RESUMEN
Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88% of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41% of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Cationes Bivalentes , Células Cultivadas , Cromatografía Líquida de Alta Presión , Ácido Edético , Humanos , Hidrólisis , Radioisótopos de Yodo , Cinética , Neprilisina/metabolismo , Concentración Osmolar , Receptores del Factor Natriurético Atrial/metabolismoRESUMEN
Space flight provides a model for the study of healthy individuals undergoing unique stresses. This review focuses on how physiological adaptations to weightlessness may affect nutrient and food requirements in space. These adaptations include reductions in body water and plasma volume, which affect the renal and cardiovascular systems and thereby fluid and electrolyte requirements. Changes in muscle mass and function may affect requirements for energy, protein and amino acids. Changes in bone mass lead to increased urinary calcium concentrations, which may increase the risk of forming renal stones. Space motion sickness may influence putative changes in gastro-intestinal-hepatic function; neurosensory alterations may affect smell and taste. Some or all of these effects may be ameliorated through the use of specially designed dietary countermeasures.
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Adaptación Fisiológica , Necesidades Nutricionales , Vuelo Espacial , HumanosRESUMEN
Postflight orthostatic intolerance is experienced by virtually all astronauts but differs greatly in degree of severity. We studied cardiovascular responses to upright posture in 40 astronauts before and after spaceflights lasting up to 16 days. We separated individuals according to their ability to remain standing without assistance for 10 min on landing day. Astronauts who could not remain standing on landing day had significantly smaller increases in plasma norepinephrine levels with standing than did those who could remain standing (105 +/- 41 vs. 340 +/- 62 pg/ml; P = 0.05). In addition, they had significantly lower standing peripheral vascular resistance (23 +/- 3 vs. 34 +/- 3 mmHg.1l-1).min; P = 0.02) and greater decreases in systolic (-28 +/- 4 vs. -11 +/- 3 mmHg; P = 0.002) and diastolic (-14 +/- 7 vs. 3 +/- 2 mmHg; P = 0.0003) pressures. The presyncopal group also had significantly lower supine (16 +/- 1 vs. 21 +/- 2 mmHg.1l-1).min; P = 0.04) and standing (23 +/- 2 vs. 32 +/- 2 mmHg.1l-1).min; P = 0.038) vascular resistance, supine (66 +/- 2 vs. 73 +/- 2 mmHg; P = 0.008) and standing (69 +/- 4 vs. 77 +/- 2 mmHg; P = 0.007) diastolic pressure, and supine (109 +/- 3 vs. 114 +/- 2 mmHg; P = 0.05) and standing (99 +/- 4 vs. 108 +/- 3 mmHg; P = 0.006) systolic pressures before flight. This is the first study to clearly document these differences among presyncopal and nonpresyncopal astronauts after spaceflight and also offer the possibility of preflight prediction of postflight susceptibility. These results clearly point to hypoadrenergic responsiveness, possibly centrally mediated, as a contributing factor in postflight orthostatic intolerance. They may provide insights into autonomic dysfunction in Earthbound patients.
Asunto(s)
Norepinefrina/metabolismo , Vuelo Espacial , Síncope Vasovagal/metabolismo , Adulto , Presión Sanguínea/fisiología , Femenino , Frecuencia Cardíaca/fisiología , Hemodinámica/fisiología , Humanos , Hipotensión Ortostática/fisiopatología , Masculino , Postura/fisiología , Pruebas de Mesa Inclinada , Resistencia Vascular/fisiologíaRESUMEN
Plasma catecholamine levels and cardiovascular responses to standing were determined in astronauts before and after several Space Shuttle missions. Blood pressure, heart rate, and cardiac output were measured and blood samples for catecholamine analyses were drawn at the end of the supine and standing periods. Supine plasma norepinephrine and epinephrine concentrations increased 34 and 65%, respectively, on landing day compared with before flight. Standing on landing day resulted in a 65 and 91% increase in plasma norepinephrine and epinephrine, respectively. Supine and standing norepinephrine levels remained elevated 3 days after landing while epinephrine levels returned to preflight levels. On landing day, supine heart rate and systolic blood pressure increased 18 and 8.9%, respectively, and standing heart rate and diastolic blood pressure were elevated by 38 and 19%, respectively. On standing, stroke volume was decreased by 26% on landing day compared with before flight. Collectively, these data indicate that the decreased orthostatic function after spaceflight results largely from the decreased stroke volume. Possible mechanisms contributing to this condition are discussed.
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Glándulas Suprarrenales/fisiología , Postura/fisiología , Vuelo Espacial , Sistema Nervioso Simpático/fisiología , Adulto , Aldosterona/sangre , Presión Sanguínea/fisiología , Peso Corporal/fisiología , Gasto Cardíaco/fisiología , Catecolaminas/sangre , Electrocardiografía , Femenino , Frecuencia Cardíaca/fisiología , Hematócrito , Hemodinámica/fisiología , Humanos , Masculino , Persona de Mediana Edad , Renina/sangre , Volumen Sistólico/fisiologíaRESUMEN
Rat osteoblasts were cultured for 4 or 5 days during a Space Shuttle mission. After 20-h treatment with 1alpha,25-dihydroxyvitamin D3, conditioned media were harvested and cellular DNA and/or RNA were fixed on board. The insulin-like growth factor binding protein (IGF BP)-3 levels in the media were three- and tenfold higher than in ground controls on the fourth and fifth flight days, as quantitated by Western ligand blotting and radioimmunoassay, respectively. The increased IGF BP-3 protein levels correlated with two- to threefold elevation of IGF BP-3 mRNA levels, obtained by reverse transcription-polymerase chain reaction. The IGF BP-5 mRNA levels in flight cultures were 33-69% lower than in ground controls. The IGF BP-4 mRNA levels in flight cultures were 75% lower than in ground controls on the fifth day but were not different on the fourth day. The glucocorticoid receptor mRNA levels in flight cultures were increased by three- to eightfold on the fourth and fifth days compared with levels in ground controls. These data suggest potential mechanisms underlying spaceflight-induced osteopenia.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Osteoblastos/metabolismo , Receptores de Glucocorticoides/metabolismo , Vuelo Espacial , Ingravidez/efectos adversos , Animales , Células Cultivadas , ADN/biosíntesis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Masculino , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Ratas WistarRESUMEN
This minireview provides an overview of known and potential gender differences in physiological responses to spaceflight. The paper covers cardiovascular and exercise physiology, barophysiology and decompression sickness, renal stone risk, immunology, neurovestibular and sensorimotor function, nutrition, pharmacotherapeutics, and reproduction. Potential health and functional impacts associated with the various physiological changes during spaceflight are discussed, and areas needing additional research are highlighted. Historically, studies of physiological responses to microgravity have not been aimed at examining gender-specific differences in the astronaut population. Insufficient data exist in most of the discipline areas at this time to draw valid conclusions about gender-specific differences in astronauts, in part due to the small ratio of women to men. The only astronaut health issue for which a large enough data set exists to allow valid conclusions to be drawn about gender differences is orthostatic intolerance following shuttle missions, in which women have a significantly higher incidence of presyncope during stand tests than do men. The most common observation across disciplines is that individual differences in physiological responses within genders are usually as large as, or larger than, differences between genders. Individual characteristics usually outweigh gender differences per se.
Asunto(s)
Caracteres Sexuales , Vuelo Espacial , Ejercicio Físico/fisiología , Femenino , Humanos , Masculino , Estados Unidos , United States National Aeronautics and Space Administration , Ingravidez/efectos adversosRESUMEN
In an effort to understand the interaction between acute postural fluid shifts and hypoxia on hormonal regulation of fluid homeostasis, the authors measured the responses to head-down tilt with and without acute exposure to normobaric hypoxia. Plasma atrial natriuretic peptide (ANP), cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), plasma aldosterone (ALD), and plasma renin activity (PRA) were measured in six healthy male volunteers who were exposed to a head-down tilt protocol during normoxia and hypoxia. The tilt protocol consisted of a 17 degrees head-up phase (30 minutes), a 28 degrees head-down phase (1 hour), and a 17 degrees head-up recovery period (2 hours, with the last hour normoxic in both experiments). Altitude equivalent to 14,828 ft was simulated by having the subjects breathe an inspired gas mixture with 13.9% oxygen. The results indicate that the postural fluid redistribution associated with a 60-minute head-down tilt induces the release of ANP and cGMP during both hypoxia and normoxia. Hypoxia increased cGMP, cAMP, ALD, and PRA throughout the protocol and significantly potentiated the increase in cGMP during head-down tilt. Hypoxia had no overall effect on the release of ANP, but appeared to attenuate the increase with head-down tilt. This study describes the acute effects of hypoxia on the endocrine response during fluid redistribution and suggests that the magnitude, but not the direction, of these changes with posture is affected by hypoxia.
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Líquidos Corporales/fisiología , Homeostasis , Hormonas/sangre , Hipoxia/fisiopatología , Postura , Adulto , Aldosterona/sangre , Factor Natriurético Atrial/sangre , AMP Cíclico/sangre , GMP Cíclico/sangre , Cabeza/fisiología , Humanos , Hipoxia/complicaciones , Masculino , Renina/sangreRESUMEN
It has been suggested that microgravity alters bone metabolism. Evidence for this phenomenon includes the negative calcium balance and decreased bone density in astronauts, as well as, inhibition of bone formation in rats flown for 2 to 3 weeks. However, the specific mechanisms that modulate these changes in microgravity are unknown. The purpose of this study was to clarify the mechanism of microgravity-induced bone demineralization using normal rat osteoblasts obtained from femur marrow cultures. The osteoblasts were cultured for 5 days during a Shuttle-Spacelab flight (STS-65). After collection of the culture medium, the cellular DNA and RNA were fixed on board. Enzyme-immunoassay of the culture medium for prostaglandin E2 (PGE2) indicated that microgravity induced a 4.5- to 136-fold increase in flight samples as compared to the ground control cultures. This increase of PGE2 production was consistent with a 3.3- to 9.5-fold elevation of inducible prostaglandin G/H synthase-2 (PGHS-2) mRNA, quantitated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA induction for the constitutive isozyme PGHS-1 was less than that for PGHS-2. The interleukin-6 (IL-6) mRNA was also increased (6.4- to 9.3-fold) in microgravity as compared to the ground controls. Since PGE2 and IL-6 are both known to play a role in osteoclast formation and bone resorption, these data provide molecular mechanisms that contribute to our understanding of microgravity-induced alterations in the bone resorption process.
Asunto(s)
Resorción Ósea/etiología , Dinoprostona/biosíntesis , Interleucina-6/biosíntesis , Osteoblastos/metabolismo , Vuelo Espacial , Ingravidez/efectos adversos , Animales , Secuencia de Bases , Biotecnología/instrumentación , Densidad Ósea , Resorción Ósea/genética , Resorción Ósea/metabolismo , Células Cultivadas , Cartilla de ADN/genética , Humanos , Interleucina-6/genética , Reacción en Cadena de la Polimerasa , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Vuelo Espacial/instrumentaciónRESUMEN
A high-performance liquid chromatographic method was developed as an alternative to automated enzymatic analysis of uric acid in human urine preserved with thymol and/or thimerosal. Uric acid (tR = 10 min) and creatinine (tR = 5 min) were separated and quantified during isocratic elution (0.025 M acetate buffer, pH 4.5) from a mu Bondapak C18 column. The uric-acid peak was identified chemically by incubating urine samples with uricase. The thymol/thimerosal peak appeared at 31 min during the washing step and did not interfere with the analysis. We validated the high-performance liquid chromatographic method for linearity, precision and accuracy, and the results were found to be excellent.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Conservadores Farmacéuticos , Timerosal , Timol , Ácido Úrico/orina , Creatinina/orina , Estabilidad de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Urine composition in astronauts during and immediately after spaceflight changes in ways that increase the renal stone-forming potential for calcium oxalate, calcium phosphate, and uric acid saturation. We examined the effect of urine volume on the risk of renal stone formation in 356 astronauts. METHODS: Renal stone-forming risk was evaluated from 24-h urine samples collected from astronauts before and after 4- to 17-d Space Shuttle flights. Urinary chemistries were performed and the relative supersaturations of calcium oxalate, brushite, sodium urate, struvite, and uric acid saturation were calculated from the biochemical results. RESULTS: Urinary supersaturation levels of stone-forming salts were inversely related to urinary output both before and after spaceflight. Urine volume > 2 L x d(-1) reduced the risk of renal-stone development without affecting urinary citrate concentrations as compared with the increased risk observed in those astronauts who excreted urine volumes < L x d(-1). CONCLUSION: Results from this study indicate that increasing daily urinary output alone is an effective countermeasure to reduce the renal stone-forming risk immediately after spaceflight. However, increasing urinary output during flight may not be entirely effective in minimizing the potential risk of renal stone formation due to the changes in the urine chemistry in astronauts exposed to microgravity. KEYWORDS: urine volume, spaceflight, renal calculi.
Asunto(s)
Astronautas , Calcio/orina , Cálculos Renales/prevención & control , Vuelo Espacial , Adulto , Análisis de Varianza , Oxalato de Calcio/análisis , Medio Ambiente Extraterrestre , Humanos , Cálculos Renales/etiología , Cálculos Renales/metabolismo , Factores de Riesgo , OrinaAsunto(s)
Endotelio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Transporte Biológico Activo , Barrera Hematoencefálica , Permeabilidad Capilar , Bovinos , División Celular , Células Cultivadas , Endotelio Vascular/citología , Regulación de la Expresión Génica , Hemorreología , Humanos , Sustancias Macromoleculares , Músculo Liso Vascular/citología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo IIRESUMEN
An operationally available diagnostic imaging capability augments spaceflight medical support by facilitating the diagnosis, monitoring and treatment of medical or surgical conditions, by improving medical outcomes and, thereby, by lowering medical mission impacts and the probability of crew evacuation due to medical causes. Microgravity-related physiological changes occurring during spaceflight can affect the genitourinary system and potentially cause conditions such as urinary retention or nephrolithiasis for which ultrasonography (U/S) would be a useful diagnostic tool. This study describes the first genitourinary ultrasound examination conducted in space, and evaluates image quality, frame rate, resolution requirements, real-time remote guidance of nonphysician crew medical officers and evaluation of on-orbit tools that can augment image acquisition. A nonphysician crew medical officer (CMO) astronaut, with minimal training in U/S, performed a self-examination of the genitourinary system onboard the International Space Station, using a Philips/ATL Model HDI-5000 ultrasound imaging unit located in the International Space Station Human Research Facility. The CMO was remotely guided by voice commands from experienced, earth-based sonographers stationed in Mission Control Center in Houston. The crewmember, with guidance, was able to acquire all of the target images. Real-time and still U/S images received at Mission Control Center in Houston were of sufficient quality for the images to be diagnostic for multiple potential genitourinary applications. Microgravity-based ultrasound imaging can provide diagnostic quality images of the retroperitoneum and pelvis, offering improved diagnosis and treatment for onboard medical contingencies. Successful completion of complex sonographic examinations can be obtained even with minimally trained nonphysician ultrasound operators, with the assistance of ground-based real-time guidance.
Asunto(s)
Pelvis/diagnóstico por imagen , Espacio Retroperitoneal/diagnóstico por imagen , Vuelo Espacial , Ingravidez , Técnicos Medios en Salud/educación , Astronautas , Estudios de Factibilidad , Humanos , Interpretación de Imagen Asistida por Computador/métodos , Radiología/educación , Consulta Remota/métodos , Ultrasonografía , Grabación en Video/métodosRESUMEN
Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Membrana Celular/enzimología , Endopeptidasas/metabolismo , Endotelio Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , Endotelio Vascular/citología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The dissociation kinetics for repressor-32P-labeled operator DNA have been examined by adding unlabeled operator DNA to trap released repressor or by adding a small volume of concentrated salt solution to shift the Kd of repressor-operator interaction. The dissociation rate constant for pLA 322-8, an operator-containing derivative of pBR 322, was 2.4 X 10(-3) s-1 in 0.15 M KCl. The dissociation rate constant at 0.15 M KCl for both lambda plac and pIQ, each of which contain two pseudooperator sequences, was approximately 6 X 10(-4) s-1. Elimination of flanking nonspecific DNA sequences by use of a 40 base pair operator-containing DNA fragment yielded a dissociation rate constant of 9.3 X 10(-3) s-1. The size and salt dependences of the rate constants suggest that dissociation occurs as a multistep process. The data for all the DNAs examined are consistent with a sliding mechanism of facilitated diffusion to/from the operator site. The ability to form a ternary complex of two operators per repressor, determined by stoichiometry measurements, and the diminished dissociation rates in the presence of intramolecular nonspecific and pseudooperator DNA sites suggest the formation of an intramolecular ternary complex. The salt dependence of the dissociation rate constant for pLA 322-8 at high salt concentrations converges with that for a 40 base pair operator. The similarity in dissociation rate constants for pLA 322-8 and a 40 base pair operator fragment under these conditions indicates a common dissociation mechanism from a primary operator site on the repressor.
Asunto(s)
ADN Bacteriano/metabolismo , Escherichia coli/genética , Regiones Operadoras Genéticas , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Escherichia coli/metabolismo , Cinética , Peso Molecular , Concentración Osmolar , Cloruro de Potasio/farmacología , Unión Proteica , Biosíntesis de ProteínasRESUMEN
The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal. Arginine reaction was quantitated by either amino acid analysis or incorporation of 14C-labeled phenylglyoxal. Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees. The correlation of the decrease in DNA binding activities with the modification of approximately 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s). For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished. This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions. These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA.
Asunto(s)
Aldehídos/farmacología , Arginina , Butanonas/farmacología , Diacetil/farmacología , Fenilglioxal/farmacología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , ADN/metabolismo , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.
Asunto(s)
Expresión Génica/fisiología , Células HeLa/citología , Células HeLa/fisiología , Hipergravedad , Transducción de Señal/fisiología , Ciclo Celular/fisiología , División Celular/fisiología , Centrifugación , AMP Cíclico/metabolismo , Genes myc/genética , Células HeLa/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Fosforilación , Timidina/farmacocinética , Células Tumorales CultivadasRESUMEN
Sep-Pak C18 extraction of human plasma and radioimmunoassay using antibodies which recognize atrial natriuretic peptide (99-128) and the prohormone sequences 1-30 and 31-67 resulted in mean values from 20 normal subjects of 26.2 (+/- 9.2), 362 (+/- 173) and 368 (+/- 160) pg/ml, respectively. A high correlation coefficient between values obtained using antibodies recognizing prohormone sequences 1-30 and 31-67 was observed (R = 0.84). Extracted plasma immunoreactivity of 1-30 and 31-67 both eluted at 46% acetonitrile. In contrast, chromatographic elution of synthetic peptides 1-30 and 31-67 was observed at 48 and 39% acetonitrile, respectively. Data suggest that the radioimmunoassay of plasma using antibodies recognizing prohormone sequences 1-30 and 31-67 may represent the measurement of a unique larger amino-terminal peptide fragment containing antigenic sites recognized by both antisera.
Asunto(s)
Factor Natriurético Atrial/inmunología , Precursores de Proteínas/inmunología , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Humanos , Péptidos/síntesis química , Péptidos/inmunología , RadioinmunoensayoRESUMEN
Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature. Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322. The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent. Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271-280]. Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs. approximately 6 for the repressor-40 base pair fragment. These data and the differences in binding parameters for the plasmids vs. the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs. Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment. These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein. The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.
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Escherichia coli/genética , Regiones Operadoras Genéticas , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Escherichia coli/metabolismo , Cinética , Conformación de Ácido Nucleico , Proteínas Represoras/aislamiento & purificación , TermodinámicaRESUMEN
Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Barrera Hematoencefálica/fisiología , Endotelio Vascular/química , Receptores de Superficie Celular/análisis , Marcadores de Afinidad , Animales , Unión Competitiva , Encéfalo/irrigación sanguínea , Bovinos , Células Cultivadas , Reactivos de Enlaces Cruzados , GMP Cíclico/metabolismo , Microcirculación/metabolismo , Péptido Natriurético Encefálico , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores del Factor Natriurético AtrialRESUMEN
PURPOSE: The metabolic and environmental factors influencing renal stone formation before, during, and after Space Shuttle flights were assessed. We established the contributing roles of dietary factors in relationship to the urinary risk factors associated with renal stone formation. MATERIALS AND METHODS: 24-hr. urine samples were collected prior to, during space flight, and following landing. Urinary and dietary factors associated with renal stone formation were analyzed and the relative urinary supersaturation of calcium oxalate, calcium phosphate (brushite), sodium urate, struvite and uric acid were calculated. RESULTS: Urinary composition changed during flight to favor the crystallization of calcium-forming salts. Factors that contributed to increased potential for stone formation during space flight were significant reductions in urinary pH and increases in urinary calcium. Urinary output and citrate, a potent inhibitor of calcium-containing stones, were slightly reduced during space flight. Dietary intakes were significantly reduced for a number of variables, including fluid, energy, protein, potassium, phosphorus and magnesium. CONCLUSIONS: This is the first in-flight characterization of the renal stone forming potential in astronauts. With the examination of urinary components and nutritional factors, it was possible to determine the factors that contributed to increased risk or protected from risk. In spite of the protective components, the negative contributions to renal stone risk predominated and resulted in a urinary environment that favored the supersaturation of stone-forming salts. Dietary and pharmacologic therapies need to be assessed to minimize the potential for renal stone formation in astronauts during/after space flight.