RESUMEN
The jaboticaba tree (Plinia peruviana (Poir.) Govaerts) is a native Brazilian species, and its fruits are very popular in Brazil. The purpose of this study was to model the diameter growth, and determine the age of jaboticaba trees in four natural populations in Southwest of Paraná State, Brazil. The knowledge of the growth dynamics of this species is essential to assist management and conservation strategies and to discover when this species was established in the region. Core samples were collected to obtain complete growth series by measurement of annual growth rings. The series were crossdated, seven growth models were adjusted, and the best model for each site and a general model were selected using statistics rules. The time series spanned periods of ~75 to ~100 years, and growth ranged between 0.27 and 0.37 cm year-1. Chapman-Richards' model showed better adherence for locations individually, and Monomolecular when grouped. Because to the age reached by the jaboticaba trees and the high density of this species in these natural populations, consequently named "jaboticabais", it was inferred that there was an anthropogenic action in the dispersion and formation of the settlements through the indigenous and caboclos that inhabited the region before 1940. Also, this is the first work to register and verify the fusion of trunks for species of the Plinia genus using double piths found in samples, a feature that must be considered to avoid overestimating the age of jaboticaba trees based solely on measuring tree diameter.
Asunto(s)
Myrtaceae , Árboles , Brasil , FrutasRESUMEN
Abstract Understanding morphological and physiological changes under different light conditions in native fruit species in juveniles' stage is important, as it indicate the appropriate environment to achieve vigorous saplings. We aimed to verify growth and morphophysiological changes under shade gradient in feijoa (Acca sellowiana (O. Berg) Burret) to achieve good quality saplings adequate to improve cultivation in orchards. The saplings were grown for twenty-one-month under four shading treatments (0%, 30%, 50%, and 80%). Growth, photosynthetic pigments, gas exchanges, chlorophyll fluorescence, and leaf anatomy parameters were evaluated. Saplings under full sun and 30% shade had higher height and diameter growth and dry mass accumulation due to higher photosynthesis rate. As main acclimatization mechanisms in feijoa saplings under 80% shade were developed larger leaf area, reduced leaf blade thickness, and enhanced quantum yield of photosystem II. Even so, the net CO2 assimilation and the electron transport rate was lower and, consequently, there was a restriction on the growth and dry mass in saplings under deep shade. Therefore, to obtain higher quality feijoa saplings, we recommend that it be carried out in full sun or up to 30% shade, to maximize the sapling vigor in nurseries and, later, this light environment can also be used in orchards for favor growth and fruit production.
Resumo A verificação de mudanças morfológicas e fisiológicas sob diferentes condições luminosas em espécies frutíferas nativas em estágio juvenil é importante, uma vez que indicam o ambiente adequado para a formação de mudas com alto vigor. Objetivou-se verificar o crescimento e as alterações morfofisiológicas sob gradiente de sombreamento em mudas de feijoa (Acca sellowiana (O. Berg) Burret) para obter mudas de boa qualidade, adequadas para fomentar os plantios da espécie em pomares. As mudas foram cultivadas por vinte e um meses sob quatro tratamentos de sombreamento (0%, 30%, 50% e 80%). Foram avaliados parâmetros de crescimento, pigmentos fotossintéticos, trocas gasosas, fluorescência da clorofila e anatomia foliar. Mudas a pleno sol e 30% de sombra apresentaram maior crescimento em altura, diâmetro e acúmulo de massa seca, devido à maior taxa de fotossíntese. Como principais mecanismos de aclimatação sob 80% de sombra, as mudas desenvolveram maior área foliar, redução da espessura do limbo foliar e aumento do rendimento quântico do fotossistema II. Mesmo assim, a assimilação líquida de CO2 e a taxa de transporte de elétrons foram menores e, consequentemente, houve restrição ao crescimento e acúmulo de massa seca das mudas no maior nível de sombreamento. Portanto, para a obtenção de mudas de feijoa de maior qualidade, recomendamos que seja realizada a pleno sol ou até 30% de sombra, para maximizar o vigor das mudas em viveiros e, posteriormente, este ambiente de luz também pode ser utilizado em pomares para favorecer o crescimento e a produção de frutos.
Asunto(s)
Myrtaceae , Feijoa , Fotosíntesis , Hojas de la Planta , Aclimatación , LuzRESUMEN
Abstract Understanding morphological and physiological changes under different light conditions in native fruit species in juveniles stage is important, as it indicate the appropriate environment to achieve vigorous saplings. We aimed to verify growth and morphophysiological changes under shade gradient in feijoa (Acca sellowiana (O. Berg) Burret) to achieve good quality saplings adequate to improve cultivation in orchards. The saplings were grown for twenty-one-month under four shading treatments (0%, 30%, 50%, and 80%). Growth, photosynthetic pigments, gas exchanges, chlorophyll fluorescence, and leaf anatomy parameters were evaluated. Saplings under full sun and 30% shade had higher height and diameter growth and dry mass accumulation due to higher photosynthesis rate. As main acclimatization mechanisms in feijoa saplings under 80% shade were developed larger leaf area, reduced leaf blade thickness, and enhanced quantum yield of photosystem II. Even so, the net CO2 assimilation and the electron transport rate was lower and, consequently, there was a restriction on the growth and dry mass in saplings under deep shade. Therefore, to obtain higher quality feijoa saplings, we recommend that it be carried out in full sun or up to 30% shade, to maximize the sapling vigor in nurseries and, later, this light environment can also be used in orchards for favor growth and fruit production.
Resumo A verificação de mudanças morfológicas e fisiológicas sob diferentes condições luminosas em espécies frutíferas nativas em estágio juvenil é importante, uma vez que indicam o ambiente adequado para a formação de mudas com alto vigor. Objetivou-se verificar o crescimento e as alterações morfofisiológicas sob gradiente de sombreamento em mudas de feijoa (Acca sellowiana (O. Berg) Burret) para obter mudas de boa qualidade, adequadas para fomentar os plantios da espécie em pomares. As mudas foram cultivadas por vinte e um meses sob quatro tratamentos de sombreamento (0%, 30%, 50% e 80%). Foram avaliados parâmetros de crescimento, pigmentos fotossintéticos, trocas gasosas, fluorescência da clorofila e anatomia foliar. Mudas a pleno sol e 30% de sombra apresentaram maior crescimento em altura, diâmetro e acúmulo de massa seca, devido à maior taxa de fotossíntese. Como principais mecanismos de aclimatação sob 80% de sombra, as mudas desenvolveram maior área foliar, redução da espessura do limbo foliar e aumento do rendimento quântico do fotossistema II. Mesmo assim, a assimilação líquida de CO2 e a taxa de transporte de elétrons foram menores e, consequentemente, houve restrição ao crescimento e acúmulo de massa seca das mudas no maior nível de sombreamento. Portanto, para a obtenção de mudas de feijoa de maior qualidade, recomendamos que seja realizada a pleno sol ou até 30% de sombra, para maximizar o vigor das mudas em viveiros e, posteriormente, este ambiente de luz também pode ser utilizado em pomares para favorecer o crescimento e a produção de frutos.
RESUMEN
Plants exposed to different light intensities generate physiological, morphological, and anatomical changes conducting to plasticity. Thus, this characteristic establishes the ability of plants to present phenotypic adjustments by the same genotype under different environmental conditions. The objective of this study was to verify the morphophysiological alterations in Campomanesia xanthocarpa (Mart.) O. Berg (guabiroba) seedlings cultivated in different shading levels. The seedlings were grown for 21 months under full sun or 30%, 50%, and 80% under shading. Growth, photosynthetic pigments, gas exchange rate, chlorophyll fluorescence, and leaf anatomy were evaluated. In all the treatments subjected to shading, plasticity mechanisms involved structural and physiological changes such as an increase in leaf area and chlorophyll content (total and Chl a), reduction in leaf thickness, and increased gas exchange and quantum yield of photosystem II. The guabiroba seedlings can be cultivated in full sun or different shading environments; even under high shading intensity (80%), the plants showed vigor similar to those produced in a sunny environment. These results confirmed our hypothesis about guabiroba acclimation capacity to shading, noteworthy information for nurseries, orchards, agroforestry systems, or forest restoration in a wide range of light environments.
Asunto(s)
Myrtaceae , Plantones , Plantones/fisiología , Clorofila , Fotosíntesis/fisiología , Hojas de la Planta/fisiologíaRESUMEN
Understanding morphological and physiological changes under different light conditions in native fruit species in juveniles' stage is important, as it indicate the appropriate environment to achieve vigorous saplings. We aimed to verify growth and morphophysiological changes under shade gradient in feijoa (Acca sellowiana (O. Berg) Burret) to achieve good quality saplings adequate to improve cultivation in orchards. The saplings were grown for twenty-one-month under four shading treatments (0%, 30%, 50%, and 80%). Growth, photosynthetic pigments, gas exchanges, chlorophyll fluorescence, and leaf anatomy parameters were evaluated. Saplings under full sun and 30% shade had higher height and diameter growth and dry mass accumulation due to higher photosynthesis rate. As main acclimatization mechanisms in feijoa saplings under 80% shade were developed larger leaf area, reduced leaf blade thickness, and enhanced quantum yield of photosystem II. Even so, the net CO2 assimilation and the electron transport rate was lower and, consequently, there was a restriction on the growth and dry mass in saplings under deep shade. Therefore, to obtain higher quality feijoa saplings, we recommend that it be carried out in full sun or up to 30% shade, to maximize the sapling vigor in nurseries and, later, this light environment can also be used in orchards for favor growth and fruit production.
Asunto(s)
Feijoa , Myrtaceae , Aclimatación , Luz , Fotosíntesis , Hojas de la PlantaRESUMEN
BACKGROUND: To enhance usage of health technology assessment (HTA) in decision-making, it is important to prioritise important barriers and facilitators to the uptake of HTA. This study aims to quantify and compare the relative importance of barriers and facilitators regarding the use of HTA in several European countries. METHODS: A survey containing two best-worst scaling (BWS) object case studies (i.e. barriers and facilitators) were conducted among 136 policy makers and HTA researchers from the Netherlands, Germany, France, and United Kingdom. Hierarchical Bayes analysis generated the mean relative importance score (RIS) for each factor and subgroup analyses assessed differences between countries. RESULTS: Six barriers (RIS≥5) and five facilitators (RIS≥6) were deemed highly important. Eleven barriers and ten facilitators differed in their importance between countries. Policy characteristics, research & researcher characteristics, and organisation & resources were particularly important to facilitate uptake of HTA, such as an explicit framework for decision-making and research of sufficient quality. CONCLUSION: The most paramount barriers and facilitators to HTA usage were quantified. For all countries it is crucial to create an explicit framework for the decision-making context to include HTA evidence. Country differences in the quality of research emphasize the need for enhanced international collaboration in HTA.
Asunto(s)
Toma de Decisiones , Política de Salud , Evaluación de la Tecnología Biomédica/métodos , Personal Administrativo , Europa (Continente) , Humanos , Cooperación Internacional , Investigación/normasRESUMEN
Polyelectrolyte complexes (PECs) represent promising materials for drug delivery and tissue engineering applications. These substances are obtained in aqueous medium without the need for crosslinking agents. PECs can be produced through the combination of oppositely charged medical grade polymers, which include the stimuli responsive ones. In this work, three-dimensional porous scaffolds were produced through the lyophilization of pH sensitive PECs made of chitosan (CS) and carrageenan (CRG). CS:CRG molar ratios of 1:1 (CSCRG1), 2:1 (CSCRG2), and 3:1 (CSCRG3) were used. The chemical compositions of the PECs, as well as their influence in the final structure of the scaffolds were meticulously studied. In addition, the pH responsiveness of the PECs in a range including the physiological pH values of 7.4 (simulating normal physiological conditions) and 4.5 (simulating inflammatory response) was assessed. Results showed that the PECs produced were stable at pH values of 7.4 and under but dissolved as the pH increased to nonphysiological values of 9 and 11. However, after dissolution, the PEC could be reprecipitated by decreasing the pH to values close to 4.5. The scaffolds obtained presented large and interconnected pores, being equally sensitive to changes in the pH. CSCRG1 scaffolds appeared to have higher hydrophilicity and therefore higher water absorption capacity. The increase in the CS:CRG molar ratios improved the scaffold mechanical properties, with CSCRG3 presenting the higher compressive modulus under wet conditions. Overall, the PEC scaffolds appear promising for tissue engineering related applications that require the use of pH responsive materials stable at physiological conditions.
Asunto(s)
Carragenina/química , Quitosano/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Concentración de Iones de Hidrógeno , PorosidadRESUMEN
Small-area analyses are increasingly valuable for health-policy planning given the observed regional variation in health care. The combination of data from different sources enables researchers to describe and analyze structural, procedural, and outcome-related variation in health care. For variation that cannot be explained by these data, the attitudes, behaviors, and preferences of important stakeholders in health care-physicians and patients-might play an explanatory role. In this paper, we outline why stakeholder preference elicitation appears to be an important addition to small-area health-care analyses. Its inclusion in various decision-making contexts may help to explain and decrease unwarranted variation in health care. At the individual level, this could happen within any decision on health service use that is taken by physicians, patients, or-within a shared decision-making context-by both. At an aggregate level, the elicitation of preferences in guideline or health program development may increase stakeholder acceptance and consistent implementation. Different approaches to the elicitation of stakeholder preferences and potential areas for application within small-area research are outlined. The role of attitudes and preferences in explaining regional variation should be acknowledged by health-care policy makers and initiatives to include these in health-care decision-making at various levels should be supported.
Asunto(s)
Actitud Frente a la Salud , Servicios de Salud Comunitaria/organización & administración , Política de Salud , Investigación sobre Servicios de Salud/organización & administración , Servicios de Salud Rural/organización & administración , Análisis de Área Pequeña , Alemania , Participación del PacienteRESUMEN
Standardization of international health economic guidelines has been repeatedly requested. In this context, an international reference case was proposed, which constitutes an agreed approach for the key elements of health economic evaluation including study perspective, comparators, source of effectiveness data, role of modeling, main (economic) outcome, source of utilities, characterizing uncertainty. It is, however, questionable whether such a reference scenario can reasonably be applied across all health care systems. Our analysis pursues the question to which degree the Institute for Quality and Efficiency in Health Care's (Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen, IQWiG) "General methods for evaluating the relation between cost and benefit" comply with the key elements of the reference case. In case of divergences, they will be described and discussed in light of the German social legislation and in consideration of current scientific evidence. In conclusion, the analysis revealed that IQWiG complied with the reference case in almost all aspects. Differences were found only with respect to the choice of main (economic) outcome and the source of utilities. These differences seem justified and well explained in the context of the German social legislation as well as in view of the weaknesses of the quality-adjusted life year (QALY) concept.
Asunto(s)
Análisis Costo-Beneficio/normas , Economía Médica/normas , Guías como Asunto , Modelos Econométricos , Garantía de la Calidad de Atención de Salud/normas , Alemania , Valores de ReferenciaRESUMEN
The characterization of health-related self-help often includes social demands, which indicate that the knowledge on this healthrelated self-help is very small. On the basis of 40 years experience in self-help, the article traces different lines of evolution in health-related self-help. The article describes the development of self-help structures and their success. It illustrates the association between self-help and self-help organisations. It demonstrates the social legitimation of work in self-help. Finally, the article describes current challenges for the work in health-related self-help. Based on the evidence of theses new challenges, for example, the increased significance of rare diseases, the growing scientific foundation of health care or the discussions on the different offers for health-related information and consultation, the article demonstrates the future trends in health-related self-help.
Asunto(s)
Atención a la Salud/métodos , Atención a la Salud/tendencias , Predicción , Autocuidado/métodos , Autocuidado/tendencias , Grupos de Autoayuda/tendencias , Alemania , HumanosRESUMEN
We report on a 50-year old man who presented with upper abdominal pain and new T-wave inversions in the anterior leads of the electrocardiogramm. Since the patient additionally had cardiovascular risk factors (positive family history, smoking, hypercholesterinemia and hypertension) he was initially thought to have an acute coronary syndrome. Moreover, he had elevated serum levels of amylase and lipase, and together with typical findings of abdominal ultrasound he was diagnosed with acute pancreatitis responsible for the patient's pain. The ECG changes persisted during the hospital stay, but had fully normalised three weeks later. Further cardiologic evaluation yielded normal results. Thus, the ECG-abnormalities could be attributed to the acute pancreatitis, which can cause ECG-pathologies mimicking an acute coronary syndrome in up to 30%. Knowledge of there findings is important in order to prevent wrong therapeutic decisions.
Asunto(s)
Dolor Abdominal/etiología , Electrocardiografía , Hipertensión/diagnóstico , Hipertrofia Ventricular Izquierda/diagnóstico , Pancreatitis/diagnóstico , Enfermedad Aguda , Diagnóstico Diferencial , Humanos , Hipertensión/complicaciones , Hipertrofia Ventricular Izquierda/complicaciones , Masculino , Persona de Mediana Edad , Pancreatitis/complicacionesRESUMEN
UNLABELLED: INTRODUCTION, AIMS, AND METHODS: Industrial countries' societies are challenged to counter the increasing costs of health care. The preponderance of cardiovascular diseases in middle-aged people demands new strategies to reduce the extremely high rates of early deaths and increased morbidity and thus to avert considerable costs. Prevention programmes are appropriate means to combine therapeutic needs with health economic goals. A review of techniques of economic evaluation is given, from which a critical appraisal of cost-effectiveness analyses in primary and secondary prevention with HMG-CoA reductase inhibitors in coronary heart disease follows. Our own analysis of cost-effectiveness of primary prevention based on the clinical results of the WOSCOP study is also presented. RESULTS AND CONCLUSIONS: Long-term treatment with HMG-CoA reductase inhibitors is effective (30% mortality reduction), safe and tolerable. With a cost-effectiveness ratio of DM15,000-25,000/life-year saved ($9000-15,000) it can be recommended for secondary prevention. The application of statins in primary prevention has also proved to be clinically effective. But with a cost-effectiveness ratio of DM330,000/life-year saved ($195,000) they cannot be generally advocated in patients with hypercholesterolaemia. However, the treatment has to be considered for high risk patients. Aspirin prevention seems to be an alternative in patients without additional risk factors.
Asunto(s)
Enfermedades Cardiovasculares/economía , Enfermedades Cardiovasculares/prevención & control , Inhibidores de Hidroximetilglutaril-CoA Reductasas/economía , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Análisis Costo-Beneficio , Humanos , Pravastatina/economía , Pravastatina/uso terapéutico , Años de Vida Ajustados por Calidad de Vida , Medición de Riesgo , Factores de Riesgo , Simvastatina/economía , Simvastatina/uso terapéuticoRESUMEN
Temperature-sensitive folding (tsf) and global-tsf-suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off-pathway aggregation, the kinetics of refolding after dilution from acid-urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants. At low temperature (10 degrees C), subunit-folding rates, measured as an increase in fluorescence, were similar for wild-type and mutants. At 25 degrees C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf-mutant background, but had no effect in the wild-type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off-pathway reaction, as revealed by temperature down-shifts after varied times during refolding at high temperature (40 degrees C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate. In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up-shifts during refolding at 25 degrees C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit-folding intermediates and were also observable during thermal unfolding of the native protein.
Asunto(s)
Bacteriófago P22/química , Glicósido Hidrolasas/química , Pliegue de Proteína , Proteínas Virales/química , Proteínas de la Cola de los Virus , Bacteriófago P22/genética , Análisis Mutacional de ADN , Genes Supresores , Glicósido Hidrolasas/efectos de los fármacos , Glicósido Hidrolasas/genética , Guanidina , Guanidinas/farmacología , Calor , Cinética , Mutación Puntual , Desnaturalización Proteica , Espectrometría de Fluorescencia , Proteínas Virales/efectos de los fármacos , Proteínas Virales/genéticaRESUMEN
One of the central problems in protein folding is how amino acid sequences within polypeptide chains direct polypeptide chain folding and avoid off-pathway aggregation both in intracellular environments and in the test tube. The tailspike protein of phage P22 is a model system for which genetic analysis has permitted mutational dissection of the role of amino acid positions in the polypeptide chain in directing its in vivo folding. Two classes of mutations that affect intracellular folding and aggregation have been characterized; temperature-sensitive folding (tsf) mutants and second-site suppressors of tsf mutants. Here we report the effects of these mutations on the in vitro refolding and aggregation pathway of the purified proteins. The tsf mutations reduced refolding yields at high temperature and increased aggregation, while second-site suppressors enhanced refolding and inhibited aggregation in the test tube. For both types of mutations, the strength of the effects observed in vitro correlated with their in vivo phenotypes. The results confirm that the mutations act intrinsically on the folding pathway of the tailspike polypeptide and not through accessory proteins.
Asunto(s)
Bacteriófago P22/metabolismo , Glicósido Hidrolasas/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Virales/metabolismo , Proteínas de la Cola de los Virus , Secuencia de Aminoácidos , Bacteriófago P22/genética , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/aislamiento & purificación , Sustancias Macromoleculares , Desnaturalización Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Termodinámica , Proteínas Virales/biosíntesis , Proteínas Virales/aislamiento & purificaciónRESUMEN
Tryptic digestion of a thermal unfolding intermediate of the phage P22 tailspike endorhamnosidase produces an N-terminally shortened protein fragment comprising amino-acid residues 108-666 [Chen, B.-L. & King, J. (1991) Biochemistry 30, 6260-6269]. In the present work, the 60-kDa C-terminal fragment was purified to homogeneity from the tryptic digest by gel-fitration chromatography. As in the case for the whole tailspike protein (72 kDa), the purified fragment was found to remain stably folded as a highly soluble, SDS-resistant, enzymatically active trimer. However, its unfolding in the presence of guanidinium chloride was accelerated at least 10-fold compared to the complete, native tailspike protein. Shortened tailspike trimers reconstituted spontaneously and with high yield after diluting a solution containing acid-urea-unfolded fragment polypeptides with neutral buffer. Upon recombinant expression of the 60-kDa polypeptide in Escherichia coli, it also assembled efficiently and formed SDS-resistant trimers. The refolding and assembly pathway of the N-terminally shortened tailspike paralleled that of the complete protein with slightly, but significantly, accelerated folding reactions, at both the subunit and the trimer levels. As found for the complete tailspike protein, yields of refolding and assembly of the 60-kDa fragments into SDS-resistant trimers decreased with increasing temperature. The refolding yield of fragments derived from a temperature-sensitive mutant (Gly244-->Arg) tailspike protein was affected in similar fashion as shown for the whole protein. We conclude that the N-terminal domain (residues 1-107) is dispensable for folding and assembly of the P22 tailspike endorhamnosidase both in vitro and in vivo.
Asunto(s)
Bacteriófago P22/enzimología , Glicósido Hidrolasas/química , Pliegue de Proteína , Proteínas de la Cola de los Virus/química , Secuencia de Aminoácidos , Clonación Molecular , Detergentes , Escherichia coli , Glicósido Hidrolasas/genética , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de la Cola de los Virus/genéticaRESUMEN
Because efficient folding in vivo and reconstitution in vitro of phage P22 tailspike protein is temperature-sensitive, and because a chaperone function of the GroE proteins for tailspike folding in vivo has been suggested by genetic observations, the interactions of purified Escherichia coli GroE proteins with phage P22 tailspikes during refolding in vitro were investigated. At elevated temperature (> 30 degrees C), in the absence of ATP, GroEL effectively trapped refolding tailspike protein and prevented reconstitution. Tailspike protein was released from GroEL by addition of ATP around 35 degrees C or without added ATP upon cooling to 25 degrees C, and native tailspike trimers were formed. In accordance with the cold release, tailspike reconstitution at < or = 25 degrees C was unaffected by GroE. No formation of native tailspike trimers was observed, when refolding was initiated at 42 degrees C in the presence of the GroE proteins and ATP or when tailspike protein was dissociated from a preformed complex with the chaperone by addition of ATP at 42 degrees C. In contrast to other GroE ligands, the tailspike polypeptide was bound by and released from GroE in similar states of folding, and the presence of GroES in addition to GroEL had no effect on reconstitution yields at any temperature. Thus, the GroE proteins may exhibit widely differing interactions even with proteins showing similarly temperature-sensitive yields of folding.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteriófago P22/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas/metabolismo , Proteínas Virales/metabolismo , Chaperonina 10 , Chaperoninas , Proteínas de Escherichia coli , Técnicas In Vitro , Sustancias Macromoleculares , Unión Proteica , Conformación Proteica , Proteínas Recombinantes , Temperatura , Proteínas Virales/ultraestructuraRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to modulate different granulocyte functions. In the present study we investigated the effect of purified and recombinant human GM-CSF, particularly on the oxidative metabolism of isolated human granulocytes. In addition, ultrastructural changes upon stimulation were evaluated. For detection of granulocyte activation the following assay systems were used: 1) lucigenin-dependent chemiluminescence (CL), 2) superoxide-dismutase (SOD) inhibitable cytochrome C-reduction (superoxide), 3) horseradish peroxidase-mediated oxidation of phenol red (hydrogen peroxide), 4) release of myeloperoxidase, 5) ultrastructural detection of hydrogen peroxide-production, and 6) scanning and transmission electron microscopy (SEM and TEM, respectively). A significant CL response was seen upon stimulation with recombinant human GM-CSF at concentrations ranging from 1 to 10(3) U/ml. The CL response started within 5-10 min with a maximum at 60-90 min and lasted more than 3 h. Thereafter granulocytes were completely deactivated to restimulation with the same mediator and with Tumor Necrosis Factor, but responded to other triggers of the oxidative burst, whereas the response to f-met-leu-phe was significantly increased. The CL signal was completely blocked by an antiserum to GM-CSF. Moreover, the response was significantly inhibited by SOD and D-Mannitol, suggesting the involvement of distinct reactive oxygen species (ROS) in generating the CL response. Significant amounts of superoxide were detected within 180 min after stimulation with GM-CSF, whereas release of hydrogen peroxide and peroxidase were only minimal as shown by functional and ultrastructural assays. Activation of granulocytes could be visualized by SEM and TEM. GM-CSF stimulated cells showed an increased adherence to the substratum developing polarized filopodia and an increased number of intracellular vesicles within 30 min after addition of the stimulus. The results clearly demonstrate that GM-CSF directly stimulates granulocytes and, particularly, their oxidative metabolism. Therefore, GM-CSF which is probably released by epidermal cells appears to be a candidate for neutrophil activation in the skin, and thereby may play a crucial role in inflammatory skin diseases.
Asunto(s)
Factores Estimulantes de Colonias/farmacología , Granulocitos/fisiología , Sustancias de Crecimiento/farmacología , Neutrófilos/fisiología , Quimiotaxis , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Granulocitos/metabolismo , Granulocitos/ultraestructura , Humanos , Peróxido de Hidrógeno/biosíntesis , Mediciones Luminiscentes , Neutrófilos/metabolismo , Neutrófilos/ultraestructura , Peroxidasa/metabolismo , Superóxidos/biosíntesisRESUMEN
Keratinocytes are capable of releasing distinct immunomodulating cytokines such as epidermal cell-derived thymocyte activating factor (ETAF) and an epidermal cell-derived natural killer cell augmenting factor (ENKAF). The present study was performed to determine whether human keratinocytes also may secrete an interleukin 3 (IL-3)-like mediator and thereby participate in the regulation of mast cell activity in the skin. Supernatants of freshly isolated human epidermal cells (EC) and malignant keratinocyte cell lines (A 431, SCC) were tested for their capacity to induce the proliferation of IL-3-dependent cell lines 32 DCL and FDCP. Human epidermal cell interleukin 3 (EC IL-3) is spontaneously released by freshly isolated EC, A 431, and squamous cell carcinoma (SCC) cells. However, both normal EC and A 431 cells produced increased levels of EC IL-3 activity when cultured in the presence of different stimulants, such as phorbol myristate acetate and lipopolysaccharide. The EC IL-3 activity was not inhibited when treated with a monoclonal anti-IL-1 or anti-IL-2-antibody. Biochemical characterization showed that human EC IL-3 has a molecular weight of 17K, elutes of DEAE-ion exchange high-performance liquid chromatography (HPLC) as one major peak at 0.36 M NaCl, and upon HPLC-chromatofocusing exhibits 3 isoelectric points of 7.8, 7.5, and 5.6. Upon reversed-phase HPLC, EC IL-3 activity eluted at about 100% acetonitrile. When highly purified EC IL-3 was labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single homogeneous band exhibiting a molecular weight of 17K was seen, which correlated with the IL-3 activity and was free of ETAF/IL-1, IL-2, and interferon activity. These data indicate that human EC synthesize an IL-3-like cytokine which is distinct from ETAF/IL-1, IL-2, and interferon and thereby may participate in the regulation of mast cell activity during inflammatory and fibrotic, as well as hypersensitivity reactions.
Asunto(s)
Carcinoma de Células Escamosas/patología , Células Epidérmicas , Interleucina-3/fisiología , Queratinas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma de Células Escamosas/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Femenino , Humanos , Interleucina-1/fisiologíaRESUMEN
In the present study we investigated the capability of human epidermal cells to generate granulocyte-activating mediators (GRAM). It could be shown that human epidermal cells as well as an epidermoid carcinoma cell line (A431) produce an epidermal cell-derived granulocyte-activating mediator (EC-GRAM) which stimulates human granulocytes to release significant levels of toxic oxygen radicals as measured by a lucigenin-dependent chemiluminescence (CL). For further characterization of EC-GRAM the A431 cell line was used. Supernatants of A431 cells usually contained maximal EC-GRAM levels within 24 h of incubation. Factor production was enhanced by bacterial lipopolysaccharide (LPS), but not by silica particles and PHA. Moreover, freeze-thaw lysates of A431 cells and extracts of heat-separated human epidermis contained significant levels of EC-GRAM. Preincubation of granulocytes with EC-GRAM resulted in an enhanced response to subsequent stimulation with the chemotactic peptide f-met-phe. In contrast EC-GRAM did not affect the response to PMA or zymosan particles. However, EC-GRAM treated granulocytes were unresponsive to restimulation with EC-GRAM. Upon high performance liquid chromatography (HPLC) gel filtration EC-GRAM eluted within two major peaks exhibiting a molecular weight of 17 kD and 44 kD.(ABSTRACT TRUNCATED AT 250 WORDS)