RESUMEN
Ice worlds are at the forefront of astrobiological interest because of the evidence of subsurface oceans. Enceladus in particular is unique among the icy moons because there are known vent systems that are likely connected to a subsurface ocean, through which the ocean water is ejected to space. An existing study has shown that sending small robots into the vents and directly sampling the ocean water is likely possible. To enable such a mission, NASA's Jet Propulsion Laboratory is developing a snake-like robot called Exobiology Extant Life Surveyor (EELS) that can navigate Enceladus' extreme surface and descend an erupting vent to capture unaltered liquid samples and potentially reach the ocean. However, navigating to and through Enceladus' environment is challenging: Because of the limitations of existing orbital reconnaissance, there is substantial uncertainty with respect to its geometry and the physical properties of the surface/vents; communication is limited, which requires highly autonomous robots to execute the mission with limited human supervision. Here, we provide an overview of the EELS project and its development effort to create a risk-aware autonomous robot to navigate these extreme ice terrains/environments. We describe the robot's architecture and the technical challenges to navigate and sense the icy environment safely and effectively. We focus on the challenges related to surface mobility, task and motion planning under uncertainty, and risk quantification. We provide initial results on mobility and risk-aware task and motion planning from field tests and simulated scenarios.
RESUMEN
This study investigates the feasibility of microalgae cultivation with the effluent (permeate) of a decentralized anaerobic membrane bioreactor (AnMBR) treating high strength domestic wastewater. Two experiments, consisting of three and two successive batch experiments with incubation times varying between 5 and 9 days, were conducted. Nutrient removal and growth of the microalgae species Acutodesmus obliquus were studied for the following culture media: (A) permeate, (B) permeate enriched with iron (Fe), magnesium (Mg), manganese (Mn), sulfur (S) and the chelating agent EDTA, (C) commercial fertilizer as control culture. Initial nutrient concentrations in the culture media ranged from 9.3 to 16.6 mg·L-1 total phosphorus (TP) and from 85.1 to 126.2 mg·L-1 total nitrogen (TN). TP reached an average removal of 97%, 98% and 99% in (A), (B) and (C) respectively. An average TN removal of 94% and 96% was achieved in (B) and (C). Starting from the third batch of the first experiment and the second batch of the second experiment, the culture with permeate (A) showed a decrease in TN removal. Further batch experiments showed the need to add iron to ensure an optimal TN removal from the permeate.
Asunto(s)
Microalgas/metabolismo , Nitrógeno/metabolismo , Fósforo/metabolismo , Eliminación de Residuos Líquidos/métodos , Aguas Residuales/química , Contaminantes del Agua/metabolismo , Reactores BiológicosRESUMEN
Understanding the reproductive and fertility concerns of teenagers and young adults with cancer (TYA) is one aspect of comprehensive age appropriate care. However, limited options for fertility preservation, coupled with vague policy recommendations, give rise to variations in information-sharing between health care professionals and TYAs, particularly as it involves sensitive discussions regarding the short- and long-term effects of cancer and treatments on fertility and reproduction. This paper presents findings from a wider evaluation at a specialist unit for TYAs with cancer. Forty people participated in semi-structured interviews, including 20 young people, parents and partners. Young people were between 2 months and 4 years from finishing treatment. Most young people received mixed levels of information on fertility and counselling before treatment. Diagnosis in the early teens meant how, and from whom, young people received information varied. Young women tended to receive incomplete information. The majority of young people were unaware of their fertility status after treatment had finished. Findings point to the inherent challenges that exist in ensuring young people aged between 13 and 25 years receive comprehensive information on their fertility and potential risk, as well as advice on how to determine their fertility status after treatment has finished.
Asunto(s)
Antineoplásicos/efectos adversos , Revelación , Necesidades y Demandas de Servicios de Salud , Infertilidad/etiología , Neoplasias/terapia , Padres , Educación del Paciente como Asunto , Radioterapia/efectos adversos , Adolescente , Adulto , Niño , Femenino , Preservación de la Fertilidad , Humanos , Masculino , Investigación Cualitativa , Adulto JovenRESUMEN
In vivo imaging of cells tagged with light-emitting probes, such as firefly luciferase or fluorescent proteins, is a powerful technology that enables a wide range of biological studies in small research animals. Reporters with emission in the red to infrared (>600 nm) are preferred due to the low absorption in tissue at these wavelengths. Modeling of photon diffusion through tissue indicates that bioluminescent cell counts as low as a few hundred can be detected subcutaneously, while approximately 10(6) cells are required to detect signals at approximately 2 cm depth in tissue. Signal-to-noise estimates show that cooled back-thinned integrating charge coupled devices (CCDs) are preferred to image-intensified CCDs for this application, mainly due to their high quantum efficiency (approximately 85%) at wavelengths >600 nm where tissue absorption is low. Instrumentation for in vivo imaging developed at Xenogen is described and several examples of images of mice with bioluminescent cells are presented.
Asunto(s)
Colorantes Fluorescentes , Luciferasas , Proteínas Luminiscentes , Animales , Diagnóstico por Imagen/métodos , Proteínas Fluorescentes Verdes , Mediciones Luminiscentes , Ratones , Ratones Endogámicos BALB C , Neoplasias/diagnóstico , Neumonía/diagnóstico , Proteína Fluorescente RojaRESUMEN
The hepatitis C virus (HCV) NS3 protein contains an amino terminal protease (NS3 aa. 1-180) and a carboxyl terminal RNA helicase (NS3 aa. 181-631). NS3 functions as a heterodimer of NS3 and NS4A (NS3/4A). NS3 helicase, a nucleic acid stimulated ATPase, can unwind RNA, DNA, and RNA:DNA duplexes, provided that at least one strand of the duplex contains a single-stranded 3' overhang (this strand of the duplex is referred to as the 3' strand). We have used 2'-O-methyl RNA (MeRNA) substrates to study the mechanism of NS3 helicase activity and to probe the relationship between its helicase and RNA-stimulated ATPase activities. NS3/4A did not unwind double-stranded (ds) MeRNA. NS3/4A unwinds hybrid RNA:MeRNA duplex containing MeRNA as the 5' strand but not hybrid duplex containing MeRNA as the 3' strand. The helicase activity of NS3/4A was 50% inhibited by 40 nM single-stranded (ss) RNA but only 35% inhibited by 320 nM ss MeRNA. Double-stranded RNA was 17 times as effective as double-stranded MeRNA in inhibiting NS3/4A helicase activity, while the apparent affinity of NS3/4A for ds MeRNA differed from ds RNA by only 2.4-fold. However ss MeRNA stimulated NS3/4A ATPase activity similar to ss RNA. These results indicate that the helicase mechanism involves 3' to 5' procession of the NS3 helicase along the 3' strand and only weak association of the enzyme with the displaced 5' strand. Further, our findings show that maximum stimulation of NS3 ATPase activity by ss nucleic acid is not directly related to procession of the helicase along the 3' strand.
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Adenosina Trifosfatasas/metabolismo , Hepacivirus/enzimología , ARN Helicasas/metabolismo , ARN Viral/fisiología , Proteínas no Estructurales Virales/metabolismo , Regiones no Traducidas 3'/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Unión Proteica , ARN Helicasas/antagonistas & inhibidores , ARN Bicatenario/metabolismo , ARN Bicatenario/farmacología , Especificidad por Sustrato , Nucleótidos de Uracilo/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
Various classes of nucleotidyl polymerases with different transcriptional roles contain a conserved core structure. Less is known, however, about the distinguishing features of these enzymes, particularly those of the RNA-dependent RNA polymerase class. The 1. 9 A resolution crystal structure of hepatitis C virus (HCV) nonstructural protein 5B (NS5B) presented here provides the first complete and detailed view of an RNA-dependent RNA polymerase. While canonical polymerase features exist in the structure, NS5B adopts a unique shape due to extensive interactions between the fingers and thumb polymerase subdomains that serve to encircle the enzyme active site. Several insertions in the fingers subdomain account for intersubdomain linkages that include two extended loops and a pair of antiparallel alpha-helices. The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway. This NS5B template:primer model also allows identification of a new structural motif involved in stabilizing the nascent base pair.
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Hepacivirus/enzimología , ARN Polimerasa Dependiente del ARN/química , Proteínas no Estructurales Virales/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Apoenzimas/química , Emparejamiento Base , Sitios de Unión , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN/química , ARN/genética , ARN/metabolismo , Proteínas Recombinantes de Fusión/química , Moldes GenéticosRESUMEN
Helicases are nucleotide triphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication. The 2.1 A resolution structure of the HCV helicase from the positive-stranded RNA hepatitis C virus reveals a molecule with distinct NTPase and RNA binding domains. The structure supports a mechanism of helicase activity involving initial recognition of the requisite 3' single-stranded region on the nucleic acid substrate by a conserved arginine-rich sequence on the RNA binding domain. Comparison of crystallographically independent molecules shows that rotation of the RNA binding domain involves conformational changes within a conserved TATPP sequence and untwisting of an extended antiparallel beta-sheet. Location of the TATPP sequence at the end of an NTPase domain beta-strand structurally homologous to the 'switch region' of many NTP-dependent enzymes offers the possibility that domain rotation is coupled to NTP hydrolysis in the helicase catalytic cycle.
Asunto(s)
ARN Nucleotidiltransferasas/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Secuencia Conservada , Cristalografía por Rayos X , ADN Helicasas/química , ADN Helicasas/metabolismo , Hidrólisis , Modelos Moleculares , Conformación Proteica , ARN Helicasas , ARN Nucleotidiltransferasas/química , ARN Viral/metabolismo , Especificidad por SustratoRESUMEN
Clot formation is a limiting factor in the use of biomaterials. We investigated the effect of surface hydrophobicity on haemostatic activation in vitro, using five polyetherurethanes of varying surface hydrophobicity (C94, C74, C54 and C34), C94 the most, and C34, the least hydrophobic, and compared them with a commercial standard pellethane. Sterilised sacks were filled with heparinised blood, rotated at 37 degrees C for 24 h and sequential samples collected into 0.103 M sodium citrate. Thrombin generation measured by thrombin-antithrombin III complexes showed a difference between the polymers at 3 h through to 6 h (P < 0.05), C94 showing the least activation and C34 the most. Factor XIIa and D-dimer levels increased between 12 (P < 0.05) and 24 h (P < 0.01) for all polyetherurethanes. The ratio of soft:hard segments (which determine hydrophobicity) of the polyetherurethanes showed a direct relationship with the degree of activation of coagulation and fibrinolysis. There was no significant increase in monocyte tissue factor expression at 5 and 105 min. Platelet function as measured by whole blood platelet aggregation showed a reduction with pellethane and C94 after 1 h using collagen, with no changes for C34, Altering surface hydrophobicity has diverse effects on haemostatic pathways, with the most hydrophobic surfaces causing least activation of coagulation but most activation of platelets.
Asunto(s)
Materiales Biocompatibles , Coagulación Sanguínea , Activación Plaquetaria , Polímeros , Poliuretanos , Adulto , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Fibrinólisis , Citometría de Flujo , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Masculino , Propiedades de SuperficieRESUMEN
Human platelet-derived growth factor (PDGF) was expressed in Escherichia coli from a high-level cytoplasmic expression vector. A cDNA fragment encoding the mature form of the human PDGF B chain (hPDGF-B) was cloned into a plasmid under transcriptional control of the inducible E. coli Tac promoter. Expression of hPDGF-B from the final construct, pTacBIq, is regulated by the lactose repressor (LacIq). Upon induction, a polypeptide of approximately 14 kDa that had the same molecular mass and immunoreactivity as authentic hPDGF-B was produced. The production of recombinant hPDGF-B was significantly increased in an E. coli strain (CAG629) defective in expression of the lon protease. Expression of hPDGF-B in the CAG629 strain accounted for approximately 1% of total cell protein. In this system, hPDGF-B is expressed as an insoluble, intracellular protein and can readily be obtained in a partially purified form after differential centrifugation. Amino acid sequence determination of the purified protein has verified that the amino-terminal portion of the recombinant PDGF is correct. After renaturation into dimers, the purified recombinant hPDGF is fully functional in assays for receptor binding and mitogenesis.
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Factor de Crecimiento Derivado de Plaquetas/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Bases , ADN/genética , Escherichia coli , Fibroblastos/efectos de los fármacos , Vectores Genéticos , Humanos , Mitosis/efectos de los fármacos , Datos de Secuencia Molecular , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesisAsunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Músculo Liso Vascular/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Encéfalo/enzimología , Bovinos , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Pulmón/enzimología , Miocardio/enzimología , Porcinos , Distribución TisularRESUMEN
The mechanisms of allosteric regulation of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum by ATP have been compared. Although both enzymes showed stimulation of ATPase activity by ATP, the cardiac enzyme did not show the plateau in ATPase activity at 10-100 microM ATP seen with the skeletal enzyme. Likewise the phosphoenzyme (EP) levels did not plateau with the cardiac enzyme as they did with the skeletal enzyme. The apparent negative cooperatively which was seen in the kinetics of ATP hydrolysis at low ATP concentrations was not due to negative cooperatively in substrate binding to either enzyme. The cardiac enzyme did show, however, much higher affinity for the ATP analog, AMPPCP, which helps explain how AMPPCP blocks ATPase activity in the cardiac enzyme and stimulates ATPase activity in the skeletal enzyme. Fluorescein isothiocyanate was used to determine if allosteric regulation takes place through site-site interactions in oligomers. The 1 to 1 ratio between AMPPCP binding sites and FITC binding sites eliminated allosteric regulation by effector sites in both enzymes. The allosteric mechanism which remained was one in which the active-site becomes an effector-site by the early departure of ADP in the reaction mechanism. The step stimulated by the binding of ATP at the active-site turned effector-site was a nonphosphorylated form of the enzyme in cardiac sarcoplasmic reticulum and a phosphorylated form in skeletal sarcoplasmic reticulum.
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ATPasas Transportadoras de Calcio/fisiología , Miocardio/enzimología , Retículo Sarcoplasmático/enzimología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Animales , Unión Competitiva , Perros , Fluoresceína-5-Isotiocianato , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Cinética , Músculos/enzimología , Tiocianatos/metabolismoRESUMEN
Most patients find difficulties coming to terms with being told that they have an incurable disease, even if it is not life-threatening. Doctors rarely have the time or the experience to answer all the questions that may arise in the patient's mind. Self-help groups can provide a range of services to help the patient. These services include initial support at the time of diagnosis, continued support, including family help, provision of information, social activities and support for research into the treatment and prevention of the disease in question.
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Enfermedades Inflamatorias del Intestino/psicología , Grupos de Autoayuda , Humanos , Enfermedades Inflamatorias del Intestino/rehabilitaciónRESUMEN
The reaction of a photoaffinity analog, 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate (BZ2ATP) with gizzard myosin is described. The incorporation of BZ2ATP into myosin is both specific and stoichiometric. About 2.2 mol BZ2ATP are incorporated/mol myosin resulting in the significant loss of EDTA(K+) ATPase activity. The Mg2+ and actin-activated ATPase activities are slightly inhibited. Addition of ATP (millimolar) during the photolysis reaction significantly inhibits incorporation of BZ2ATP into myosin. Our data show that the label is mainly incorporated into the heavy chain of myosin with some label in the 20-kDa light chain. Limited proteolysis of radioactively labeled myosin subfragment 1 with trypsin reveals the presence of radioactivity mainly in the 50-kDa fragment and some in the 29-kDa and 25-kDa fragments. However, our data on the ATP-sensitive incorporation of BZ2ATP into the tryptic fragments suggest that the 50-kDa peptide, not the 29-kDa peptide, may be located at or around the active site.
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Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Marcadores de Afinidad , Molleja de las Aves/metabolismo , Miosinas/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión , Activación Enzimática/efectos de los fármacos , Molleja de las Aves/enzimología , Hidrólisis , Fosforilación , Fotoquímica , Tripsina , PavosRESUMEN
The nucleotide binding domain of the active site of the Ca,Mg-ATPase of cardiac sarcoplasmic reticulum (SR) has been isolated using fluorescein isothiocyanate (FITC) as an active site label and sequenced. After removal of non-specifically incorporated FITC with hydroxylamine, the amount of label incorporated was stoichiometric with residual ATPase activity, demonstrating that the label was incorporated uniquely at the active site. The SR was succinylated before digestion by trypsin in order to obtain a peptide of sufficient length to determine if the cardiac SR ATPase is a candidate for the unidentified cDNA clone recently sequenced by MacLennan et al. (Nature 316: 696-700, 1985). The sequence of the labeled SR peptide, obtained by affinity chromatography on a FITC antibody column, was T S M S K M F K G P E V I D R. This sequence was identical with that predicted by the unidentified clone and is significantly different from the sequence reported by Kirley et al. (Biochem. Biophys. Res. Commun. 130: 732-738, 1985) for a FITC labeled peptide isolated from cardiac SR.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+) , ATPasas Transportadoras de Calcio , Miocardio/enzimología , Retículo Sarcoplasmático/enzimología , Animales , Sitios de Unión , Perros , Fluoresceína-5-Isotiocianato , Fluoresceínas , Nucleótidos/metabolismo , Fragmentos de Péptidos/análisis , TiocianatosRESUMEN
Four mechanisms for the allosteric regulation of the calcium and magnesium ion activated adenosinetriphosphatase (Ca,Mg-ATPase) of sarcoplasmic reticulum were examined. Negative cooperativity in substrate binding was not supported by 3H-labeled 5'-adenylyl methylenediphosphate (AMPPCP) binding, which was best fit by a single class of sites. Although calcium had no effect on the absence of cooperativity, it did increase the affinity of the enzyme for AMPPCP. Allosteric regulation via an effector site for AMPPCP or ATP on the same ATPase chain was eliminated by the stoichiometry of ATP and AMPPCP binding, 1 mol of site per mole of enzyme. The possibility that AMPPCP acts at an effector site was eliminated by showing that it competitively inhibits the rate of phosphoenzyme formation. Allosteric regulation of kinetics via site-site interaction in an oligomer was eliminated by showing that the inhibition of ATPase activity by fluorescein isothiocyanate is linearly dependent upon its incorporation into the sarcoplasmic reticulum. The fourth mechanism considered was stimulation of ATPase activity by the binding of ATP or AMPPCP at the active site after departure of ADP but before the departure of inorganic phosphate. This hypothesis was supported by site stoichiometry and by the observation that AMPPCP or ATP stimulates v/EP, the rate of ATP hydrolysis for a given level of phosphoenzyme. Computer simulation of this branched monomeric model could duplicate all experimental observations made with AMPPCP and ATP as allosteric regulators. The condition that the affinity of ATP binding to the enzyme be reduced when it is phosphorylated, which is required by the computer model, was confirmed experimentally.
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Adenosina Trifosfato/análogos & derivados , ATPasa de Ca(2+) y Mg(2+)/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Retículo Sarcoplasmático/enzimología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Regulación Alostérica , Animales , Fluoresceína-5-Isotiocianato , Fluoresceínas/farmacología , Colorantes Fluorescentes/farmacología , Cinética , Matemática , Ratones , Músculos/enzimología , Unión Proteica , Tiocianatos/farmacologíaRESUMEN
Two main candidates have been proposed for the role of relaxant neurotransmitter in the intestine: (a) the purine nucleotide, 5'-adenosine triphosphate (ATP) and (b) the neuropeptide, vasoactive intestinal peptide (VIP). The candidacy of VIP is favored by its precise location in nerve fibers that innervate circular smooth muscle and tenia coli. We have used a photoaffinity analog of ATP, 3'-O-(4-Benzoyl)benzoyl ATP, that binds irreversibly to ATP receptors and inactivates them in the presence of light, and a specific VIP antiserum to examine the claims of VIP and ATP as relaxant neurotransmitters in tenia coli of the guinea pig. Both VIP and ATP caused dose-dependent, tetrodotoxin-insensitive relaxation of tenia coli. The effect of ATP was equipotent to that of its stable isostere alpha, beta-methylene ATP and resistant to degradation by adenosine deaminase, indicating interaction of ATP with purinergic-P2 receptors. Photoactivated 3'-O-(4-Benzoyl) benzoyl adenosine triphosphate selectively inhibited relaxation induced by ATP but had no effect on relaxation induced by VIP or by field (i.e., neural) stimulation. Vasoactive intestinal peptide antiserum (final dilution 1:60), on the other hand, inhibited relaxation caused by VIP and by field stimulation but had no effect on relaxation caused by ATP. Neither normal rabbit serum nor preneutralized VIP antiserum had any effect on relaxation induced by ATP, VIP, or field stimulation. Inhibition of neurally induced relaxation by VIP antiserum ranged from 52% +/- 7% (p less than 0.01) at the lowest frequency of stimulation to 15% +/- 4% (p less than 0.01) at the highest frequency, consistent with competitive interaction between antiserum and neurally released VIP. Near-maximal field stimulation at 1 Hz caused an eightfold (800% +/- 49%, p less than 0.01) increase in VIP release into the bathing medium. The results favor VIP (and probably peptide histidine isoleucine, a relaxant homologue known to be cosynthesized with VIP) as the main neural mediator of relaxation in tenia coli.
Asunto(s)
Intestino Delgado/fisiología , Neurotransmisores , Nucleótidos de Purina/fisiología , Péptido Intestinal Vasoactivo/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/fisiología , Animales , Relación Dosis-Respuesta a Droga , Cobayas , Relajación Muscular , Neurotransmisores/fisiología , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Two main candidates, adenosine 5'-triphosphate (ATP) and vasoactive intestinal peptide (VIP), have been proposed as inhibitory transmitters at neuromuscular junctions in the gut. We have used a photoaffinity analogue of ATP, 3'-O-(4-benzoyl)benzoyl ATP or BzATP, that binds covalently to ATP receptors and inactivates them in the presence of light and a specific high-affinity VIP antiserum in order to examine the contributions of ATP and VIP to neurally induced relaxation in circular smooth muscle of the gastric fundus of the guinea pig. VIP and ATP caused dose-dependent relaxation; the effect of ATP was equal to that of its stable isostere, alpha, beta-methylene ATP, and was resistant to degradation by adenosine deaminase, indicating interaction of ATP with purinergic P2-receptors. Relaxation induced by VIP was selectively inhibited by VIP antiserum (final dilution 1:120), while that induced by ATP was selectively inhibited by photoactivated BzATP. Relaxation induced by electrical field (i.e., neural) stimulation was inhibited by VIP antiserum only; photoactivated BzATP had no effect. Inhibition of neurally induced relaxation ranged from 86% (P less than 0.01) at the lowest frequencies to 34% (P less than 0.01) at the highest frequencies. Maximal field stimulation caused an 11-fold increase in VIP release from intramural neurons. The results strongly favor VIP as the neural mediator of gastric relaxation.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso/fisiología , Estómago/fisiología , Péptido Intestinal Vasoactivo/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Marcadores de Afinidad/farmacología , Animales , Complejo Antígeno-Anticuerpo , Estimulación Eléctrica , Cobayas , Sueros Inmunes , Técnicas In Vitro , Cinética , Músculo Liso/inervación , Estómago/inervaciónRESUMEN
The ATP analog, 3'-O-(4-benzoyl)benzoic adenosine triphosphate (BzATP) was an effective photoaffinity analog of ATP for labeling the sarcoplasmic reticulum Ca,Mg-ATPase. In contrast to 8-azido-ATP and arylazido-ATP, BzATP produced significant inhibition of ATPase activity. The incorporation of [alpha-32P]BzATP was restricted to the Ca,Mg-ATPase at 250 microM BzATP and the incorporation was antagonized by ATP. We conclude that the photodependent incorporation of BzATP is restricted to the active site because the inhibition of ATPase activity was stoichiometrically related to analog incorporation. Resolution of the tryptic fragments produced from photolabeled Ca,Mg-ATPase demonstrated that fragments A and B were labeled in a ratio of about 2:1. Both fragments must, therefore, contain portions of the active site.