RESUMO
Species interactions, and their fitness consequences, vary across the geographic range of a coevolutionary relationship. This spatial heterogeneity in reciprocal selection is predicted to generate a geographic mosaic of local adaptation, wherein coevolutionary traits are phenotypically variable from one location to the next. Under this framework, allopatric populations should lack variation in coevolutionary traits due to the absence of reciprocal selection. We examine phenotypic variation in tetrodotoxin (TTX) toxicity of the Rough-Skinned Newt (Taricha granulosa) in regions of allopatry with its TTX-resistant predator, the Common Garter Snake (Thamnophis sirtalis). In sympatry, geographic patterns of phenotypic exaggeration in toxicity and toxin-resistance are closely correlated in prey and predator, implying that reciprocal selection drives phenotypic variation in coevolutionary traits. Therefore, in allopatry with TTX-resistant predators, we expect to find uniformly low levels of newt toxicity. We characterized TTX toxicity in northwestern North America, including the Alaskan panhandle where Ta. granulosa occur in allopatry with Th. sirtalis. First, we used microsatellite markers to estimate population genetic structure and determine if any phenotypic variation in toxicity might be explained by historical divergence. We found northern populations of Ta. granulosa generally lacked population structure in a pattern consistent with northern range expansion after the Pleistocene. Next, we chose a cluster of sites in Alaska, which uniformly lacked genetic divergence, to test for phenotypic divergence in toxicity. As predicted, overall levels of newt toxicity were low; however, we also detected unexpected among- and within-population variation in toxicity. Most notably, a small number of individuals contained large doses of TTX that rival means of toxic populations in sympatry with Th. sirtalis. Phenotypic variation in toxicity, despite limited neutral genetic divergence, suggests that factors other than reciprocal selection with Th. sirtalis likely contribute to geographic patterns of toxicity in Ta. granulosa.
RESUMO
Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu(2+), which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca(2+)-dependent, and CaM-binding domain is localized to 35-70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca(2+) signaling and cyclophilin activity in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Sinalização do Cálcio/fisiologia , Calmodulina/metabolismo , Ciclofilinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sinalização do Cálcio/efeitos dos fármacos , Calmodulina/genética , Cobre/farmacologia , Ciclofilinas/genética , Estrutura Terciária de ProteínaRESUMO
Females, by mating with more than one male in their lifetime, may reduce their risk of receiving sperm from genetically incompatible sires or increase their prospects of obtaining sperm from genetically superior sires. Although there is evidence of both kinds of genetic benefits in crickets, their relative importance remains unclear, and the extent to which experimentally manipulated levels of polyandry in the laboratory correspond to those that occur in nature remain unknown. We measured lifetime polyandry of free-living female decorated crickets, Gryllodes sigillatus, and conducted an experiment to determine whether polyandry leads to an increase in offspring viability. We experimentally manipulated both the levels of polyandry and opportunities for females to select among males, randomly allocating the offspring of experimental females to high-food-stress or low-food-stress regimes to complete their development. Females exhibited a high degree of polyandry, mating on average with more than seven different males during their lifetime and up to as many as 15. Polyandry had no effect on either the developmental time or survival of offspring. However, polyandrous females produced significantly heavier sons than those of monandrous females, although there was no difference in the adult mass of daughters. There was no significant interaction between mating treatment and offspring nutritional regimen in their effects on offspring mass, suggesting that benefits accruing to female polyandry are independent of the environment in which offspring develop. The sex difference in the extent to which male and female offspring benefit via their mother's polyandry may reflect possible differences in the fitness returns from sons and daughters. The larger mass gain shown by sons of polyandrous females probably leads to their increased reproductive success, either because of their increased success in sperm competition or because of their increased life span.
Assuntos
Gryllidae/anatomia & histologia , Gryllidae/fisiologia , Inseminação/fisiologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Evolução Biológica , Feminino , Masculino , Fatores Sexuais , Comportamento Sexual AnimalRESUMO
Calmodulin is a regulatory protein activated during Ca2+ signalling. We have isolated a cDNA, designated LeCBDGK (Lycopersicon esculentum calmodulin-binding diacylglycerol kinase) encoding a novel calmodulin-binding protein with sequence similarity to diacylglycerol kinases from animals. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid. We delineated the calmodulin-binding domain to approximately 25 residues near the C-terminus of LeCBDGK. We have also isolated a second diacylglycerol kinase cDNA, designated LeDGK1, identical to LeCBDGK, except that it lacks the calmodulin-binding domain. Both recombinant LeCBDGK and LeDGK1 were catalytically active in vitro. Anti-DGK antiserum detected two immunoreactive proteins associated with microsomal and plasma membrane fractions from cell suspensions. The higher molecular weight immunoreactive protein was also present in soluble extracts and bound to calmodulin-agarose in the presence of calcium, demonstrating that native LeCBDGK is a calmodulin-binding protein. In the presence of calcium, LeCBDGK associated with membrane cell fractions in vitro, but calmodulin antagonists disrupted this association, suggesting a possible role of calcium in the recruitment of LeCBDGK from soluble to membrane cell fractions. Native LeCBDGK and calmodulin co-immunoprecipitated from tomato soluble cell extracts, suggesting their interaction in vivo. The same gene encodes both LeCBDGK and LeDGK1 and the calmodulin-binding domain of LeCBDGK is encoded by a separate exon. Thus, alternative transcript splicing leads to calmodulin-binding and non-binding forms of diacylglycerol kinases in tomato. Possible roles of LeCBDGK and LeDGK1 in calcium and lipid signalling are discussed.
Assuntos
Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sinalização do Cálcio , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Frações Subcelulares/enzimologiaRESUMO
Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Plantas/enzimologia , Isoformas de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , ATPases Transportadoras de Cálcio/metabolismo , Ativação EnzimáticaRESUMO
Agricultural productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. Overexpression of a vacuolar Na+/H+ antiport from Arabidopsis thaliana in Arabidopsis plants promotes sustained growth and development in soil watered with up to 200 millimolar sodium chloride. This salinity tolerance was correlated with higher-than-normal levels of AtNHX1 transcripts, protein, and vacuolar Na+/H+ (sodium/proton) antiport activity. These results demonstrate the feasibility of engineering salt tolerance in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/fisiologia , Proteínas de Transporte de Cátions , Cloreto de Sódio/toxicidade , Trocadores de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Expressão Gênica , Hidrogênio/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Trocadores de Sódio-Hidrogênio/genética , Vacúolos/metabolismoRESUMO
The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.
Assuntos
Arabidopsis/genética , Cálcio/metabolismo , Calmodulina/metabolismo , Glutamato Descarboxilase/genética , Isoenzimas/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glutamato Descarboxilase/metabolismo , Isoenzimas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Wild-type and GTPase-deficient recombinant TGalpha1 were used along patch-clamp techniques to study the role of heterotrimeric G proteins in the regulation of the hyperpolarized active tomato plasma membrane Ca2+ channel. Recombinant alpha-subunits induced an increase in channel activity as shown by the increase in channel events and the mean open probability of the channel. Our results suggest a membrane-delimited pathway involving heterotrimeric G proteins in Ca2+ channel activation.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Membrana Celular/fisiologia , Guanosina Trifosfato/metabolismo , Solanum lycopersicum , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
This report describes the cloning and characterization of a plant cDNA coding for a protein which shows high amino acid sequence similarity with prohibitin, whose gene is associated with antiproliferative activity in mammalian cells. Arabidopsis thaliana and Nicotiana tabacum prohibitin complete cDNAs were isolated, and the expression pattern of prohibitin was examined using polyclonal antibodies raised against the Arabidopsis recombinant prohibitin expressed in Escherichia coli. A single immunoreactive protein was detected in various plant species and in all Arabidopsis organs examined. Subcellular fractionation using tobacco leaves revealed prohibitin in a mitochondrial-enriched fraction. Phylogenetic conservation of prohibitin's amino acid sequence and subcellular localization suggests a similar function in plants, yeast and mammals.
Assuntos
Antineoplásicos , Genes de Plantas , Proteínas de Plantas/genética , Proteínas/genética , Proteínas Repressoras , Sequência de Aminoácidos , Arabidopsis/genética , Compartimento Celular , Sequência Conservada , DNA Complementar/genética , Mitocôndrias/química , Dados de Sequência Molecular , Plantas Tóxicas , Proibitinas , Proteínas/imunologia , RNA Mensageiro/genética , RNA de Plantas/genética , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Frações Subcelulares/química , Distribuição Tecidual , Nicotiana/genéticaRESUMO
The authors developed a new apparatus for extracting nitrogen or other inert gases from blood by flushing (sparging) the specimen with another gas. To investigate the utility of the new methodology, the apparatus was used in conjunction with a mass spectrometer to measure the blood N2 content of healthy normobaric, non-smoking, adult volunteers; the mean was found to be 11.7 microliters/ml +/- 0.9 microliter. This compares closely with values cited in the literature. The within-subject variation for repeat samples taken several weeks apart was significantly (P < 0.003) less than the variation between different subjects, suggesting that there may be true differences in N2 content between different individuals. These data must be considered preliminary, a larger study is needed to investigate population differences in detail. The advantages of the new method are discussed.
Assuntos
Gasometria/métodos , Nitrogênio/sangue , Adulto , Gasometria/instrumentação , Computadores , Desenho de Equipamento , Humanos , Gases Nobres/sangueRESUMO
PURPOSE: Provision of general anaesthesia in areas remote from the operating room creates many difficult challenges especially if required for repeated radiotherapy in the prone position. This case illustrates these problems and some innovative solutions. CLINICAL FEATURES: A nine-year-old girl with medulloblastoma became extremely distressed whenever cobalt radiotherapy was attempted. Sedation with midazolam and high dose propofol infusion failed to achieve satisfactory conditions and caused concerns regarding airway management. The patient received a total of 37 endotracheal anaesthetics in the prone position using isoflurane in oxygen. Activated charcoal was used to scavenge anaesthetic vapors and adequate gas supplies were assured by connecting an "H' size tank to the oxygen pipeline inlet of the anaesthesia machine. Measurement of isoflurane in exhaust gases using gas chromatography confirmed the effectiveness of scavenging. No serious complications occurred related to repeated anaesthesia. CONCLUSION: The methods and equipment described permitted safe delivery of repeated inhalation general anaesthesia for radiotherapy. The same methods could be applied to anaesthesia in other remote locations.
Assuntos
Anestesia por Inalação/métodos , Radioisótopos de Cobalto/uso terapêutico , Criança , Feminino , Humanos , Intubação Intratraqueal , Meduloblastoma/radioterapiaRESUMO
A novel technique for the measurement of gas solubility and gas content in liquids and suspensions is described. Saturation of the liquid and subsequent extraction of the test gas both took place in a specially modified gastight syringe. The test gas was extracted from the saturated liquid by bubbling an inert carrier gas through the liquid ("sparging"). All gas exiting the apparatus was directed toward a mass spectrometer that measured the volume of extracted test gas in the presence of the carrier. The technique was used to measure the solubilities of nitrogen, oxygen, carbon monoxide, carbon dioxide, and nitrous oxide in olive oil at 37 degrees C. The Bunsen solubility coefficients so obtained are in good agreement with those obtained by classic techniques.
Assuntos
Gases Nobres/química , Dióxido de Carbono/química , Nitrogênio/química , Oxigênio/química , SolubilidadeRESUMO
To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0-7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (Vmax = 30 micromol of CO2 min-1 mg of protein-1) by the addition of exogenous calmodulin (K0.5 = 15 nM) in the presence of calcium (K0.5 = 0.8 microM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (Km = 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.
Assuntos
Anticorpos Monoclonais/farmacologia , Cálcio/farmacologia , Calmodulina/farmacologia , Glutamato Descarboxilase/metabolismo , Plantas/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Calmodulina/metabolismo , Clonagem Molecular , Ativação Enzimática , Escherichia coli , Glutamato Descarboxilase/imunologia , Glutamato Descarboxilase/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência MolecularRESUMO
We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.
Assuntos
Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Glutamato Descarboxilase/química , Glutamato Descarboxilase/metabolismo , Plantas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cálcio/farmacologia , Calmodulina/química , Calmodulina/isolamento & purificação , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/isolamento & purificação , Clonagem Molecular , Escherichia coli , Glutamato Descarboxilase/isolamento & purificação , Glutationa Transferase/biossíntese , Glutationa Transferase/isolamento & purificação , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfon-amide and trifluoperazine. With saturating CaM or Ca2+, the concentrations of Ca2+ and CaM required for half-maximal stimulation were about 7 to 11 [mu]M and 25 nM, respectively. The effect of Ca2+ and CaM appeared to be through a 2.4-fold stimulation of Vmax and a 55% reduction in Km. The results suggested that GAD is activated via Ca2+ signal transduction.
RESUMO
The identity of a soluble 62-kD Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the alpha-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Fabaceae/enzimologia , Glutamato Descarboxilase/metabolismo , Plantas Medicinais , Sequência de Aminoácidos , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/isolamento & purificação , Ativação Enzimática , Fabaceae/genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/isolamento & purificação , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Raízes de Plantas/química , Análise de Sequência , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Addition of l-[U-(14)C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-(14)C]glutamate, and (c) efflux of [(14)C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H(+)/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H(+)/l-Glu symport may involve both H(+) consumption during 4-aminobutyrate production and ATP-driven H(+) efflux.
RESUMO
HYPOTHESIS: Impaired baroreflex-induced release of noradrenaline during angiotensin converting enzyme (ACE) inhibition may interfere with orthostatic responses of blood pressure. OBJECTIVE: To compare blood pressure and heart rate responses of hypertensive patients to upright posture over a 12 h period before and after quinapril therapy. DESIGN: Four weeks of placebo was given to all patients. If subject's sitting diastolic blood pressure was between 100 and 115 mmHg, patients received 2.5, 5, 10 or 20 mg quinapril bid for four more weeks in a double-blind randomized fashion. SETTING: Ambulatory hypertension clinic with admission to clinical investigation unit overnight for interventions. PATIENTS: Uncomplicated essential hypertensives of English or Irish ancestry. Twenty-five eligible patients completed the study while three withdrew early. INTERVENTIONS: Supine and erect blood pressures and heart rates were recorded for each patient at 1 to 2 h intervals over a 12 h period following ingestion of placebo capsule and again after initial acute dose of quinapril the next day. Blood was sampled for quinapril and quinaprilat concentrations. Procedure was repeated after four weeks of quinapril therapy following morning dose of drug. MAIN RESULTS: Orthostatic response of diastolic pressure was reduced while maintenance of systolic was impaired 1 to 4 h following an acute dose of quinapril. Orthostatic cardioacceleration was maintained. Responses were largely restored after four weeks of chronic therapy. CONCLUSIONS: A possible initial impairment of baroreflex-mediated peripheral vasoconstriction by ACE inhibition may be offset by compensatory changes to vascular reactivity.
Assuntos
Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Isoquinolinas/uso terapêutico , Tetra-Hidroisoquinolinas , Cápsulas , Creatinina/sangue , Método Duplo-Cego , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacologia , Masculino , Pessoa de Meia-Idade , Postura , Potássio/urina , Quinapril , Sódio/urinaRESUMO
HYPOTHESIS: Reduction of plasma angiotensin II inhibits release of noradrenaline in response to a sympathetic nervous system stimulus. OBJECTIVE: To compare heart rate and plasma catecholamine responses to the Valsalva maneuvre before and after inhibition of angiotensin converting enzyme. DESIGN: Four weeks of placebo (all patients). If sitting diastolic blood pressure was found to be between 100 and 115 mmHg, then double-blind randomization to receive 2.5, 5, 10 or 20 mg of quinapril bid for a further four weeks. SETTING: Ambulatory hypertension clinic with admission to clinical investigation unit overnight for interventions. PATIENTS: Uncomplicated essential hypertensives of English-Irish ancestry. Twenty-five eligible patients completed the study, while three additional patients withdrew early. INTERVENTIONS: Blood pressure and heart rate were monitored weekly during placebo, and biweekly during active treatment. Patients were rehearsed in performance of the Valsalva maneuvre. At the end of the placebo and active therapy phases, each patient performed standard Valsalva maneuvres immediately before and 2 h after the morning dose of placebo or active drug. Heart rate was recorded continuously throughout the maneuvre, while blood was sampled for catecholamine determinations prior to the start of straining and again approximately 10 s following the end of straining. MAIN RESULTS: Heart rate responses to the Valsalva maneuvre were not affected by acute administration of quinapril, but a slight attenuation of the tachycardic response was seen after four weeks of therapy (P less than 0.05). An increase in plasma noradrenaline concentration associated with the straining phase of the Valsalva maneuvre was markedly attenuated after both acute and chronic quinapril therapy (P less than 0.01). CONCLUSION: By blocking biosynthesis of angiotensin II, converting enzyme inhibition may attenuate baroreceptor-stimulated release of noradrenaline.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Frequência Cardíaca/fisiologia , Hipertensão/tratamento farmacológico , Isoquinolinas/uso terapêutico , Norepinefrina/sangue , Sistema Nervoso Simpático/fisiologia , Tetra-Hidroisoquinolinas , Manobra de Valsalva , Epinefrina/sangue , Feminino , Humanos , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , QuinaprilRESUMO
An attempt was made to establish whether cardioselective beta-blockade could counteract the stimulation by hydrochlorothiazide of the renin-angiotensin and sympathetic nervous systems and to what extent such actions contributed to the antihypertensive effect of bevantolol. The hemodynamic and neurohumoral responses of 21 thiazide resistant hypertensives who had received sequential chronic therapy with hydrochlorothiazide, hydrochlorothiazide combined with bevantolol and bevantolol monotherapy were compared. In these patients, bevantolol had a negative chronotropic effect and appeared, when administered alone, to induce an overall lowering of sympathetic nervous system activity without inhibiting the reflex responses of peripheral vascular resistance to postural change or lowered heart rate. When bevantolol and hydrochlorothiazide were administered together, sympathetic activity appeared to be maintained, possibly as a reflex response to volume depletion but vascular resistance did not appear to be responsive to baroreceptor stimulation. Diminished vascular reactivity induced by the hydrochlorothiazide is suspected to be a contributory factor. Inhibition of thiazide stimulated renin release by bevantolol may contribute to the antihypertensive effect of the combined therapy.