de Inmunización , Masculino , Pruebas de Neutralización , Manifestaciones Cutáneas , Pruebas Cutáneas , Viruela/inmunología , Factores de TiempoRESUMEN
A dosage equal to or greater than approximately 3.4 Dex (decimal exponent, log(10)) weanling mouse intracerebral 50% lethal dose (LD(50)) was sufficient to elicit a yellow fever antibody response, as determined by the plaque neutralization (PN) test, in better than 90% of vaccinated rhesus monkeys. Lower dosages were progressively less effective in terms of PN titers and the PN and hemagglutination-inhibition serological conversion rates observed. A dose of between 3.4 and 4.2 Dex weanling mouse intracerebral LD(50), or one-tenth to one times the dosage recommended for man, provided an optimal antibody response in monkeys. In rhesus monkeys, in contrast to the findings for man, pre-existing yellow fever antibody did not interfere with the antibody response to yellow fever vaccine. The PN test was felt to be a more sensitive and specific indicator of yellow fever antibody in rhesus monkeys after vaccination than the hemagglutination inhibition or complement fixation tests.
Asunto(s)
Formación de Anticuerpos , Vacunas Virales/administración & dosificación , Virus de la Fiebre Amarilla/inmunología , Animales , Antígenos Virales , Arbovirus/inmunología , Línea Celular/microbiología , Pruebas de Fijación del Complemento , Haplorrinos , Pruebas de Inhibición de Hemaglutinación , Inyecciones Intramusculares , Riñón , Dosificación Letal Mediana , Macaca , Ratones , Pruebas de Neutralización , Vacunación , Cultivo de VirusAsunto(s)
Pruebas de Inhibición de Hemaglutinación/normas , Hemaglutininas Virales/aislamiento & purificación , Virus Vaccinia/inmunología , Animales , Línea Celular , Embrión de Pollo , Técnicas de Cultivo , Efecto Citopatogénico Viral , Fibroblastos , Haplorrinos , Pruebas de Hemaglutinación , Sueros Inmunes , Macaca , Métodos , Conejos , Refrigeración , Tensoactivos , Temperatura , Virus Vaccinia/crecimiento & desarrolloAsunto(s)
Células Cultivadas/microbiología , Virus ARN , Animales , Anticuerpos Antivirales/análisis , Encéfalo , Línea Celular , Reacciones Cruzadas , Efecto Citopatogénico Viral , Técnica del Anticuerpo Fluorescente , Haplorrinos , Riñón , Macaca , Virus del Sarampión/aislamiento & purificación , Pruebas de Neutralización , Virus ARN/clasificación , Virus ARN/crecimiento & desarrollo , Virus ARN/inmunología , Virus ARN/aislamiento & purificación , Conejos , Serotipificación , Bazo , Cultivo de VirusAsunto(s)
Encefalitis/veterinaria , Virus del Sarampión/patogenicidad , Enfermedades de los Monos/microbiología , Animales , Línea Celular , Cricetinae , Efecto Citopatogénico Viral , Modelos Animales de Enfermedad , Eritrocitos/inmunología , Haplorrinos/inmunología , Hemabsorción , Técnica de Placa Hemolítica , Calor , Riñón , Macaca , Virus del Sarampión/crecimiento & desarrollo , Virus del Sarampión/aislamiento & purificación , Ensayo de Placa Viral , Virulencia , Cultivo de Virus , Replicación ViralAsunto(s)
Antígenos/análisis , Staphylococcus/inmunología , Aerobiosis , Anaerobiosis , Animales , Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Pared Celular/análisis , Pared Celular/inmunología , Transporte de Electrón , Inmunoquímica , Inmunodifusión , Inmunoelectroforesis , Consumo de Oxígeno , Peptidoglicano/análisis , Pronasa , Conejos/inmunología , Staphylococcus/análisis , Staphylococcus/crecimiento & desarrollo , Staphylococcus/metabolismo , Ácidos Teicoicos/análisisAsunto(s)
Encéfalo/microbiología , Células Cultivadas/microbiología , Virus/inmunología , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Línea Celular , Sistema Nervioso Central/inmunología , Reacciones Cruzadas , Efecto Citopatogénico Viral , Virus de la Encefalitis/aislamiento & purificación , Haplorrinos , Sueros Inmunes , Inmunización , Riñón , Macaca , Virus del Sarampión/aislamiento & purificación , Pruebas de Neutralización , Conejos , Serotipificación , Virosis/inmunología , Virus/aislamiento & purificaciónRESUMEN
Neutralization of vaccinia virus with immune rabbit serum occurred optimally when incubated at 37 C for 16 to 24 hr in the plaque neutralization test employing the MA-104 embryonic rhesus monkey kidney cell line.
Asunto(s)
Pruebas de Neutralización , Virus Vaccinia/inmunología , Animales , Línea Celular , Haplorrinos , Sueros Inmunes , Riñón , Cinética , Conejos , Temperatura , Virus Vaccinia/crecimiento & desarrolloRESUMEN
Quantitation of soluble residual host protein in chicken embryo-derived vaccines was performed rapidly, economically, and accurately by radial immunodiffusion.
Asunto(s)
Inmunodifusión , Proteínas/análisis , Vacunas Virales/análisis , Animales , Embrión de Pollo , Humanos , Hipersensibilidad/prevención & control , Sueros Inmunes , Inmunoelectroforesis , Vacunas contra la Influenza/análisis , Vacuna Antisarampión/análisis , Métodos , Ovalbúmina , Conejos , Vacuna contra Viruela/análisis , Vacunas Virales/normas , Fiebre AmarillaRESUMEN
The use of three diluents (i.e., 0.01 m phosphate-buffered saline, PBS; PBS with 0.2% gelatin, PBS/GEL; and PBS with 0.4% bovine plasma albumin) and three methods (i.e., the standard tube macro-procedure, TUBE; the manual microtechnique, MANUAL; and the semiautomatic microtechnique, AUTO) were statistically compared for their reproducibility and sensitivity in determining hemagglutinin (HA) and hemagglutination-inhibition (HI) antibody titers. In the HA test, analyses of between-cell variances of the different methods showed the AUTO microtiter procedure to be more reproducible than the standard TUBE method. The MANUAL microtiter procedure was the least reproducible. In the HI test, the TUBE method was the most reproducible. No significant difference in the reproducibility of the diluents was observed in either the HA or HI test. When a comparison of the sensitivity of test methods and diluents was made for determining HA titers, the AUTO microtiter procedure and PBS/GEL diluent appeared to be the method and diluent of choice. Evaluation of another instrument, the autopipetter, which standardizes the volume of diluent to be added in the microtechnique, suggests that the reproducibility of the AUTO microtiter procedure might be further increased.
Asunto(s)
Anticuerpos/análisis , Autoanálisis , Pruebas de Inhibición de Hemaglutinación/normas , Pruebas de Hemaglutinación/normas , Técnicas de Dilución del Indicador/instrumentación , Infecciones por Orthomyxoviridae/inmunología , Albúmina Sérica Bovina , Análisis de Varianza , Animales , Antígenos , Automatización , Líquidos Corporales , Tampones (Química) , Pollos , Eritrocitos/inmunología , Membranas Extraembrionarias , Gelatina , Pruebas de Inhibición de Hemaglutinación/instrumentación , Pruebas de Hemaglutinación/instrumentación , Masculino , Métodos , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/sangre , Fosfatos , Cloruro de Sodio , Soluciones , PorcinosRESUMEN
Seven Hong Kong variant strains were compared for their capacity to produce haemagglutinin (HA). From the results of 9 serial egg passages made at 33 degrees C and 36 degrees C, the A2/Aichi/2/68 strain was found suitable as the vaccine production strain for the 1968-69 monovalent influenza vaccine manufactured in the United States of America.Results of initial experiments comparing the HA production by 2 Hong Kong variant recombinants and by the A2/Aichi/2/68 strain showed no essential difference among these strains. When these experiments were repeated starting with viruses that had undergone the same number of egg passages from isolation, the mean HA and chicken cell agglutination titres of the 3 recombinant strains used in these studies were only 1.5 times greater than those of the A2/Aichi/2/68 strain. By comparison, the HA producing capacity of A0/PR/8/34 was almost 6 times that of the A2/Aichi/2/68 strain.