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NMR spectroscopy was used to measure the rates of the first and second substitution reactions between iodoalkane (R = Me, 1-butyl) and DABCO in methanol, acetonitrile and DMSO. Most of the reactions were recorded at three different temperatures, which permitted calculation of the activation parameters from Eyring and Arrhenius plots. Additionally, the reaction rate and heat of reaction for 1-iodobutane + DABCO in acetonitrile and DMSO were also measured using calorimetry. To help interpret experimental results, ab initio calculations were performed on the reactant, product, and transition state entities to understand structures, reaction enthalpies and activation parameters. Markov chain Monte Carlo statistical sampling was used to determine a distribution of kinetic rates with respect to the uncertainties in measured concentrations and correlations between parameters imposed by a kinetics model. The reactions with 1-iodobutane are found to be slower in all cases compared to reactions under similar conditions for iodomethane. This is due to steric crowding around the reaction centre for the larger butyl group compared to methyl which results in a larger activation energy for the reaction.
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Numerous algorithms have been developed to determine the source characteristics for an atmospheric radionuclide release, e.g., (Bieringer et al., 2017). This study compares three models that have been applied to the data collected by the International Monitoring System operated by the Comprehensive Nuclear-Test-Ban Treaty Organization Preparatory Commission to estimate source event parameters. Each model uses a different approach to estimate the parameters. A deterministic model uses a possible source region (PSR) approach (Ringbom et al., 2014) that is based on the correlation between predicted and measured sample values. A model (now called BAYEST) developed at Pacific Northwest National Laboratory uses a Bayesian formulation (Eslinger et al., 2019, 2020; Eslinger and Schrom, 2016). The FREAR model uses a different Bayesian formulation (De Meutter and Hoffman, 2020; De Meutter et al., 2021a, 2021b). The performance of the three source-location models is evaluated with 100 synthetic release cases for the single xenon isotope, 133Xe. The release cases resulted in detections in a fictitious network with 120 noble gas samplers. All three source-location models use the same sampling data. The two Bayesian models yield more accurate location estimates than the deterministic PSR model, with FREAR having slightly better location performance than BAYEST. Samplers with collection periods of 3, 6, 8, 12, and 24-h were used. Results from BAYEST show that location accuracy improves with each reduction in sample collection length. The BAYEST model is slightly better for estimating the start time of the release. The PSR model has about the same spread in start times as the FREAR model, but the PSR results have a better average start time. The Bayesian source-location algorithms give more accurate results than the PSR approach, and provide release magnitude estimates, while the base PSR model does not estimate the release magnitude. This investigation demonstrates that a reasonably dense sampling grid will sometimes yield poor location and time estimates regardless of the model. The poor estimates generally coincide with cases where there is a much larger distance between the release point and the first detecting sampler than the average sampler spacing.
Assuntos
Poluentes Radioativos do Ar , Monitoramento de Radiação , Poluentes Radioativos do Ar/análise , Monitoramento de Radiação/métodos , Teorema de Bayes , Radioisótopos de Xenônio/análise , AlgoritmosRESUMO
Design of an efficient monitoring network requires information on the type and size of releases to be detected, the accuracy and reliability of the measuring equipment, and the desired network performance. This work provides a scientific basis for optimizing or minimizing networks of 133Xe samplers to achieve a desired performance level for different levels of release. The approach of this work varies the density of sampling locations to find optimal location subsets, and to explore the properties of variations of those subsets - how crucial is a specific subset; are substitutions problematic? The choice of possible station locations is arbitrary but constrained to some extent by the location of islands, land masses, difficult topography (mountains, etc.) and the places where infrastructure exists to run and support a sampler. Performance is evaluated using hypothetical releases and atmospheric transport models that cover an entire year. Three network performance metrics are calculated: the probability of detecting the releases, the expected number of stations to detect the releases, and the expected number of samples that detect the releases. The quantitative measures support picking optimal or near-optimal network of a specific station density. If a detection probability of 90% (high) was desired for a design basis release of 1014 Bq (1% of 133Xe production from a 1 kt explosion), then a very high density would be required using today's sampling and measurement technology. If the design basis release were raised to 1015 Bq, then the station density could be lowered by a factor of 3. To achieve a location goal of three station detections on average, posited here for the first time, would also require very high station density for a release of 1014 Bq.
Assuntos
Monitoramento de Radiação , Explosões , Reprodutibilidade dos TestesRESUMO
We present a dataset of structural relaxations of bulk ternary transition metal dichalcogenides (TMDs) computed via plane-wave density functional theory (DFT). We examined combinations of up to two chalcogenides with seven transition metals from groups 4-6 in octahedral (1T) or trigonal prismatic (2H) coordination. The full dataset consists of 672 unique stoichiometries, with a total of 50,337 individual configurations generated during structural relaxation. Our motivations for building this dataset are (1) to develop a training set for the generation of machine and deep learning models and (2) to obtain structural minima over a range of stoichiometries to support future electronic analyses. We provide the dataset as individual VASP xml files as well as all configurations encountered during relaxations collated into an ASE database with the corresponding total energy and atomic forces. In this report, we discuss the dataset in more detail and highlight interesting structural and electronic features of the relaxed structures.
RESUMO
Optimization of thermal transport across the interface of two different materials is critical to micro-/nanoscale electronic, photonic, and phononic devices. Although several examples of compositional intermixing at the interfaces having a positive effect on interfacial thermal conductance (ITC) have been reported, an optimum arrangement has not yet been determined because of the large number of potential atomic configurations and the significant computational cost of evaluation. On the other hand, computation-driven materials design efforts are rising in popularity and importance. Yet, the scalability and transferability of machine learning models remain as challenges in creating a complete pipeline for the simulation and analysis of large molecular systems. In this work we present a scalable Bayesian optimization framework, which leverages dynamic spawning of jobs through the Message Passing Interface (MPI) to run multiple parallel molecular dynamics simulations within a parent MPI job to optimize heat transfer at the silicon and aluminum (Si/Al) interface. We found a maximum of 50% increase in the ITC when introducing a two-layer intermixed region that consists of a higher percentage of Si. Because of the random nature of the intermixing, the magnitude of increase in the ITC varies. We observed that both homogeneity/heterogeneity of the intermixing and the intrinsic stochastic nature of molecular dynamics simulations account for the variance in ITC.
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Predicting source or background radionuclide emissions is limited by the effort needed to run gas/aerosol atmospheric transport models (ATMs). A high-performance surrogate model is developed for the HYSPLIT4 (NOAA) ATM to accelerate transport simulation through model reduction, code optimization, and improved scaling on high performance computing systems. The surrogate model parameters are a grid of short-duration transport simulations stored offline. The surrogate model then predicts the path of a plume of radionuclide particles emitted from a source, or the field of sources which may have contributed to a detected signal, more efficiently than direct simulation by HYSPLIT4. Termed the Atmospheric Transport Model Surrogate (ATaMS), this suite of capabilities forms a basis to accelerate workflows for probabilistic source prediction and estimation of the radionuclide atmospheric background.
Assuntos
Monitoramento de Radiação , Radioisótopos/isolamento & purificação , Aerossóis , Simulação por Computador , Estudos RetrospectivosRESUMO
HIV/AIDS is having a devastating impact on South Africa and particularly on poor communities. Empowerment of communities has been identified as an important step towards mitigating the consequences and helping communities to overcome the challenges presented. Participatory Action Research (PAR) has been identified as a useful methodology for the purpose of facilitating empowerment. This study explores the challenges involved in implementing PAR in the context of HIV/AIDS and poverty. In this article, the author describes a PAR project that took place in 2003/ 2004 with a group of five Xhosa speaking people living with HIV/AIDS in Masiphumelele, Cape Town. The aims of the study were to: 1. Create an opportunity for the participants to engage in a participatory process aimed at self-awareness and empowerment. 2. To record and analyse this process with the intention of producing insight into the use of PAR in the context of poverty and HIV/AIDS and to identify the challenges involved. The findings of this study highlight some important insights into the process of engaging people in the PAR process and the experiences of HIV positive people living in the context of poverty. The study explores the challenges involved in the process of empowerment and examines the process of "transferring" power and control from the researcher to the participants. Challenges were uncovered both from the point of view of the researcher who had to "let go of control" and participants who had to take on control. Participants struggled with issues of low self-efficacy and learned helplessness. Fluctuations in health also contributed towards alternating periods of hope and despair and these problems had an impact on their motivation to participate in the study. Lack of motivation to participate is a challenge highlighted in the literature and explored in this study. Participation is necessary for a study of this nature to be of benefit to the community, but unfortunately those most in need were found to be least likely to participate. The study also critically examines the research process that was conducted and highlights the positive and negative contribution of the process towards empowerment. Certain aspects of the research process, including the contracting process, were identified as being problematic as they emphasize the power and control of the researcher rather than the participants. Recommendations for future research include: Promoting participation among the disempowered; the Contracting process and Power relations in PAR.
Assuntos
Infecções por HIV , Pobreza/etnologia , Pobreza/psicologia , Poder Psicológico , Enfermagem Transcultural/métodos , Adulto , Feminino , Infecções por HIV/etnologia , Infecções por HIV/enfermagem , Infecções por HIV/psicologia , Humanos , Masculino , Pesquisa Metodológica em Enfermagem , África do Sul , Adulto JovemRESUMO
Algorithms that estimate the location and magnitude of an atmospheric release using remotely sampled air concentrations typically involve a single chemical or radioactive isotope. A new Bayesian algorithm is presented that makes discrimination between possible types of releases (e.g., nuclear explosion, nuclear power plant, or medical isotope production facility) an integral part of the analysis for samples that contain multiple isotopes. Algorithm performance is demonstrated using synthetic data and correctly discriminated between most release-type hypotheses, with higher accuracy when data are available on three or more isotopes.
Assuntos
Poluentes Radioativos do Ar/análise , Monitoramento de Radiação/métodos , Liberação Nociva de Radioativos/classificação , Radioisótopos de Xenônio/análise , Algoritmos , Teorema de Bayes , Explosões , Resíduos de Serviços de Saúde , Centrais Nucleares , Monitoramento de Radiação/instrumentaçãoRESUMO
Vasopressin and its analogue 1-deamino-8-D-arginine vasopressin (DDAVP) are known to raise plasma von Willebrand factor (vWF) levels. DDAVP is used as a hemostatic agent for the treatment of von Willebrand's disease. However, its cellular mechanisms of action have not been elucidated. DDAVP, a specific agonist for the vasopressin V2 receptor (V2R), exerts its antidiuretic effect via a rise in cAMP in kidney collecting ducts. We tested the hypothesis that DDAVP induces vWF secretion by binding to V2R and activating cAMP-mediated signaling in endothelial cells. vWF secretion from human umbilical vein endothelial cells (HUVECs) can be mediated by cAMP, but DDAVP is ineffective, presumably due to the absence of V2R. We report that DDAVP stimulates vWF secretion in a cAMP-dependent manner in HUVECs after transfection of the V2R. In addition, vasopressin and DDAVP induce vWF secretion in human lung microvascular endothelial cells (HMVEC-L). These cells (but not HUVECs) express endogenous V2R, as shown by RT-PCR. Vasopressin-induced vWF secretion is mimicked by DDAVP and inhibited by the selective V2R antagonist SR121463B. It is mediated by cAMP, since it is inhibited by the protein kinase A inhibitor Rp-8CPT-cAMPS. These results indicate that vasopressin induces cAMP-mediated vWF secretion by a direct effect on endothelial cells. They also demonstrate functional expression of V2R in endothelial cells, and provide a cellular mechanism for the hemostatic effects of DDAVP.
Assuntos
AMP Cíclico/fisiologia , Desamino Arginina Vasopressina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Receptores de Vasopressinas/fisiologia , Fator de von Willebrand/metabolismo , Arginina Vasopressina/farmacologia , Células Cultivadas , Endotélio Vascular/metabolismo , Humanos , Pulmão/metabolismo , RNA Mensageiro/análise , Receptores de Vasopressinas/análise , Receptores de Vasopressinas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In X-linked nephrogenic diabetes insipidus (NDI) the urine of male patients is not concentrated after the administration of the antidiuretic hormone arginine-vasopressin. This disease is due to mutations in the V2 receptor gene that maps to chromosome region Xq28. In 1969, Bode and Crawford suggested that most NDI patients in North America shared common ancestors of Ulster Scot immigrants who arrived in Halifax in 1761 on the ship Hopewell. A link between this family and a large Utah kindred was also suggested. DNA was obtained from 17 affected male patients from the "Hopewell" kindred and from four additional families from Nova Scotia and New Brunswick who shared the same Xq28 NDI haplotype. The Utah kindred and two families (Q2, Q3) from Quebec were also studied. The "Hopewell" mutation, W71X, is a single base substitution (G-->A) that changes codon 71 from TGG (tryptophan) to TGA (stop). The W71X mutation was found in affected members of the Hopewell and of the four satellite families. The W71X mutation is the cause of X-linked NDI for the largest number of related male patients living in North America. Other families (Utah, Q2 and Q3) that are historically and ethnically unrelated bear other mutations in the V2 receptor gene.
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Diabetes Insípido/genética , Receptores de Vasopressinas/genética , Sequência de Aminoácidos , Sequência de Bases , Feminino , Genes , Haplótipos , Humanos , Masculino , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Linhagem , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Cromossomo XRESUMO
The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Central to its antidiuretic action in mammals is the exocytotic insertion of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the apical membrane of principal cells, an event initiated by an increase in cAMP and activation of protein kinase A. Water is then reabsorbed from the hypotonic urine of the collecting duct. The water channels aquaporin-3 (AQP3) and aquaporin-4 (AQP4), which are constitutively present in the basolateral membrane, allow the exit of water from the cell into the hypertonic interstitium. Withdrawal of the hormone leads to endocytotic retrieval of AQP2 from the cell membrane. The hormone-induced rapid redistribution between the interior of the cell and the cell membrane establishes the basis for the short term regulation of water permeability. In addition water channels (AQP2 and 3) of principal cells are regulated at the level of expression (long term regulation). This review summarizes the current knowledge on the molecular mechanisms underlying the short and long term regulation of water channels in principal cells. In the first part special emphasis is placed on the proteins involved in short term regulation of AQP2 (SNARE proteins, Rab proteins, cytoskeletal proteins, G proteins, protein kinase A anchoring proteins and endocytotic proteins). In the second part, physiological and pathophysiological stimuli determining the long term regulation are discussed.
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Aquaporinas/fisiologia , Arginina Vasopressina/fisiologia , Túbulos Renais Coletores/fisiologia , Animais , HumanosRESUMO
The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels.
Assuntos
Proteínas de Ligação ao GTP/análise , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo , Membrana Celular/análise , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Ligação ao GTP/imunologia , Soros Imunes , Immunoblotting , Substâncias Macromoleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Gravidez , RatosRESUMO
In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H(+)-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H(+)-production after stimulation with either histamine, forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE2 (10(-9)-10(-7) M) or somatostatin (10(-9)-10(-6) M). PGE2 inhibited histamine- and forskolin-stimulated [14C]aminopyrine accumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE2 and somatostatin on histamine- and forskolin-stimulated H(+)-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32P]NAD+, pertussis toxin catalysed the incorporation of [32P]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi alpha). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32P]ADP ribosylation of the 41,000 molecular weight protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Toxina Adenilato Ciclase , Dinoprostona/antagonistas & inibidores , Células Parietais Gástricas/efeitos dos fármacos , Toxina Pertussis , Somatostatina/antagonistas & inibidores , Fatores de Virulência de Bordetella/farmacologia , Difosfato de Adenosina/metabolismo , Aminopirina/análise , Animais , Colforsina/farmacologia , Dinoprostona/farmacologia , Feminino , Proteínas de Ligação ao GTP/antagonistas & inibidores , Histamina/farmacologia , Células Parietais Gástricas/metabolismo , Prótons , Ratos , Ratos Endogâmicos , Somatostatina/farmacologiaRESUMO
Nephrogenic diabetes insipidus (NDI) is characterized by resistance of the kidney to the action of arginine-vasopressin (AVP); it may be due to genetic or acquired causes. Recent advances in molecular genetics have allowed the identification of the genes involved in congenital NDI. While inactivating mutations of the vasopressin V2 receptor are responsible for X-linked NDI, autosomal recessive NDI is caused by inactivating mutations of the vasopressin-regulated water channel aquaporin-2 (AQP-2). About 70 different mutations of the V2 receptor have been reported, most of them missense mutations. The functionally characterized mutants show a loss of function due to defects in their synthesis, processing, intracellular transport, AVP binding, or interaction with the G protein/adenylyl cyclase system. Thirteen different mutations of the AQP-2 gene have been reported. Functional studies of three AQP-2 mutations reveal impaired cellular routing as the main defect. The great number of different mutations with various functional defects hinders the development of a specific therapy. Gene therapy may, however, eventually become applicable to the congenital forms of NDI. At present all gene-therapeutic approaches lack safety and efficiency, which is of particular relevance in a disease that is treatable by an adequate water intake. The progress with regard to the molecular basis of antidiuresis contributes to the understanding of acquired forms of NDI on a molecular level. Recent data show that lithium dramatically reduces the expression of AQP-2. Likewise, hypokalemia reduces the expression of this water channel. The exact mechanisms leading to this reduced expression of AQP-2 remain to be determined.
Assuntos
Aquaporinas , Diabetes Insípido Nefrogênico/etiologia , Sequência de Aminoácidos , Aquaporina 2 , Aquaporina 6 , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/terapia , Genes Recessivos , Ligação Genética , Humanos , Canais Iônicos/genética , Canais Iônicos/fisiologia , Dados de Sequência Molecular , Mutação/genética , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/fisiologia , Vasopressinas/fisiologia , Cromossomo XRESUMO
The mutation of the type-2 vasopressin receptor (V2R) apparently responsible for X-linked congenital nephrogenic diabetes insipidus (CNDI) in the Q3 family consists of a T to C transition in codon 113, causing the change of Arg-113 to Trp. Arg-113 is located in the putative first extracellular loop of the V2R next to a frequently conserved Cys thought to interact via a disulfide bridge with a Cys of the second extracellular loop. The present study explored whether this mutation may account for the CNDI phenotype. The mutation was excised from the genomic DNA of a Q3 patient and introduced into the V2R cDNA, which was then placed into an expression plasmid and transfected into COS cells for transient expression and murine L cells for stable expression. Studies with L cells expressing similar levels of wild type and Q3 receptors showed that the mutant receptor has a 20-fold reduced affinity for arginine vasopressin (AVP) and stimulates adenylyl cyclase with an EC50 that is increased by a factor of about 60-fold. The same shift in the EC50 for adenylyl cyclase stimulation was obtained when deamino[8-D-Arg]vasopressin was substituted for AVP. Studies with COS cells revealed that at equal levels of transfected DNA, the mutant receptor is expressed at lower levels (about 20%) than the wild type receptor, indicating that the mutation hinders the transport of the receptor to the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenilil Ciclases/fisiologia , Arginina Vasopressina/metabolismo , Diabetes Insípido Nefrogênico/genética , Mutação Puntual , Receptores de Vasopressinas/genética , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Desamino Arginina Vasopressina/metabolismo , Desamino Arginina Vasopressina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Células L , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.
Assuntos
Azidas/metabolismo , Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Guanosina Trifosfato/análogos & derivados , Toxina Pertussis , Hipófise/fisiologia , Receptores Muscarínicos/fisiologia , Fatores de Virulência de Bordetella/farmacologia , Marcadores de Afinidade , Animais , Canais de Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Linhagem Celular , Di-Hidropiridinas/farmacologia , Condutividade Elétrica , Guanosina Trifosfato/metabolismo , Peso Molecular , Fotoquímica , Hipófise/efeitos dos fármacos , Prolactina/metabolismo , RatosRESUMO
According to the 'shuttle hypothesis', vasopressin increases the water permeability of renal epithelial cells by exocytotic fusion of vesicles containing the water channel AQP-CD with the apical plasma membrane, whereas withdrawal of vasopressin results in endocytotic uptake of AQP-CD. The proteins involved in the redistribution of AQP-CD have not been identified. With a panel of monoclonal antibodies, we detected Rab3-, Rab5a- and synaptobrevin II-like proteins in a kidney preparation enriched in AQP-CD-containing vesicles. The synaptobrevin II-like proteins is not identical with the ubiquitous cellubrevin. Rab3- and synaptobrevin II- but not Rab5a-like proteins were co-enriched with AQP-CD. The data suggest that the proteins involved in hormonal regulation of water permeability in kidney epithelial cells are identical or similar to those involved in regulated exocytosis in secretory cells.
Assuntos
Aquaporinas , Proteínas de Ligação ao GTP/análise , Canais Iônicos/análise , Medula Renal/metabolismo , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Vasopressinas/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos , Aquaporina 2 , Aquaporina 6 , Eletroforese em Gel de Poliacrilamida , Endocitose , Epitélio/metabolismo , GTP Fosfo-Hidrolases/análise , Proteínas de Ligação ao GTP/isolamento & purificação , Soros Imunes , Immunoblotting , Canais Iônicos/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Peptídeos/síntese química , Peptídeos/imunologia , Proteínas R-SNARE , Ratos , Proteínas rab3 de Ligação ao GTP , Proteínas rab5 de Ligação ao GTPRESUMO
Incubation of membranes of human erythrocytes and platelets but not of human neutrophils with [32P]NAD leads to covalent modification of various membrane proteins and of added albumin. In membranes of all three cell types, pertussis toxin (PT), in the presence of NAD, specifically labelled a 40 kDa peptide, i.e. the alpha-subunit of a guanine nucleotide-binding protein. This effect of PT was slightly reduced by NADP, whereas modification of other membrane proteins and of albumin was largely suppressed, independent of whether PT was present or not. Labelling of cytosolic proteins in the presence of NAD was marginal; only in neutrophil cytosol, PT modified a 40 kDa peptide. Membranes of erythrocytes and platelets exhibited NAD-degrading activity, which was inhibited by NADP. The data suggest a high substrate specificity of PT for NAD. Inhibition of endogenous enzymes by NADP may prove useful for the evaluation of PT substrates.
Assuntos
Proteínas de Membrana , NADP/farmacologia , NAD/farmacologia , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Plaquetas/enzimologia , Plaquetas/metabolismo , Fenômenos Químicos , Química , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Neutrófilos/metabolismoRESUMO
In the chain of events by which chemotactic peptides stimulate NADPH oxidase-catalyzed superoxide formation in human neutrophils, the involvements of a pertussis toxin-sensitive guanine nucleotide-binding protein (N-protein), mobilization of intracellular calcium and protein kinase C stimulation have been proposed. Superoxide formation was studied in membranes from human neutrophils; NADPH oxidase was stimulated by arachidonic acid in the presence of neutrophil cytosol. Fluoride and stable GTP analogues, such as GTP gamma S and GppNHp, which all activate N-proteins, enhanced NADPH oxidase activity up to 4-fold. GDP beta S inhibited the effect of GTP gamma S. These data suggest that NADPH oxidase is regulated by an N-protein, independent of an elevation of the cytoplasmic calcium concentration.
Assuntos
Nucleotídeos de Guanina/farmacologia , NADH NADPH Oxirredutases/sangue , Neutrófilos/enzimologia , Membrana Celular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Humanos , NADPH Oxidases , Superóxidos/sangue , Tionucleotídeos/farmacologiaRESUMO
The hydrodynamic properties of the Gi2 alpha-subunit in human neutrophil cytosol were analyzed. The S20,w value was 3.3 S at 30 degrees C and 4.1 S at 4 degrees C. Reconstitution of the cytosolic alpha-subunit with beta gamma-complex purified from porcine brain resulted in an S20,w value of 4.0 S at 30 degrees C and 4.2 S at 4 degrees C. Treatment of cytosol with G-protein-activating agents, GTP gamma S and MgCl2, decreased the S20,w value to 2.4 S at 30 degrees C and 2.9 S at 4 degrees C. Our results indicate that the cytosolic alpha-subunit of neutrophils is an inactive monomeric species at 30 degrees C capable of interacting with G-protein beta gamma-complex and responsive to treatment with activating agents leading to the two activated forms, alpha at 4 degrees C and alpha at 30 degrees C.