RESUMO
PURPOSE: Trauma-related death is used as a parameter to evaluate the quality of trauma care and identify cases in which mortality could have been prevented under optimal trauma care conditions. The aim of this study was to identify trauma-related preventable death (TRPD) within our institute by an external expert panel and to evaluate inter-panel reliability. METHODS: Trauma-related deaths between the 1st of January 2020 and the 1st of February 2022 at the Amsterdam University Medical Centre were identified. The severely injured patients (injury severity score ≥ 16) were enrolled for preventability analysis by an external multidisciplinary panel, consisting of a trauma surgeon, anaesthesiologist, emergency physician, neurosurgeon, and forensic physician. Case descriptions were provided, and panellists were asked to classify deaths as non-preventable, potentially preventable, and preventable. Agreements between the five observers were assessed by Fleiss kappa statistics. RESULTS: In total 95 trauma-related deaths were identified. Of which 36 fatalities were included for analysis, the mean age was 55.3 years (± 24.5), 69.4% were male and 88.9% suffered blunt trauma. The mean injury severity score was 35.3 (± 15.3). Interobserver agreement within the external panel was moderate for survivability (Fleiss kappa 0.474) but low for categorical preventable death classification (Fleiss kappa 0.298). Most of the disagreements were between non-preventable or potentially preventable with care that could have been improved. CONCLUSION: Multidisciplinary panel review has a moderate inter-observer agreement regarding survivability and low agreement regarding categorical preventable death classification. A valid definition and classification of TRPD is required to improve inter-observer agreement and quality of trauma care.
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Development of ready-to-use biomaterials and scaffolds is vital for further advancement of scaffold-based tissue engineering in clinical practice. Scaffolds need to mimic 3D ultrastructure, have adequate mechanical strength, are biocompatible, non-immunogenic and need to promote tissue regeneration in vivo. Although decellularization of native tissues seems promising to deliver scaffolds that meet these criteria, adequate decellularization of hard, poorly penetrable and poorly diffusible tissues remains challenging whilst being a very time-consuming process. In this study, a method to decellularize hard, dense tissues using supercritical carbon-dioxide preceded by a freeze/thaw cycle and followed by several washing steps is presented, demonstrating decellularisation efficiency and substantially reduced production/handling time. Additionally, supercritical carbon-dioxide treatment was used as sterilization method, further reducing the time required to produce the final scaffold. Histological evaluation showed that, after fine-tuning of the process, a partially acellular scaffold was obtained, with preservation of glycosaminoglycans and collagen fibers, albeit that the amount of residual dsDNA was still higher then chemically decellularized tissue. Biomechanical properties of the scaffold were similar to the native, non-decellularized tissue. After sterilization with supercritical carbon-dioxide the simulated functional outcome was more similar to native trachea, when compared to sterilization using gamma irradiation. Thus, decellularization and sterilization using supercritical carbon-dioxide with washing steps is an effective method for dense cartilaginous materials, and tuneable to meet different demands in other applications, but further optimization may be required. STATEMENT OF SIGNIFICANCE: Further advancement of the use of tissue engineered tracheal constructs is restricted by the lack of the ideal scaffold. Decellularized trachea is considered a promising scaffold, but the hard, poorly diffusible tissue remains challenging while forming a very time consumable process. Decellularization using supercritical carbon dioxide (scCO2) seems promising, resulting in efficient removal of cellular material while reducing production and handling time. Addition of scCO2 as a sterilization method resulted in further time reduction while improving functional outcome in comparison with traditional sterilization methods. This study presents an promising alternative method for decellularization and sterilization of dense materials, which can be tuned to meet different demands in other applications.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Dióxido de Carbono/química , Esterilização/métodos , Materiais Biocompatíveis , Matriz ExtracelularRESUMO
INTRODUCTION: Twenty years ago the Dutch trauma care system was reformed by the designating 11 level one Regional trauma centres (RTCs) to organise trauma care. The RTCs set up the Dutch National Trauma Registry (DNTR) to evaluate epidemiology, patient distribution, resource use and quality of care. In this study we describe the DNTR, the incidence and main characteristics of Dutch acutely admitted trauma patients, and evaluate the value of including all acute trauma admissions compared to more stringent criteria applied by the national trauma registries of the United Kingdom and Germany. METHODS: The DNTR includes all injured patients treated at the ED within 48 hours after trauma and consecutively followed by direct admission, transfers to another hospital or death at the ED. DNTR data on admission years 2007-2018 were extracted to describe the maturation of the registry. Data from 2018 was used to describe the incidence rate and patient characteristics. Inclusion criteria of the Trauma Audit and Research (TARN) and the Deutsche Gesellschaft für Unfallchirurgie (DGU) were applied on 2018 DNTR data. RESULTS: Since its start in 2007 a total of 865,460 trauma cases have been registered in the DNTR. Hospital participation increased from 64% to 98%. In 2018, a total of 77,529 patients were included, the median age was 64 years, 50% males. Severely injured patients with an ISS≥16, accounted for 6% of all admissions, of which 70% was treated at designated RTCs. Patients with an ISS≤ 15were treated at non-RTCs in 80% of cases. Application of DGU or TARN inclusion criteria, resulted in inclusion of respectively 5% and 32% of the DNTR patients. Particularly children, elderly and patients admitted at non-RTCs are left out. Moreover, 50% of ISS≥16 and 68% of the fatal cases did not meet DGU inclusion criteria CONCLUSION: The DNTR has evolved into a comprehensive well-structured nationwide population-based trauma register. With 80,000 inclusions annually, the DNTR has become one of the largest trauma databases in Europe The registries strength lies in the broad inclusion criteria which enables studies on the burden of injury and the quality and efficiency of the entire trauma care system, encompassing all trauma-receiving hospitals.
Assuntos
Centros de Traumatologia , Ferimentos e Lesões , Idoso , Criança , Europa (Continente) , Feminino , Alemanha , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Sistema de Registros , Reino Unido , Ferimentos e Lesões/epidemiologia , Ferimentos e Lesões/terapiaAssuntos
Transplante de Rim/estatística & dados numéricos , Rim , Doadores de Tecidos , Obtenção de Tecidos e Órgãos/organização & administração , Adulto , Idoso , Morte Encefálica , Cadáver , Reanimação Cardiopulmonar , Causas de Morte , Sobrevivência de Enxerto , Parada Cardíaca , Humanos , Transplante de Rim/fisiologia , Pessoa de Meia-Idade , Países Baixos , Preservação de Órgãos/métodos , Preservação de Órgãos/estatística & dados numéricosRESUMO
In this study, the short-term outcome of renal transplants from non-heart-beating donors (NHBD) preserved by machine perfusion (MP) is evaluated and compared to preservation by cold storage (CS). Twenty-two NHBD kidneys were procured during 1993 and 1994 after in situ perfusion with histidine-tryptophan ketoglutarate and preserved by continuous perfusion using University of Wisconsin organ preservation solution for MP as a perfusate. Between 1980 and 1992, 57 NHBD kidneys were procured and preserved by CS. Donors in the MP group sustained increased first warm ischemia times (WIT1) (P < 0.1) and recipients in the MP group suffered longer anastomosis time, worse HLA-DR mismatch, and more initial use of cyclosporin as immunosuppressant; all these factors are known to be deleterious to short-term outcome. Despite these unfavorable conditions, delayed function (DF) rate was decreased in the MP group, although not significantly. However, when considering only kidneys with WIT1 > or = 45 min, short-term outcome was significantly better in the MP group (P < 0.05). We conclude that MP is superior for the preservation of NHBD kidneys, especially after prolonged warm ischemia.
Assuntos
Transplante de Rim , Preservação de Órgãos , Adulto , Temperatura Baixa , Coração/fisiopatologia , Humanos , Pessoa de Meia-Idade , Perfusão , Doadores de TecidosRESUMO
Treatment of proximal femoral fractures is difficult. A retrospective analysis of 92 patients treated with a custom-made extended gamma nail with a mean follow up of 4 months is presented. The early results of this new method are promising. The indications for the use of the long gamma nail were intertrochanteric fractures with extension to the subtrochanteric region, imminent and pathologic fractures of the intertrochanteric and subtrochanteric region, mostly in elderly patients, proximal femoral fractures, and combination injuries in multiple trauma patients and delayed unions. In 67 cases the patients were primarily treated with a long Gamma nail. In 25 cases the nail was used after the failure of initial treatment. Complications occurred in 32 patients; however, 88 patients were able to walk at the end of the study. The advantage of this new method is that with a closed surgical technique full weight bearing directly after the operation is achieved. The long Gamma nail provides a procedure by which most complex fractures in the proximal femur can be managed.
Assuntos
Pinos Ortopédicos , Fixação Intramedular de Fraturas/métodos , Fraturas do Quadril/cirurgia , Idoso , Pinos Ortopédicos/efeitos adversos , Feminino , Fixação Intramedular de Fraturas/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Suporte de CargaRESUMO
A Dictyostelium discoideum mutant defective in folate chemotaxis has been analysed using biochemical, behavioural, and genetic methods. A subset of the cell-surface folate binding sites appeared to be locked in a high-affinity state from which folate dissociated extremely slowly. Changes in cell area and motility induced by step increases in folate required 10- to 100-fold higher concentrations than in the wild type. Folate-stimulated cyclic GMP production was also altered. Chemotactic responses to cyclic AMP as well as cyclic AMP-stimulated cyclic GMP production were normal. The mutation responsible for the chemotaxis defect, termed folA1000, was localized to linkage group IV. The alterations in folate binding and sensitivity to folate co-localized with the folA1000 mutation. We conclude that the folA1000 mutation arrests the folate chemotaxis receptor in a high affinity state that can only poorly transduce folate binding into chemotactic responses.
Assuntos
Quimiotaxia , Dictyostelium/fisiologia , Mutação , Ácido Fólico/metabolismoRESUMO
The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with Kd values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 10(-5) M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10-100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTP gamma S binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.
Assuntos
Células Quimiorreceptoras/metabolismo , Fatores Quimiotáticos/metabolismo , Dipeptídeos/metabolismo , Lactamas/metabolismo , Mixomicetos/crescimento & desenvolvimento , Quimiotaxia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Dictyostelium/citologia , Cinética , Luz , Peso Molecular , Mixomicetos/análise , Espalhamento de RadiaçãoRESUMO
GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTP gamma S greater than GTP greater than 8-N3-GTP; the binding of GTP gamma S and GTP, but not of 8-N3-GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (Kd = 2.6 microM, Bmax = 85 nM). More detailed studies with [35S]GTP gamma S showed heterogeneous binding composed of two forms of binding sites with respectively high (Kd = 0.2 microM) and low (Kd = 6.3 microM) affinity. cAMP derivatives enhanced GTP gamma S binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTP gamma S binding showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTP gamma S binding, but eliminated the cAMP stimulation of GTP and GTP gamma S binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.
Assuntos
Dictyostelium/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Toxina Pertussis , Receptores de AMP Cíclico/fisiologia , Tionucleotídeos/metabolismo , Fatores de Virulência de Bordetella/farmacologia , Membrana Celular/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Dictyostelium/efeitos dos fármacos , Nucleotídeos de Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato) , CinéticaRESUMO
The response of guanylate cyclase to addition of extracellular stimuli is well documented. Here we report for the first time the response of guanylate cyclase to removal of stimuli. Three methods were employed to terminate rapidly a stimulus of folic acid. (1) Addition of a highly active folate deaminase preparation, or (2) 12-fold dilution of the stimulated cell suspension, or (3) addition of an excess concentration of a non-agonistic derivative of folic acid, i.e., 2-deaminofolic acid, which chases the folate agonist from its cell-surface receptors. Accumulation of cGMP terminated instantaneously upon addition of deaminase, but degradation of the synthesized cGMP was not observed until 10-12 s after stimulation. Also in a cGMP phosphodiesterase-lacking 'streamer' mutant an instantaneous termination of further cGMP accumulation was observed upon stimulus removal. This suggests that the termination of cGMP accumulation is due to inactivation of guanylate cyclase instead of a steady state of cGMP synthesis and degradation. Further accumulation of cGMP was approx. 75% reduced upon dilution of a cell suspension after stimulation with both agonists. Stimulation by 300 nM folic acid or by 30 nM N10-methylfolic acid (a more potent agonist) yielded identical results. However, upon addition of deaminofolic acid the accumulation of cGMP continued normally if the cells had been stimulated with N10-methylfolic acid, but only slightly in the case of a folic acid stimulus. The effect of stimulus duration on desensitization was monitored; it was observed that 50% desensitization was induced by stimulation for 1 s, while 4 s was sufficient for maximal desensitization. Short stimuli were observed to elicit high levels of desensitization without much excitation of guanylate cyclase. A desensitization-like process was observed at the level of the folate-binding chemotactic receptors as well. Relationships between the cGMP response data and folic acid receptor kinetics are discussed.
Assuntos
Proteínas de Transporte/metabolismo , Dictyostelium/metabolismo , Guanilato Ciclase/metabolismo , Proteínas de Protozoários , Receptores de Superfície Celular , Aminoidrolases/farmacologia , GMP Cíclico/biossíntese , Receptores de Folato com Âncoras de GPI , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Cinética , Metotrexato/análogos & derivados , Metotrexato/metabolismo , Metotrexato/farmacologia , Conformação ProteicaRESUMO
In a wide range of cell types, stimulus-response coupling is accompanied by a rise in cytoplasmic pH (pHi). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pHi. About 1.5 min after stimulation, pHi starts increasing from pHi approximately 7.45 to pHi approximately 7.60, as is revealed independently by two different pH null-point methods. The rise in pHi is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pHi is accomplished by the DES-sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.
Assuntos
AMP Cíclico/fisiologia , Dictyostelium/fisiologia , Concentração de Íons de Hidrogênio , Diferenciação Celular , Quimiotaxia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Citoplasma/fisiologia , Tionucleotídeos/farmacologia , Fatores de TempoRESUMO
In Dictyostelium discoideum cells, extracellular cAMP induces the rapid (within 2 s) activation of guanylate cyclase, which is followed by complete desensitization after about 10 s. cAMP binding to these cells is heterogeneous, showing a subclass of fast dissociating sites coupled to adenylate cyclase (A-sites) and a subclass of slowly dissociating sites coupled to guanylate cyclase (B-sites). The kinetics of the B-sites were further investigated on a seconds time scale. Statistical analysis of the association of [3H]cAMP to the B-sites and dissociation of the complex revealed that the receptor can exist in three states which interconvert according to the following scheme. (formula; see text). cAMP binds to the BF-state (off-rate 2.5 s) which rapidly (t1/2 = 3 s) converts to the BS-state (off-rate 15 s) and subsequently (without a detectable delay) into the BSS-state (off-rate 150 s). In membranes, both the BS- and BSS-states are converted to the BF-state by GTP and GDP, suggesting the involvement of a G-protein. Densensitized cells show a 80% reduction of the formation of the BSS-state, but no reduction of the BF- or BS-state. These data are combined into a model in which the transitions of the B-sites are mediated by a G-protein; activation of the G-protein and guanylate cyclase is associated with the transition of the BS- to the BSS-state of the receptor, whereas desensitization is associated with the inhibition of this transition.
Assuntos
Dictyostelium/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanilato Ciclase/metabolismo , Receptores de AMP Cíclico/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/enzimologia , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Cinética , Matemática , TrítioRESUMO
Recently, we demonstrated the presence of multiple folate-binding sites on the cell surface of Dictyostelium discoideum. These sites were divided into two major classes, with different ligand specificities (A and B). Each major class consists of several interconvertible subtypes. In the present report, the ability of 13 folate analogs to activate both adenylate and guanylate cyclase in pre- as well as postaggregative cells is examined. The patterns of correlation between binding and activation data indicate that guanylate cyclase activation is mediated by the B-sites in both developmental stages (P less than 0.001). In postaggregative cells, adenylate cyclase also seems to be activated by the B-sites (P less than 0.001). In contrast, adenylate cyclase activation in preaggregative cells was well correlated with the specificity of A-sites (P less than 0.01). Remarkably, the potencies of activation were less affected by molecular modifications than the binding affinities were, as suggested by a slope of 0.4 in a plot of K0.5 values of activation vs. binding. This observation argues against the existence of a transduction mechanism in which the response is proportional to receptor occupancy. For the B-receptor, however, the degree of receptor occupancy appears to determine the response. The existence of folic acid antagonists is demonstrated, some of which are specific for either A-sites coupled to adenylate cyclase or for B-sites coupled to guanylate cyclase.
Assuntos
Proteínas de Transporte/metabolismo , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Dictyostelium/metabolismo , Ácido Fólico/farmacologia , Receptores de Superfície Celular , Adenilil Ciclases/metabolismo , Dictyostelium/enzimologia , Ativação Enzimática , Receptores de Folato com Âncoras de GPI , Ácido Fólico/análogos & derivados , Antagonistas do Ácido Fólico/análise , Guanilato Ciclase/metabolismo , CinéticaRESUMO
In membrane preparations from D. discoideum cells GTP-binding activity is observed. The lack of GTP binding to intact cells suggests that the binding sites are localized inside the cell. The GTP-binding activity also remains in the particulate fraction in the presence of 1 mM Ca++. This excludes membrane-associated microtubuli to be responsible for the observed GTP binding. Scatchard analysis suggests the existence of one type of binding site (Kd = 2.6 microM and 3.6 X 10(5) sites per cell). The kinetics of association as well as dissociation, however, suggest that GTP binding is more complex than binding to a single type of site. GDP and guanylyl imidodiphosphate are potent competitors of GTP binding (respectively 5- and 10-fold worse than GTP) while GMP, cGMP and several adenine nucleotides are ineffective up to 1 mM. The chemoattractants cAMP and folic acid both increase the equilibrium binding level of GTP, while dissociation of GTP is accelerated. These data indicate the functional coupling between cell surface receptors and G-proteins.
Assuntos
AMP Cíclico/farmacologia , Dictyostelium/metabolismo , Ácido Fólico/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Guanosina Difosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , CinéticaRESUMO
D. discoideum cells contain surface receptors for the chemoattractant cAMP which are composed of fast and slowly dissociating binding sites with half-lifes of respectively about 1 s and 15 s (Van Haastert and De Wit, J. Biol. Chem. 259, 13321-13328). In membranes prepared by shearing the cells through a Nucleopore filter, ATP has no effect on cAMP-binding at equilibrium, but the number of slowly dissociating sites is increased about 2-fold by ATP while their apparent affinity and off-rate are not altered by ATP. The effect of ATP is stimulated about 3-fold by Ca2+ with a half maximal effect at 100 microM Ca2+. The tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases this Ca2+-sensitivity of the ATP effect to about 0.2 microM Ca2+. These data suggest that a specific subpopulation of cAMP receptors in membranes from D. discoideum is altered by the action of protein kinase C.
Assuntos
Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Forbóis/farmacologia , Proteínas Quinases/metabolismo , Receptores de AMP Cíclico/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , AMP Cíclico/metabolismo , Dictyostelium , Nucleotídeos de Guanina/farmacologia , Cinética , Proteína Quinase C , Receptores de AMP Cíclico/efeitos dos fármacosRESUMO
Dictyostelium discoideum cells show 2 distinct classes of cell surface binding sites for folates. One type is non-specific, i.e., binds folic acid (FA), 2-deaminofolic acid (DAFA), and methotrexate (MTX) with similar affinity (K0.5 congruent to 140 nM). Scatchard analysis of this non-specific binding type suggests either heterogeneity or negative cooperativity. Isolated D. discoideum membranes show similar binding characteristics. Guanine nucleotides changed the binding levels of [3H]MTX. In the presence of 0.1 mM GTP, the number of binding sites remains unchanged, while the affinity decreases. GDP and guanylyl imidodiphosphate (GPPNP) are required at about 20-fold higher concentration than GTP, which elicits a half-maximal effect at 15 microM. Other guanine and adenine nucleotides are ineffective up to 1 mM. These results suggests that the non-specific cell surface receptor for folic acid interacts with a guanine nucleotide regulatory (G-) protein.
Assuntos
Proteínas de Transporte/metabolismo , Dictyostelium/metabolismo , Nucleotídeos de Guanina/farmacologia , Receptores de Superfície Celular , Ligação Competitiva , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Receptores de Folato com Âncoras de GPI , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Cinética , Metotrexato/metabolismoRESUMO
The binding of [3H]cAMP to Dictyostelium discoideum cells was analyzed on a seconds time scale under both equilibrium and nonequilibrium conditions. The binding of [3H]cAMP increases rapidly to a maximum obtained at about 6 s, which is followed by a decrease to an equilibrium value reached at about 45 s. This decrease of [3H]cAMP binding is not the result of ligand degradation or isotope dilution by cAMP secretion but is due to a transition of high-affinity binding to low-affinity binding. Analysis of the dissociation rate of [3H]cAMP from the binding sites indicates that these high- and low-affinity binding sites are both fast dissociating with a half-life of about 1 s. In addition, these dissociation experiments reveal a third binding type which is slowly dissociating with a half-life of about 15 s. The number and affinity of these slowly dissociating sites does not change during the incubation with [3H]cAMP. The drugs caffeine and chlorpromazine do not change the total number of binding sites, but they change the ratio of the three binding types. In the presence of 10 mM caffeine almost all binding sites are in the low affinity conformation, while in the presence of 0.1 mM chlorpromazine the ratio is shifted to both the high-affinity type and slowly dissociating type. The results indicate that the cAMP-binding activity of D. discoideum cells is heterogeneous. In the absence of cAMP about 4% of the sites are slowly dissociating with Kd = 12.5 nM, about 40% are fast dissociating with high affinity (Kd = 60 nM), and about 60% are fast dissociating with low affinity (Kd = 450 nM). During the binding reaction the number of slowly dissociating sites does not change. The number of high-affinity sites decreases to a minimum of about 10% with a concomitant increase of low-affinity sites to about 90%. This transition of binding types shows first-order kinetics with a half-life of about 9 s. A half-maximal transition is induced by 12.5 nM cAMP.
Assuntos
Dictyostelium/metabolismo , Receptores de AMP Cíclico/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Cinética , Matemática , Modelos Biológicos , TrítioRESUMO
The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and RP-cAMPN(CH3)2. We have investigated the agonistic and antagonistic activities of these compounds in four cAMP-dependent reactions: activation of the cellular slime mold Dictyostelium discoideum via its cell surface cAMP receptor, and phosphorylation by cAMP-dependent protein kinases type I, type II (both mammalian enzymes), and type D (derived from D. discoideum). The results show that 1) the compounds (SP)-cAMPS and (SP)-cAMPN(CH3)2 are (mostly full) agonists for the four proteins. Half-maximal activation is at micromolar concentrations (0.8-7 microM). 2) (RP)-cAMPS is a full antagonist for the cell surface receptor and protein kinases type I and II, with apparent inhibition constants between 0.8 and 8 microM. This compound is a partial agonist for protein kinase type D, where it induces maximally 50% activation of the enzyme if compared with cAMP. 3) (RP)-cAMPN(CH3)2 is a full antagonist for the cell surface receptor, and for protein kinase type II. This compound is a partial agonist for protein kinase type I (at least 50% activation if compared with cAMP), and inactive for protein kinase type D. This derivative is at least 25-fold less active as an antagonist than (RP)-cAMPS. 4) The activity of mixtures of different concentrations of the antagonist (RP)-cAMPS with different concentrations of cAMP reveals that the compound is a competitive antagonist of cAMP at micromolar concentrations.