RESUMEN
Guided by structure-based design, we synthesized two novel series of potent inhibitors of BACE1 and generated extensive SAR around both the prime and non-prime side binding pockets. The key feature of both series is a cyclic amine motif specifically crafted to achieve interactions with both the flap and with the S2' pocket.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Imidazolidinas/síntesis química , Imidazolidinas/farmacología , Piperazinas/síntesis química , Piperazinas/farmacología , Animales , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Humanos , Imidazolidinas/química , Ratones , Ratones Transgénicos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Piperazinas/química , Relación Estructura-ActividadRESUMEN
Based on a lead compound identified from the patent literature, we developed patentably novel BACE-1 inhibitors by introducing a cyclic amine scaffold as embodied by 1a and 1b. Extensive SAR studies assessed a variety of isophthalamide replacements including substituted pyrrolidinones and ultimately led to the identification of 11. Due to its favorable overall profile, 11 has been extensively profiled in various in vivo settings.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Pirrolidinas/química , Pirrolidinas/farmacocinética , Administración Oral , Amidas/química , Amidas/farmacocinética , Amidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Ratones , Ratones Transgénicos , Modelos Moleculares , Fenoles/química , Inhibidores de Proteasas/farmacología , Pirrolidinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Based on lead compound 1 identified from the patent literature, we developed novel patentable BACE-1 inhibitors by introducing a cyclic amine scaffold. Extensive SAR studies on both pyrrolidines and piperidines ultimately led to inhibitor 2f, one of the most potent inhibitors synthesized to date.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Piperidinas/química , Piperidinas/farmacología , Inhibidores de Proteasas/química , Pirrolidinas/química , Pirrolidinas/farmacología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Línea Celular , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Piperidinas/síntesis química , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Pirrolidinas/síntesis química , Relación Estructura-ActividadRESUMEN
G-protein-gated inwardly rectifying K(+) (GIRK) channels are widely expressed in the brain and are activated by at least eight different neurotransmitters. As K(+) channels, they drive the transmembrane potential toward E(K) when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits (GIRK1-4 or Kir 3.1-4), with GIRK1 being the most unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However, more recent data indicate that native channels can be either homo- or heterotetrameric complexes composed of several GIRK subunit combinations. The functional implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK channels formed by the co-assembly of GIRK2 and GIRK3, the most abundant GIRK subunits found in the mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at the single channel level was an approximately 5-fold lower sensitivity to activation by Gbetagamma. Complexes containing only GIRK2 and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels composed of GIRK2 and GIRK3 subunits exist in brain.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/química , Canales de Potasio/metabolismo , Animales , Encéfalo/metabolismo , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Activación del Canal Iónico , Sustancias Macromoleculares , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Canales de Potasio/genética , Pruebas de Precipitina , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , TransfecciónRESUMEN
GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine-activated potassium current (IKACh) channel in pacemaker cells of the heart. The channel is activated by direct binding of G-protein Gbetagamma subunits. The GIRK1 subunit is atypical in the GIRK family in having a unique ( approximately 125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flag-tagged GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surface localization. Furthermore, GIRK1 homomultimers reside in core-glycosylated and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [35S]Methionine pulse-labeling experiments demonstrated that GIRK4 associates with GIRK1 either during or shortly after subunit synthesis. Mutant and chimeric channel subunits were utilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of Delta374-501 resulted in an intracellular GIRK1 subunit that produced normal IKACh-like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis of the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in the processing and cell surface localization of IKACh in vivo.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Activación del Canal Iónico , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Células COS , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Glicosilación , Atrios Cardíacos/metabolismo , Ratones , Ratones Noqueados , Canales de Potasio/genética , Canales de Potasio/fisiología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The cardiac G protein-gated K+ channel, IKACh, is directly activated by G protein beta gamma subunits (Gbeta gamma). IKAChis composed of two inward rectifier K+ channel subunits, GIRK1 and GIRK4. Gbeta gamma binds to both GIRK1 and GIRK4 subunits of the heteromultimeric IKACh. Here we delineate the Gbeta gamma binding regions of IKACh by studying direct Gbeta gamma interaction with native purified IKACh, competition of this interaction with peptides derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis of regions implicated in Gbeta gamma binding, and functional expression of mutated subunits in mammalian cells. Only two GIRK4 peptides, containing amino acids 209-225 or 226-245, effectively competed for Gbeta gamma binding. A single point mutation introduced into GIRK4 at position 216 (C216T) dramatically reduced the potency of the peptide in inhibiting Gbeta gamma binding and Gbeta gamma activation of expressed GIRK1/GIRK4(C216T) channels. Conversion of 5 amino acids in GIRK4 (226-245) to the corresponding amino acids found in the G protein-insensitive IRK1 channel, completely abolished peptide inhibition of Gbeta gamma binding to IKACh and Gbeta gamma activation of GIRK1/mutant GIRK4 channels. We conclude from this data that Gbeta gamma binding to GIRK4 is critical for IKACh activation.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Células COS , Bovinos , Cricetinae , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Proteínas de Unión al GTP/antagonistas & inhibidores , Datos de Secuencia Molecular , Miocardio/metabolismo , Canales de Potasio/agonistas , Canales de Potasio/química , Unión ProteicaRESUMEN
Co-expression of clones encoding Kir6.2, a K+ inward rectifier, and SUR1, a sulfonylurea receptor, reconstitutes elementary features of ATP-sensitive K+ (KATP) channels. However, the precise kinetic properties of Kir6.2/SUR1 clones remain unknown. Herein, intraburst kinetics of Kir6.2/SUR1 channel activity, heterologously co-expressed in COS cells, displayed mean closed times from 0.7 +/- 0.1 to 0.4 +/- 0.03 msec, and from 0.4 +/- 0.1 to 2.0 +/- 0.2 msec, and mean open times from 1.9 +/- 0.4 to 4.5 +/- 0.8 msec, and from 12.1 +/- 2.4 to 5.0 +/- 0.2 msec between -100 and -20 mV, and +20 to +80 mV, respectively. Burst duration for Kir6.2/SUR1 activity was 17. 9 +/- 1.8 msec with 5.6 +/- 1.5 closings per burst. Burst kinetics of the Kir6.2/SUR1 activity could be fitted by a four-state kinetic model defining transitions between one open and three closed states with forward and backward rate constants of 1905 +/- 77 and 322 +/- 27 sec-1 for intraburst, 61.8 +/- 6.6 and 23.9 +/- 5.8 sec-1 for interburst, 12.4 +/- 6.0 and 13.6 +/- 2.9 sec-1 for intercluster events, respectively. Intraburst kinetic properties of Kir6.2/SUR1 clones were essentially indistinguishable from pancreatic or cardiac KATP channel phenotypes, indicating that intraburst kinetics per se were insufficient to classify recombinant Kir6.2/SUR1 amongst native KATP channels. Yet, burst kinetic behavior of Kir6.2/SUR1 although similar to pancreatic, was different from that of cardiac KATP channels. Thus, expression of Kir6.2/SUR1 proteins away from the pancreatic micro-environment, confers the burst kinetic identity of pancreatic, but not cardiac KATP channels. This study reports the kinetic properties of Kir6.2/SUR1 clones which could serve in the further characterization of novel KATP channel clones.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Receptores de Droga/metabolismo , Compuestos de Sulfonilurea/metabolismo , Animales , Células COS , Línea Celular , Clonación Molecular , Cobayas , Técnicas In Vitro , Cinética , Miocardio/metabolismo , Páncreas/metabolismo , Canales de Potasio/genética , Receptores de Droga/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores de SulfonilureasRESUMEN
Homozygous weaver mice are profoundly ataxic because of the loss of granule cell neurons during cerebellar development. This granule cell loss appears to be caused by a genetic defect in the pore region (Gly156-->Ser) of the heterotrimeric guanine nucleotide-binding protein (G protein)-gated inwardly rectifying potassium (K+) channel subunit (GIRK2). A related subunit, GIRK1, associates with GIRK2 to constitute a neuronal G protein-gated inward rectifier K+ channel. The weaver allele of the GIRK2 subunit (wvGIRK2) caused loss of K+ selectivity when expressed either as wvGIRK2 homomultimers or as GIRK1-wvGIRK2 heteromultimers. The mutation also let to loss of sensitivity to G protein betagamma dimers. Expression of wvGIRK2 subunits let to increased cell death, presumably as a result of basal nonselective channel opening.
Asunto(s)
Cerebelo/metabolismo , Proteínas de Unión al GTP/fisiología , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Elementos sin Sentido (Genética) , Células CHO , Muerte Celular , Línea Celular , Cerebelo/citología , Cricetinae , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Potenciales de la Membrana , Ratones , Ratones Mutantes Neurológicos , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/metabolismo , Oocitos/citología , Técnicas de Placa-Clamp , Mutación Puntual , Canales de Potasio/genética , TransfecciónRESUMEN
GIRK1 and CIR are G-protein activated inward rectifier K+ channel subunits that combine to form the heteromultimer IKACh, the G beta gamma-activated atrial K channel responsible for the vagal slowing of heart rate. Epitope-tagged channel subunits were constructed by the introduction of distinct six amino acid epitopes into the C-termini or putative extracellular domains of GIRK1 and CIR. Carboxyl-terminal tagged subunits were activated by purified G beta gamma subunits in inside-out patches when expressed in Cos cells. Interestingly, insertion of three amino acids into the putative extracellular domain of GIRK1 resulted in an inactive subunit that acted as a dominant negative subunit when coexpressed with wild type GIRK1 and CIR in Xenopus oocytes. The epitope-tagged CIR-AU1 subunit coimmunoprecipitated GIRK1-AU5 from metabolically labeled Cos cells. Immunofluorescence labeling of Cos cells localized GIRK1-AU5 to internal cytoskeletal structures that co-stained with antibodies against the intermediate filament protein, vimentin. CIR-AU1 localized primarily to the plasma membrane. Double immunofluorescence labeling showed that GIRK1-AU5 plasma membrane staining was detectable only when coexpressed with CIR-AU1.
Asunto(s)
Epítopos/análisis , Canales de Potasio de Rectificación Interna , Canales de Potasio/inmunología , Animales , Células COS , Técnica del Anticuerpo Fluorescente , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Oocitos/metabolismo , Canales de Potasio/análisis , Pruebas de Precipitina , Transfección , Xenopus laevisRESUMEN
Previous studies have demonstrated that the alpha 2A-adrenergic receptor (alpha 2A AR) incorporates [3H]palmitate and that replacement of Cys442 by Ala or Ser eliminates detectable acylation without perturbing coupling to pertussis toxin-sensitive GTP-binding proteins (Kennedy, M. E., and Limbird, L. E. (1993) J. Biol. Chem. 268, 8003-8011) or, as shown here, without perturbing agonist-dependent receptor phosphorylation, in contrast to the consequences of eliminating beta 2-adrenergic receptor acylation. As a first step in revealing the functional role for this post-translational modification at the alpha 2A AR, we explored sequences in the alpha 2AAR which confer alpha 2AAR acylation and whether or not [3H]palmitoylation of the alpha 2AAR is dynamic. Deletion of the 7 terminal amino acids distal to Cys442 of the alpha 2AAR did not eliminate detectable [3H]palmitoylation of the alpha 2AAR, whereas truncation to Leu441 did, indicating both that Cys442 is the likely site for acylation and that sequences distal to Cys442 are not required for acylation at Cys442. Since mutation of sequences proximal to Cys442 altered overall receptor structure, based on markedly reduced detectable adrenergic receptor binding, proximal motifs required for palmitoylation of the alpha 2AAR could not be explored further. When the turnover of [35S]Met/Cys-labeled alpha 2AAR was compared with the turnover of the [3H]palmitate-labeled alpha 2AAR, it was of interest that agonist treatment accelerated the half-life of decay of the [3H]palmitate-labeled alpha 2AAR without detectable receptor down-regulation, providing evidence that the acylation of the alpha 2AAR may be a dynamic process.
Asunto(s)
Palmitatos/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Acilación , Agonistas alfa-Adrenérgicos/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Tartrato de Brimonidina , Cartilla de ADN/química , Técnicas In Vitro , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Quinoxalinas/farmacología , Receptores Adrenérgicos alfa/química , Proteínas Recombinantes , Relación Estructura-Actividad , Porcinos , Yohimbina/farmacologíaAsunto(s)
Síndrome Pulmonar por Hantavirus/prevención & control , Exposición Profesional/efectos adversos , Animales , Canadá/epidemiología , Orthohantavirus/aislamiento & purificación , Síndrome Pulmonar por Hantavirus/epidemiología , Humanos , Peromyscus/virología , Factores de Riesgo , Control de RoedoresRESUMEN
The aim of this study was to investigate the effects of ultrasound irradiation on a biodegradable drug delivery system. Microporous, disk-shaped specimens of a 50-50% copolymer of polylactic and polyglycolic acids were gel cast from an acetone solution. A protein was incorporated in these specimens, which were then immersed in phosphate buffered saline and subjected to ultrasound irradiation every second day. The investigation was performed in two phases: in the first, a study was performed for 58 days to determine if ultrasonic irradiation affected the kinetics of protein release from the specimens. In the second phase, effects of frequency and duration of the ultrasound signal on the degradation of the implant were studied for 40 days. The results indicate that ultrasound irradiation resulted in almost a threefold increase in protein elution from the specimens. Both the ultrasound frequency and signal duration affected the molecular weight loss and mass loss, and changed the overall degradation kinetics of the polymer.
Asunto(s)
Materiales Biocompatibles/efectos de la radiación , Ácido Láctico , Ácido Poliglicólico , Polímeros/efectos de la radiación , Materiales Biocompatibles/química , Implantes de Medicamentos , Microscopía Electrónica de Rastreo , Peso Molecular , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , Temperatura , Inhibidores de Tripsina/química , UltrasonidoRESUMEN
The alpha 2A-adrenergic receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.
Asunto(s)
Compartimento Celular , Polaridad Celular , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Análisis Mutacional de ADN , Perros , Proteínas de Unión al GTP/genética , Glicosilación , Aparato de Golgi/metabolismo , Riñón/citología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Receptores Adrenérgicos alfa 2/genética , Receptores de Péptidos/metabolismo , Eliminación de SecuenciaRESUMEN
The alpha 2A-adrenergic receptor (alpha 2AAR) is coupled to a variety of effectors via pertussis toxin-sensitive GTP-binding proteins. Like most members of the G-protein-coupled receptor superfamily, the primary structure of the alpha 2AAR possesses a putative consensus sequence for palmitoylation in the COOH terminus at Cys-442. This study demonstrates that the alpha 2AAR incorporates [3H] palmitic acid in metabolic labeling studies and that mutation of Cys-442 to Ala or Ser eliminates detectable 3H-palmitoylation. However, mutation of Cys-442 does not alter adrenergic ligand specificity or allosteric modulation by amphipathic agents, such as amiloride analogs. Since reports in the literature suggest that a homologous mutation in the beta 2-adrenergic receptor attenuates coupling to Gs (O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569) whereas chemical removal of palmitate from bovine rhodopsin enhances coupling to Gt (Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123), we examined if mutation of Cys-442 and parallel loss of detectable palmitoylation alter alpha 2AAR coupling to G-proteins. Several independent cell lines of Madin-Darby canine kidney II cells expressing wild-type (Cys-442) or mutant (Ala-442 and Ser-442) alpha 2AARs were established. Metabolic labeling of Madin-Darby canine kidney cells expressing wild-type (Cys-442) or mutant (Ala-442) alpha 2AARs with [3H]palmitic acid indicated that only wild-type Cys-442-containing receptors incorporated [3H]palmitate, monitored following isolation of the alpha 2AAR detergent extracts using yohimbine-agarose chromatography. Receptor-G-protein coupling was assessed by evaluating sensitivity of receptor-agonist interactions to guanine nucleotides in competition for [3H]yohimbine antagonist binding, guanyl-5'-yl imidotrisphosphate sensitivity of pertussis toxin-sensitive p-[125I]iodoclonidine agonist binding, and agonist-stimulated guanosine 5'-O-(thiotriphosphate) binding. Using all three approaches, no detectable change in alpha 2AAR-G-protein coupling was apparent, in contrast to apparent opposite effects on the beta 2-adrenergic receptor-Gs and rhodopsin-Gt coupling reported previously by others. One interpretation is that this conserved cysteine may play differing roles at different receptor-G-protein interfaces. Alternatively, this shared structural motif may play a role in not yet investigated pathways, such as receptor expression, turnover, and localization.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Ácidos Palmíticos/metabolismo , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Acilación , Alanina , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía de Afinidad , Cisteína , Perros , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanilil Imidodifosfato/farmacología , Riñón , Cinética , Mutagénesis Sitio-Dirigida , Ácido Palmítico , Receptores Adrenérgicos beta/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serina , Porcinos , Transfección , Yohimbina/metabolismoRESUMEN
The recent emphasis on gloving has resulted in accounts of healthcare workers washing gloves instead of changing them. This study evaluated the efficacy of soap and three germicidal agents in decontaminating latex and vinyl medical gloves that were experimentally contaminated with Staphylococcus aureus. Gloves were tested for perforations before and after the glove-washing procedure. Results of this study demonstrated that contamination was reduced from the glove surfaces. Although the routine washing of gloved hands cannot be recommended, it may be carried out under limited circumstances.
Asunto(s)
Desinfectantes , Guantes Quirúrgicos , Staphylococcus aureus/fisiología , Desinfección/métodos , Seguridad de Equipos , Estudios de Evaluación como AsuntoRESUMEN
We have provided a detailed protocol for the synthesis of a yohimbine-agarose matrix that has been shown to be effective for isolation of the alpha 2A-adrenergic receptor from human platelet and purification of the alpha 2A-adrenergic receptor to apparent homogeneity from porcine brain cortex using chromatography on only two sequential yohimbine-agarose columns. In addition, this affinity matrix also interacts with alpha 2 receptors of the alpha 2B subtype extracted from cultured NG108-15 cells. Finally, this affinity matrix has proven useful for monitoring posttranslational modifications of the receptor in digitonin extracts of metabolically labeled cells. Thus, this affinity matrix can be exploited for the purification of multiple alpha 2-adrenergic receptor subtypes on both a macro- and microscale and should be of value to any laboratory exploring the molecular basis for alpha 2-adrenergic functions.
Asunto(s)
Plaquetas/metabolismo , Receptores Adrenérgicos alfa/aislamiento & purificación , Sefarosa/análogos & derivados , Yohimbina/análogos & derivados , Agonistas alfa-Adrenérgicos/metabolismo , Fraccionamiento Celular/métodos , Cromatografía de Afinidad/métodos , Digitonina , Humanos , Indicadores y Reactivos , Ensayo de Unión Radioligante/métodos , Receptores Adrenérgicos alfa/análisis , Receptores Adrenérgicos alfa/metabolismo , Sefarosa/síntesis química , Tritio , Yohimbina/sangre , Yohimbina/síntesis químicaRESUMEN
Platelets metabolize arachidonic acid to thromboxane A2, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide-derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human--hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and its gene regulation.
Asunto(s)
Plaquetas/enzimología , Mapeo Cromosómico , ADN de Neoplasias/aislamiento & purificación , Leucemia Eritroblástica Aguda/enzimología , Prostaglandina-Endoperóxido Sintasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Chlorocebus aethiops , Cromosomas Humanos Par 9 , Clonación Molecular , Regulación Neoplásica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Células Tumorales CultivadasRESUMEN
The activities of 10 formulations as mycobactericidal agents in Mycobacterium tuberculosis-contaminated suspensions (suspension test) and stainless steel surfaces (carrier test) were investigated with sputum as the organic load. The quaternary ammonium compound, chlorhexidine gluconate, and an iodophor were ineffective in all tests. Ethanol (70%) was effective against M. tuberculosis only in suspension in the absence of sputum. Povidone-iodine was not as efficacious when the test organism was dried on a surface as it was in suspension, and its activity was further reduced in the presence of sputum. Sodium hypochlorite required a higher concentration of available chlorine to achieve an effective level of disinfection than did sodium dichloroisocyanurate. Phenol (5%) was effective under all test conditions, producing at least a 4-log10 reduction in CFU. The undiluted glutaraldehyde-phenate solution was effective against M. tuberculosis and a second test organism, Mycobacterium smegmatis, even in the presence of dried sputum, whereas the diluted solution (1:16) was only effective against M. smegmatis in the suspension test. A solution of 2% glutaraldehyde was effective against M. tuberculosis. This investigation presents tuberculocidal efficacy data generated by methods simulating actual practices of routine disinfection.
Asunto(s)
Desinfectantes/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Técnicas Bacteriológicas , Evaluación Preclínica de Medicamentos , Farmacorresistencia Microbiana , Humanos , Técnicas In Vitro , Esputo/microbiología , Tuberculosis/prevención & control , Tuberculosis/transmisiónRESUMEN
The efficacy of 14 disinfectants against Listeria innocua and two strains of Listeria monocytogenes in the presence of organic matter was studied. Quantitative efficacy tests were used. Many of the disinfectants tested were not as effective on Listeria spp. when the test organisms were dried onto the surface of steel disks (carrier tests) as they were when the organisms were placed in suspension (suspension test). The presence of whole serum and milk (2% fat) further reduced the disinfectant capacities of most of the formulations studied. Only three disinfectants (povidone-iodine, chlorhexidine gluconate, and glutaraldehyde) were effective in the carrier test in the presence of serum; however, all three were ineffective when challenged with milk (2% fat). Only one solution, sodium dichloroisocyanurate, was effective in the presence of milk. All but four formulations (chloramine-T, phosphoric acid, an iodophor, and formaldehyde) were effective in the suspension tests, regardless of the organic load. L. monocytogenes was observed to be slightly more resistant to disinfection than L. innocua was. There was no difference in disinfectant susceptibility between the two strains of L. monocytogenes. These findings emphasize the need for caution in selecting an appropriate disinfectant for use on contaminated surfaces, particularly in the presence of organic material.