RESUMEN

Petunia (Petunia hybrida) pollen requires flavonols (Fl) to germinate. Adding kaempferol to Fl-deficient pollen causes rapid and synchronous germination and tube outgrowth. We exploited this system to identify genes responsive to Fls and to examine the changes in gene expression that occur during the first 0.5 h of pollen germination. We used a subtracted library and differential screening to identify 22 petunia germinating pollen clones. All but two were expressed exclusively in pollen and half of the clones were rare or low abundance cDNAs. RNA gel-blot analysis showed that the steady-state transcript levels of all the clones were increased in response to kaempferol. The sequences showing the greatest response to kaempferol encode proteins that have regulatory or signaling functions and include S/D4, a leucine-rich repeat protein, S/D1, a LIM-domain protein, and D14, a putative Zn finger protein with a heme-binding site. Eight of the clones were novel including S/D10, a cDNA only expressed very late in pollen development and highly up-regulated during the first 0.5 h of germination. The translation product of the S/D3 cDNA shares some features with a neuropeptide that regulates guidance and growth in the tips of extending axons. This study confirmed that the bulk of pollen mRNA accumulates well before germination, but that specific sequences are transcribed during the earliest moments of Fl-induced pollen germination.

Asunto(s)

Flavonoides , Germinación/efectos de los fármacos , Quempferoles , Proteínas de Plantas/genética , Polen/fisiología , Quercetina/análogos & derivados , Solanaceae/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Quercetina/farmacología , ARN Mensajero/genética , Secuencias Repetitivas de Aminoácido , Solanaceae/fisiología

RESUMEN

In an earlier study, we have demonstrated that by mutating five amino acid residues to those conserved in the opioid receptors, the OFQ receptor could be converted to a functional receptor that bound many opioid alkaloids with nanomolar affinities. Surprisingly, when the reciprocal mutations, Lys-214 --> Ala (TM5), Ile-277 --> Val/His-278 --> Gln/Ile-279 --> Val (TM6), and Ile-304 --> Thr (TM7), are introduced in the delta receptor, neither the individual mutations nor their various combinations significantly reduce the binding affinities of opioid alkaloids tested. However, these mutations cause profound alterations in the functional characteristics of the mutant receptors as measured in guanosine 5'-3-O-(thio)triphosphate binding assays. Some agonists become antagonists at some constructs as they lose their ability to activate them. Some alkaloid antagonists are transformed into agonists at other constructs, but their agonistic effects can still be blocked by the peptide antagonist TIPP. Even the delta inverse agonist 7-benzylidenenaltrexone becomes an agonist at the mutant containing both the Ile-277 --> Val/His-278 --> Gln/Ile-279 --> Val and Ile-304 --> Thr mutations. Thus, although the mutated residues are thought to be part of the binding pocket, they are critically involved in the control of the delta receptor activation process. These findings shed light on some of the structural bases of ligand efficacy. They are also compatible with the hypothesis that a ligand may achieve high affinity binding in several different ways, each having different effects on receptor activation.

Asunto(s)

Antagonistas de Narcóticos , Narcóticos/farmacología , Receptores Opioides/agonistas , Animales , Ligandos , Mutación , Ratas , Receptores Opioides/genética , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides delta/genética , Transducción de Señal/genética , Relación Estructura-Actividad , Receptor de Nociceptina

RESUMEN

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They exist in planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA in Escherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that the k(cat)/K(m) values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

Asunto(s)

Galactosiltransferasas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Escherichia coli/genética , Galactosiltransferasas/aislamiento & purificación , Galactosiltransferasas/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Desarrollo de la Planta , Plantas/enzimología , Polen/enzimología , Polen/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura , Distribución Tisular

RESUMEN

Chronic testing of the Nimbus/UOP Axial Flow Pump was performed on 22 calves for periods of implantation ranging from 27 to 226 days (average, 74 days). The following parameters were measured: plasma free hemoglobin, blood and plasma viscosity, erythrocyte deformability and mechanical fragility, oxygen delivery index (ODI), blood cell counts, hematocrit, hemoglobin, blood urea nitrogen, creatinine, bilirubin, total protein, fibrinogen, and plasma osmolality. Most of the above parameters were stable during the full course of support. Compared with baseline, statistically significant differences during the entire period of implantation were only found in: hematocrit (p<0.001), hemoglobin (p<0.005), red blood cell (RBC) count (p<0.001), and whole blood viscosity (p<0.01). Plasma viscosity and ODI were mostly stable during the period of implantation. In some animals, an acute increase in fibrinogen concentration, plasma and blood viscosity, and a decrease in ODI were found to be early signs of the onset of infection. A small (10%) decrease in deformability of RBCs was found during the first 2 weeks after implantation. This alteration in RBC deformability was highly correlated (r = 0.793) with changes in total plasma protein concentration that fell more than 15% (p<0.001) during the same period. Mechanical fragility of RBCs was found to be slightly increased after implantation. Plasma free hemoglobin remained close to baseline level (p>0.2). After the first 2 weeks of the postoperative period, pump performing parameters for all animals were consistent and stable. In general, the Nimbus/UOP Axial Flow Pump demonstrated basic reliability and biocompatibility and did not produce significant alterations in the mechanical properties of blood or animal health status. The pump provided adequate hemodynamics and was well tolerated by the experimental animal for periods as long as 7.5 months. Monitoring rheologic parameters of blood is very helpful for evaluation of health during heart-assist device application.

Asunto(s)

Corazón Auxiliar , Hemorreología/normas , Ensayo de Materiales , Monitoreo Fisiológico/normas , Animales , Velocidad del Flujo Sanguíneo , Proteínas Sanguíneas , Viscosidad Sanguínea , Bovinos , Deformación Eritrocítica , Fibrinógeno/metabolismo , Factores de Tiempo

RESUMEN

Nimbus Inc. (Rancho Cordova, CA) and the University of Pittsburgh have completed the second year of development of a totally implanted axial flow blood pump under the National Institutes of Health Innovative Ventricular Assist System Program. The focus this year has been on completing pump hydraulic development and addressing the development of the other key system components. Having demonstrated satisfactory pump hydraulic and biocompatibility performance, pump development has focused on design features that improve pump manufacturability. A controller featuring full redundancy has been designed and is in the breadboard test phase. Initial printed circuit layout of this circuit has shown it to be appropriately sized at 5 x 6 cm to be compatible with implantation. A completely implantable system requires the use of a transcutaneous energy transformer system (TETS) and a diagnostic telemetry system. The TETS power circuitry has been redesigned incorporating an improved, more reliable operating topography. A telemetry circuit is undergoing characterization testing. Closed loop speed control algorithms are being tested in vitro and in vivo with good success. Eleven in vivo tests were conducted with durations from 1 to 195 days. Endurance pumps have passed the 6 month interval with minimal bearing wear. All aspects of the program continue to function under formal quality assurance.

Asunto(s)

Corazón Auxiliar , Humanos , Diseño de Prótesis

RESUMEN

Flavonols are small (C15) plant-specific molecules that are required for petunia and maize pollen to germinate. They exist in two chemical forms: the aglycone or glycosyl conjugates. Flavonol-deficient pollen is biochemically complemented by flavonol aglycones but not by the glycosylated forms that accumulate in wild type (WT) pollen. Coincident with the biochemical induction of germination, the added flavonol aglycone is rapidly converted to a galactoside and then to a glucosyl galactoside (diglycoside) that is identical to the compound present in WT pollen. A flavonol 3-O-galactosyltransferase (F3GalTase) activity has been identified that controls the formation of glycosylated flavonols in pollen. Importantly, this enzyme also catalyzes the reverse reaction, i.e. the production of the flavonol aglycone from the galactoside and UDP (Fig. 1). F3GalTase/RevGalTase therefore has the potential to control the level of the bioactive flavonol species and as a result, pollen germination.

Asunto(s)

Flavonoides/metabolismo , Fenómenos Fisiológicos de las Plantas , Polen/fisiología , Flavonoles , Glicosilación

RESUMEN

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the delta receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.

Asunto(s)

Mutagénesis Sitio-Dirigida , Péptidos Opioides/metabolismo , Receptores Opioides/genética , Receptores Opioides/metabolismo , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Encefalinas/metabolismo , Encefalinas/farmacología , Ligandos , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacología , Ratas , Receptores Opioides/agonistas , Receptor de Nociceptina , Nociceptina

RESUMEN

Nimbus and the University of Pittsburgh (UOP) have continued the development of a totally implanted axial flow blood pump under the National Institutes of Health (NIH) Innovative Ventricular Assist System (IVAS) program. This 62 cc device has an overall length of 84 mm and an outer diameter of 34.5 mm. The inner diameter of the blood pump is 12 mm. It is being designed to be a totally implanted permanent device. A key achievement during the past year was the completion of the Model 2 pump design. Ten of these pumps have been fabricated and are being used to conduct in vitro and in vivo experiments to evaluate the performance of different materials and hydraulic components. Efforts for optimizing the closed loop speed control have continued using mathematical modeling, computer simulations, and in vitro and in vivo testing. New hydraulic blade designs have been tested using computational fluid dynamics (CFD) and flow visualization. A second generation motor was designed with improved efficiency. To support the new motor, a new motor controller fabricated as a surface mount PC board has been completed. The program is now operating under a formal QA system.

Asunto(s)

Corazón Auxiliar , Animales , Fenómenos Biomecánicos , Ingeniería Biomédica , Estudios de Evaluación como Asunto , Humanos , Técnicas In Vitro , Diseño de Prótesis

RESUMEN

This paper summarizes the authors' in vivo experience to date with an implantable axial flow blood pump designed for long-term ventricular support. This small, valveless pump with blood-lubricated bearings has been implanted in six calves (83 +/- 6 kg) as a left ventricular assist device (LVAS). The left ventricle and descending thoracic aorta were cannulated by left thoracotomy, and the pump was placed in a subcutaneous pocket below the costal margin. Animals remained hemodynamically stable throughout the course of support during partial left ventricular bypass. Five animals were killed after 15, 27, 52, 57, and 181 days. The longest survivor (181 days) demonstrated normal pump function at the time death. Pump speed was maintained at 10,100 +/- 100 rpm, with an average pump flow rate of 4.9 +/- 0.5 L/min under resting physiologic conditions. Average plasma free hemoglobin was 17.4 +/- 7.5 mg/dl. Renal, hepatic, and hematologic indices remained within physiologic range in all of these animals, except during the immediate postoperative period. Histopathologic analyses of major organs after death revealed small renal cortical infarcts in five of six animals; the remaining organs were normal. These animal studies support the feasibility of this small implanted axial flow pump for long-term ventricular assistance.

Asunto(s)

Corazón Auxiliar , Animales , Aorta Torácica/fisiología , Velocidad del Flujo Sanguíneo , Bovinos , Estudios de Factibilidad , Corazón Auxiliar/efectos adversos , Hematócrito , Hemodinámica/fisiología , Hemoglobinas/metabolismo , Corteza Renal/patología , Lubrificación , Toracotomía , Tromboembolia/etiología

RESUMEN

This paper summarizes the authors' in vivo experience in evaluating a miniature centrifugal blood pump designed for pediatric/neonatal ventricular support. Left ventricular bypass was accomplished in two adult sheep and five juvenile lambs (5.5-80.0 kg) via either central (left ventricle to carotid artery) or peripheral (jugular vein to carotid artery) cannulation. Animals were weaned from mechanical ventilation and continuously monitored. Hemodynamic parameters remained within a normal range over the duration of the bypass. Two of five lambs were electively killed at 8, and 76 hours; the remaining three lambs died from respiratory complications at 33, 44, and 156 hours. There were no mechanical complications, and blood seal integrity was confirmed beyond 6 days. The pump speed was maintained at 3,000-4,500 rpm with pump flow rates between 0.4-1.5 L/min. Average plasma free hemoglobin was below 20 mg/dl in the five lamb experiments. Renal, hepatic, and hematologic indices also remained within physiologic ranges. Histopathologic analyses of major organs revealed renal cortical infarctions in two of five lambs. Examination of the pump surfaces after explant indicated small areas of thrombus in the housing adjacent to the outflow ports in two experiments. These encouraging results support further testing and refinement of this miniature centrifugal pump.

Asunto(s)

Corazón Auxiliar/normas , Animales , Recuento de Células Sanguíneas , Nitrógeno de la Urea Sanguínea , Centrifugación , Niño , Creatinina/sangre , Puente Cardíaco Izquierdo , Corazón Auxiliar/efectos adversos , Hematócrito , Hemodinámica/fisiología , Hemoglobinas/análisis , Hemoglobinas/metabolismo , Humanos , Recién Nacido , Complicaciones Posoperatorias/mortalidad , Ovinos , Trombosis/etiología , Trombosis/fisiopatología

RESUMEN

We prospectively evaluated 15 adult cancer patients being treated with adrenocorticosteroids (steroids) to determine the frequency and time course of "steroid myopathy." Nine (60%) developed clinically detectable proximal muscle weakness that, in six, was severe enough to interfere with activities of daily living. Proximal muscle weakness developed within 15 days in eight of nine patients and was significantly related to the cumulative dose of steroid. Eight of nine patients with proximal muscle weakness, and two of six without such weakness, experienced a significant decline in respiratory function, leading to symptomatic dyspnea in four patients of the former group. In three patients who could be followed for more than 3 months off steroids, there was either improvement or resolution of the weakness and, when present, of the respiratory impairment. Steroid myopathy is a common complication among cancer patients receiving steroids. It can often affect respiratory function even when proximal limb muscles remain strong. Clinical recognition is important since steroid myopathy can lead to increased morbidity and may be reversible with reduction or discontinuation of steroids.

Asunto(s)

Antineoplásicos Hormonales/efectos adversos , Dexametasona/efectos adversos , Enfermedades Musculares/inducido químicamente , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Hormonales/uso terapéutico , Dexametasona/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Debilidad Muscular , Músculos/fisiopatología , Enfermedades Musculares/fisiopatología , Estudios Prospectivos , Músculos Respiratorios/fisiopatología

RESUMEN

Structural elements of the rat mu-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the mu, delta, and kappa receptors (Asn150 to Ala, His297 to Ala, and Tyr326 to Phe) and two designed to test for mu/delta selectivity (Ile196 to Val and Val202 to Ile). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H-labeled D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150 in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, beta-endorphin1-31, JOM-13, deltorphin II, dynorphin1-13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of mu, delta, and kappa agonists and antagonists. Altering Val202 to Ile in TM4 produced no change on ligand affinity, but Ile196 to Val resulted in a four- to fivefold decreased affinity for the mu agonists morphine and DAMGO, with no change in the binding affinities of kappa and delta ligands.

Asunto(s)

Residuos de Medicamentos/metabolismo , Receptores Opioides mu/metabolismo , Animales , Unión Competitiva , Células COS , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Etilcetociclazocina/metabolismo , Inmunohistoquímica , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Ratas , Receptores Opioides mu/genética

RESUMEN

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

Asunto(s)

Mutación Puntual , Receptores Opioides/genética , Analgésicos/metabolismo , Animales , Benzomorfanos/metabolismo , Dinorfinas/metabolismo , Modelos Moleculares , Naltrexona/análogos & derivados , Naltrexona/metabolismo , Antagonistas de Narcóticos/metabolismo , Ratas , Receptores Opioides/metabolismo , Receptor de Nociceptina

RESUMEN

Proline is highly conserved in the presumed transmembrane alpha-helices of seven-transmembrane helix-containing, G protein coupled receptors. Unique properties of this imino acid have led to speculations of structural and perhaps dynamic importance for seven-transmembrane helix-containing receptor function. To avoid potentially deleterious consequences of proline-directed mutagenesis, substitutions were made in the X residue of X-Pro peptide bonds (where X is the residue on the amino-terminal side of proline), which may influence static geometries and potential agonist-induced conformational changes at the X-Pro peptide bond. In the fifth helix, Ile205 was substituted with either an alanine (I205A) or a tyrosine (I205Y). Similarly, in the sixth helix, Leu286 was substituted with either an alanine (L286A) or a tyrosine (L286Y). Mutant I205A demonstrated subtle changes in D1 pharmacology and signal transduction. The I205Y and L286Y mutations produced comparatively drastic impairments in both binding and signal transduction. Remarkably, the L286A mutation resulted in constitutive activity characterized by elevated basal signal transduction and increased agonist potencies. In addition, (R)-(+)-SCH23390, a classical antagonist at the wild-type D1 receptor, behaved as a partial agonist at L286A. This is the first report of a constitutively active receptor resulting from this point mutation and the first report of a constitutively active mutant dopamine receptor. These results are discussed in terms of binding pocket geometry and potential mechanisms of signal transduction.

Asunto(s)

Mutagénesis Sitio-Dirigida , Prolina/fisiología , Receptores de Dopamina D1/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Células COS/metabolismo , Células COS/fisiología , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Relación Estructura-Actividad

RESUMEN

Patients with gynecologic malignancies may develop metastases throughout the neuraxis. Cervix-related carcinomatous meningitis is a distinctly unusual clinical event with only two previous cases reported in the English medical literature. We review clinical, radiographic, and pathologic findings of a woman with advanced adenocarcinoma of the uterine cervix, whose course was complicated by leptomeningeal metastases. Carcinomatous meningitis occurs in the setting of rapidly advancing systemic disease and represents a terminal complication of cervical cancer.

Asunto(s)

Adenocarcinoma/secundario , Neoplasias Meníngeas/secundario , Meningitis/etiología , Neoplasias del Cuello Uterino/patología , Adenocarcinoma/complicaciones , Adenocarcinoma/diagnóstico , Adulto , Nervios Craneales/patología , Resultado Fatal , Femenino , Humanos , Neoplasias Meníngeas/complicaciones , Neoplasias Meníngeas/diagnóstico , Tomografía Computarizada por Rayos X , Neoplasias del Cuello Uterino/complicaciones

RESUMEN

OBJECTIVES: Research has described benefits of physical, athletic, and avocational activity on improving self-esteem, quality of life, and locus of control in persons with disabilities. The objective of this study was to identify meaningful components of the experience of sea kayaking as described by persons with spinal cord injury (SCI). METHOD: Three subjects with SCI who had participated in recreational kayaking were interviewed. Qualitative research methods included strategies from Guba's model for rigor in qualitative research, Spradley's interviewing guidelines, and Good's method of semantic network analysis. Three interviews of approximately 45 min in length were conducted with each subject. Initial interviews began with a single question: "Tell me about sea kayaking." Subsequent questions contained only concepts and terms used in the subjects' responses. RESULTS: The subjects valued the novelty, challenge, safety, sociability, and natural environment aspects of sea kayaking. Perceptions of the self as able in the eyes of others and the need for support in pursuit of outdoor leisure activities were themes that figured prominently in the subjects' discourse. CONCLUSION: Subjects' comments indicate that meaningful time use and the construction of an identity after injury are linked. This link has also been suggested in the rehabilitation literature. This information suggests the use of therapeutic intervention that supports a person's adjustment to an irreversible SCI.

Asunto(s)

Adaptación Psicológica , Terapia Ocupacional/métodos , Recreación , Traumatismos de la Médula Espinal/rehabilitación , Adulto , Femenino , Humanos , Control Interno-Externo , Masculino , Calidad de Vida , Autoimagen , Traumatismos de la Médula Espinal/psicología

RESUMEN

BACKGROUND: We are developing a miniaturized centrifugal blood pump for use as a temporary cardiac assist device in neonatal and pediatric sized patients. This pump has a very low priming volume of 13 mL. A small motor stator has also been designed, which resulted in a device that can be placed very close to the patient, thereby minimizing overall circuit volume. METHODS: Testing to date has included in vitro hemodynamic performance, in vitro hemolysis generation, and in vivo evaluation in 5 lambs weighing 5.5 to 21 kg. Two lambs underwent peripheral cannulation from external jugular vein to carotid artery, whereas 3 others were cannulated from left atrium to carotid artery. RESULTS: In vitro data demonstrated pump capacity spanning 0.3 to 3.0 L/min and very low hemolysis generation at these conditions. In vivo, the pump functioned satisfactorily for periods up to 148 hours, and the bypass appeared to be well tolerated by the animals. Plasma free hemoglobin levels remained less than 25 mg/dL during all animal experiments. All devices were thrombus-free at explantation. CONCLUSIONS: We conclude that this device has merit as an alternative to current oversized systems used for neonatal and pediatric cardiac assistance. In addition, a chronic neonatal lamb model in which to evaluate pediatric circulatory assist devices has been developed successfully.

Asunto(s)

Corazón Auxiliar , Animales , Niño , Preescolar , Diseño de Equipo , Corazón Auxiliar/efectos adversos , Hemodinámica , Humanos , Técnicas In Vitro , Lactante , Recién Nacido , Ovinos

RESUMEN

Dopamine receptors belong to the seven transmembrane helix-containing, G protein-coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate-114 in helix 3 and two helix 5 serines (194 and 197) of the D2 receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site-directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D2 receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine-198 with an alanine selectively disrupted [3H]N-0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine-411 or the helix 6 leucine-387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine-411 in helix 7 and leucine-387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D2 receptor.

Asunto(s)

Receptores Dopaminérgicos/fisiología , Transducción de Señal , Agua/farmacología , Secuencia de Aminoácidos , Línea Celular , AMP Cíclico/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ensayo de Unión Radioligante , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Dopaminérgicos/genética

RESUMEN

The opioid peptides are derived from three prohormone precursors referred to as proopiomelanocortin (POMC), proenkephalin (ProEnk) and prodynorphin (ProDyn). Following specific cleavage, several biologically active peptides are generated that can bind to the mu, delta and kappa receptors. The present study examines the receptor binding affinities of the POMC, ProEnk and ProDyn peptides to the cloned mu, delta and kappa receptors expressed transiently in transfected COS-1 cells. Consistent with previous findings using brain homogenates, competition studies demonstrate that no opioid peptide family can be exclusively associated with a specific opioid receptor type. Short ProEnk peptides, such as Leu- and Met-enkephalin are selective for delta, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to micro, delta and kappa. Similarly, Peptide E, the BAM peptides, and metorphamide have a high affinity for all three opioid receptor types. While dynorphin A peptides and alpha- and beta-neoendorphin have a preference for kappa, they also bind the cloned delta and mu receptors. Our findings do not easily fit a simple 'message-address' model where the Try-Gly-Gly-Phe core is extended and this gradually alters selectivity. Rather, the pattern appears more discontinuous, and would fit better with the idea of two similar but distinct cores; a Try-Gly-Gly-Phe Met- or Leu core that is necessary and sufficient for mu and delta but not kappa and a Tyr-Gly-Gly-Phe-Met or Leu core with an Arg-X extension that is equally necessary and sufficient for kappa.

Asunto(s)

Péptidos Opioides/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Línea Celular , Datos de Secuencia Molecular , Ensayo de Unión Radioligante

RESUMEN

Wild-type petunia pollen accumulates high levels of flavonol 3-O-glycosides. Pollen from conditionally male-fertile petunia has no flavonols and is unable to germinate. Pollen function is restored both in vivo and in vitro by providing flavonol aglycones, but not flavonol glycosides, to the pollen. In the present study, incubation of an in vitro suspension of conditionally male-fertile pollen with kaempferol or quercetin resulted in the accumulation of kaempferol and quercetin 3-O-glycosides in the pollen. We identified two glycosyltransferase activities associated with the intact pollen grain that catalyze the formation of a gametophyte-specific class of flavonol glycosides. Feeding studies showed that product formation was highly specific for flavonols with an unsubstituted 3-hydroxyl group and was not dependent on an external source of UDP-hexose. Ultraviolet spectral analysis, fast atom bombardment mass spectrometry, 1H-nuclear magnetic resonance, and 13C-nuclear magnetic resonance identified the products as kaempferol and quercetin 3-O-(2"- O-beta-D-glucopyranosyl)-beta-D-galactopyranoside, identical with the flavonol 3-O-glycosides present in wild-type pollen. The sugars are linked in a 1-->2 configuration that results in a pollen-specific class of compounds. To retain both glycosyltransferase activities in a cell-free extract, it was necessary to add triton X-100, suggesting that one or both of the proteins may be associated with a pollen membrane. A model for flavonol glycoside biosynthesis and uptake into the pollen is discussed in terms of the germination requirement for flavonols.

Asunto(s)

Flavonoides/metabolismo , Glicósidos/biosíntesis , Glicosiltransferasas/metabolismo , Plantas/enzimología , Polen/enzimología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Catálisis , Sistema Libre de Células , Cromatografía Líquida de Alta Presión , Flavonoles , Glicósidos/química , Datos de Secuencia Molecular , Especificidad por Sustrato