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ATP-receptors (P2X2, P2X3, P2X4 & P2X7) are found in neurohypophysial terminals (NHT). These purinergic receptor subtypes are known to be cation selective. Here we confirm that both sodium (Na+ ) and calcium (Ca2+ ) are permeable through these NHT purinergic receptors, but to varying degrees (91% vs. 9%, respectively). Furthermore, extracellular calcium inhibits the ATP-current magnitude. Thus, the objective of this study was to determine the effects of extracellular Na+ vs. Ca2+ on ATP-induced vasopressin (AVP) release from populations of rat isolated NHT. ATP (200 µM) perfused exogenously for 2 minutes in Normal Locke's buffer caused an initial transient increase in AVP release followed by a sustained increase in AVP release which lasted for the duration of the ATP exposure. Replacing extracellular NaCl with NMDG-Cl had no apparent effect on the ATP-induced transient increase in AVP release but abolished the sustained AVP release induced by ATP. Furthermore, removal of extracellular calcium resulted in no ATP-induced transient increase in AVP release, but had no effect on the delayed, sustained increase in AVP release. The ATP-induced calcium-dependent transient increase in AVP release was >95% inhibited by 10 µM of the P2X purinergic receptor antagonist PPADS, a dose sufficient to block P2X2 and P2X3 receptors but not P2X4 or P2X7 receptors. Interestingly, the ATP-induced calcium-independent, sodium-dependent sustained increase in AVP release was not affected by 10 µM PPADS. The ATP-induced calcium-dependent transient increase in AVP release was not affected by the P2X7 receptor antagonist BBG (100 nM). However, the ATP-induced sodium-dependent sustained AVP release was inhibited by 50%. Therefore, these results show that rat isolated NHT exhibit a biphasic response to exogenous ATP that is differentially dependent on extracellular calcium and sodium. Furthermore, the initial transient release appears to be through P2X2 and/or P2X3 receptors and the sustained release is through a P2X7 receptor. This article is protected by copyright. All rights reserved.
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BACKGROUND: Virtual reality-based simulators offer a cost-effective and efficient alternative to traditional medical training and planning. Developing a simulator that enables the training of medical skills and also supports recognition of errors made by the trainee is a challenge. The first step in developing such a system consists of error identification in the real procedure, in order to ensure that the training environment covers the most significant errors that can occur. This paper focuses on identifying the main system requirements for an interactive simulator for training bilateral sagittal split osteotomy (BSSO). METHODS: An approach is proposed based on failure mode and effects analysis (FMEA), a risk analysis method that is well structured and already an approved technique in other domains. RESULTS: Based on the FMEA results, a BSSO training simulator is currently being developed, which centres upon the main critical steps of the procedure (sawing and splitting) and their main errors. CONCLUSIONS: FMEA seems to be a suitable tool in the design phase of developing medical simulators. Herein, it serves as a communication medium for knowledge transfer between the medical experts and the system developers. The method encourages a reflective process and allows identification of the most important elements and scenarios that need to be trained.
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Competencia Clínica , Instrucción por Computador/instrumentación , Errores Médicos/prevención & control , Modelos Biológicos , Osteotomía Sagital de Rama Mandibular/instrumentación , Robótica/instrumentación , Interfaz Usuario-Computador , Simulación por Computador , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Errores Médicos/clasificación , Osteotomía Sagital de Rama Mandibular/métodosRESUMEN
ATP-induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC(50) of 50 µm in somata versus 9.6 µm in terminals). The ATP-induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC(50) of 3.6 µm in somata versus 11.6 µm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP-γ-S and benzoyl-benzoyl-ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC(50) , inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones.
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Adenosina Trifosfato/fisiología , Sistema Hipotálamo-Hipofisario/fisiología , Neuronas/fisiología , Terminales Presinápticos/fisiología , Receptores Purinérgicos P2X/fisiología , Adenosina Trifosfato/agonistas , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Animales , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/citología , Agonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Ratas , Ratas Sprague-Dawley , Suramina/farmacologíaRESUMEN
Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca(2+) currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback-mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically-stimulated neuropeptide release. Intact neurohypophyses (NH) from wild-type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25-s bursts (3 V at 39 Hz) separated by 21-s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically-stimulated AVP release. A similar inhibition by suramin was observed in electrically-stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically-stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically-stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this 'selective' antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive-feedback mechanism to 'differentially' modulate neuropeptide release from central nervous system terminals.
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Adenosina Trifosfato/fisiología , Arginina Vasopresina/metabolismo , Oxitocina/metabolismo , Neurohipófisis/metabolismo , Receptores Purinérgicos P2X2/fisiología , Receptores Purinérgicos P2X3/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Estimulación Eléctrica/métodos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Neurohipófisis/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2X/farmacología , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X3/genética , Suramina/farmacologíaRESUMEN
In spite of improvements in cementing technique, migration of tibial component remains a problem in total knee arthroplasty. This study compares the primary stability of tibial components using two different cementing techniques with roentgen stereophotogrammetric analysis (RSA) in vitro. A total of 20 tibia specimens were matched into two groups, 10 specimens per group. Cementing technique was randomized to each group. In the first group only the base and in the second group the base and stem were cemented. The implants and the tibial metaphysis were marked with markers for the RSA analysis. All specimens were tested with an axial load of 2,000 N for 1,000 and 10,000 cycles and RSA analysis was performed. Endpoints for radiosterometric analysis were maximum total point motion, maximum subsidence, lift off, rotation and translation along the x-, y-, and z-axes. After 1,000 and 10,000 cycles, no significant differences could be found, but two tibial components of the surface cementing group showed a migration of more than 2 mm defined as failure compared to six failed tibial components in the full cementing group (P = 0.068). This higher number of failed arthroplasties in the fully cemented prosthesis group demonstrates a disadvantageous load distribution in the tibia apophysis which can cause an early component loosening.
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Artroplastia de Reemplazo de Rodilla/instrumentación , Cementación/métodos , Análisis de Falla de Equipo , Tibia , Anciano , Anciano de 80 o más Años , Cadáver , Femenino , Humanos , Prótesis de la Rodilla , Masculino , FotogrametríaRESUMEN
OBJECTIVE: To evaluate the relative value of three stress testing modalities for establishing the presence of coronary artery disease in women presenting with chest pain. BACKGROUND: Echocardiographic testing modalities have the potential to be more effective than standard electrocardiographic stress testing (EST), but they require prospective evaluation. METHODS: Patients with no cardiac history and at least two cardiac risk factors were randomly assigned to one of three initial diagnostic strategies: treadmill EST (n=54), treadmill echocardiographic stress testing (ExE) (n=57) or dobutamine stress echocardiography (DSE) (n=47). Patients were followed prospectively for 28.1+/-14.2 months. The nature of the presenting chest pain was established clinically. RESULTS: Twelve patients (7.6%) were determined to have had cardiac chest pain, 128 patients (81.0%) received a diagnosis of noncardiac chest pain and 18 patients (11.4%) had indeterminate results. The echocardiographic testing modalities were associated with fewer indeterminate results than was EST (two of 104 [1.9%] versus 13 of 54 [24.1%]). All modalities were highly effective in excluding cardiac chest pain, with negative predictive values of 91.3%, 83.7% and 88.4%, respectively, for EST, DSE and ExE. The proportion of cases for which both definitive and accurate results were provided was 92.9% for DSE, 82.4% for ExE and 67.3% for EST. CONCLUSIONS: The results support the favourable prognosis of women presenting with chest pain syndrome and the reliability of negative results obtained with any of the testing modalities. Echocardiographic testing modalities are more likely than EST to provide both definitive and accurate results and would, therefore, seem to be the superior primary noninvasive testing modality in this patient population.
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Cardiotónicos , Dolor en el Pecho/diagnóstico , Dobutamina , Ecocardiografía de Estrés , Electrocardiografía , Prueba de Esfuerzo , Adulto , Anciano , Enfermedad de la Arteria Coronaria/diagnóstico , Femenino , Humanos , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de Riesgo , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To review older patients' perceptions of their medical care before hospital admission and to determine whether there are common perceptions family physicians should address after discharge. DESIGN: Semistructured interviews with qualitative analysis. SETTING: Inpatient geriatric rehabilitation and assessment unit. PARTICIPANTS: Community-living seniors admitted from home or transferred from acute care hospitals. METHOD: Consecutively admitted patients were interviewed within a week of admission. Participants were asked open-ended and Likert-type questions. Responses were analyzed to uncover recurrent themes and descriptive statistics. MAIN FINDINGS: Patients thought physicians' personalities and ability to communicate were important factors in their satisfaction with care received. Loyalty to a physician was an important theme and might have made patients minimize their concerns about care. Most patients were confident in being discharged back into the care of their family physicians. CONCLUSION: Physicians' personalities and communication skills affected whether patients were satisfied with care. Older patients are loyal to their family physicians; they did not identify any issues for family physicians to address with them after discharge.
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Medicina Familiar y Comunitaria , Satisfacción del Paciente , Relaciones Médico-Paciente , Anciano , Anciano de 80 o más Años , Comunicación , Femenino , Geriatría , Encuestas de Atención de la Salud , Humanos , Masculino , Admisión del Paciente , Personalidad , Competencia Profesional , Centros de RehabilitaciónRESUMEN
The analysis of the genetic basis of phenotypic traits is moving towards the complex diseases prevalent in wealthy populations. There is an increasing requirement for the detection of different types of sequence variation, particularly single-nucleotide polymorphisms (SNPs). SNPs occur about once every 100 to 300 bases. High-density SNP maps will help to identify the multiple genes associated with complex diseases such as cancer, diabetes, vascular disease, and some forms of mental illness.
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ADN/análisis , Polimorfismo de Nucleótido Simple , ADN/genética , Predisposición Genética a la Enfermedad/genética , HumanosRESUMEN
BACKGROUND: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. RESULTS: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. CONCLUSIONS: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.
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Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol. Such apparent import inefficiency is very marked in guinea pig (Cavia porcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homo sapiens) hepatocytes, for example. In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p. Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected Deltapex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e. Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not. In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomyces cerevisiae, whereas AGT-Lys-Lys-Leu was not. These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s. They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu.
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Peroxisomas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catalasa/metabolismo , Células Cultivadas , Clonación Molecular , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Peroxisomas/enzimología , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Acute ethanol (EtOH) exposure reduces the evoked release of vasopressin (AVP) and oxytocin (OT) from excised neurohypophyses and from dissociated neurohypophysial terminals of the rat. METHODS AND RESULTS: Rats placed on a diet that maintained blood levels of 30 mM EtOH for 20 to 40 days developed tolerance to acute EtOH inhibition of release. In the presence of 10 mM EtOH, high (50 mM) K+-induced release of AVP from isolated neurohypophysial terminals of EtOH-naive rats was reduced by 77.7+/-1.4%, whereas in the chronic EtOH group, release was reduced by only 9.4+/-8.7%. Similar tolerance was evident during acute challenge with 75 mM EtOH, as well as for release of OT from isolated terminals. Animals treated with an intraperitoneal injection of EtOH and sacrificed 90 min postinjection did not exhibit the reduced EtOH inhibition of release from dissociated terminals during a 75 mM EtOH acute challenge. CONCLUSIONS: The altered component responsible for the tolerance to inhibition of release resides in the isolated terminal, because tolerance measured in vitro from intact neurohypophyses was similar to that seen in isolated terminals. The failure of EtOH-injected animals to exhibit reduced inhibition of release in response to an acute EtOH challenge indicates that short-term elevated blood alcohol level does not induce this tolerance. The finding of tolerance to EtOH-induced inhibition of release from the intact neurohypophysis and isolated terminals provides a physiological preparation in which to examine the molecular targets of acute drug action modified after chronic exposure to the drug.
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Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Oxitocina/metabolismo , Neurohipófisis/efectos de los fármacos , Vasopresinas/metabolismo , Animales , Depresores del Sistema Nervioso Central/sangre , Etanol/sangre , Masculino , Neurohipófisis/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The early stages of Venezuelan equine encephalitis virus (VEE) pathogenesis in the mouse model have been examined using a genetic approach. Disease progression of a molecularly cloned single-site mutant was compared with that of the parental virus to determine the step in the VEE pathogenetic sequence at which the mutant was blocked. Assuming that such a block constitutes a genetic screen, isolates from different tissues thought to be distal to the block in the VEE pathogenetic sequence were analyzed to determine the pathogenetic step at which revertants of the mutant were selected. Directed mutation and analysis of reversion in vivo provide two powerful genetic tools for the dissection of the wild-type VEE pathogenetic sequence. Virus from the parental virulent clone, V3000, first replicated in the draining lymph node after subcutaneous inoculation in the left rear footpad. Movement of a cloned avirulent mutant, V3010 (E2 76 Glu to Lys), to the draining lymph node was impaired, replication in the node was delayed, and spread beyond the draining lymph node was sporadic. Serum, contralateral lymph node, spleen, and brain isolates from V3010 inoculated animals were invariably revertant with respect to sequence at E2 76 and/or virulence in mice. Revertants isolated from serum and contralateral lymph node retained the V3010 E2 Lys 76 mutation but also contained a second-site mutation, Glu to Lys at E2 116. Modification of the V3010 clone by addition of the second-site mutation at E2 116 produced a virus that bypassed the V3010 block at the draining lymph node but that did not possess full wild-type capacity for replication in the central nervous system or for induction of mortality. A control construct containing only the E2 116 reverting mutation on the V3000 background was identical to V3000 in terms of early pathogenetic steps and virulence. Therefore, analysis of mutant replication and reversion in vivo suggested (1) that the earliest steps in VEE pathogenesis are transit to the draining lymph node and replication at that site, (2) that the mutation in V3010 impairs transit to the draining lymph node and blocks dissemination to other tissues, and (3) that reversion can overcome the block without restoring full virulence.
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Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/virología , Mutación Puntual/genética , Supresión Genética/genética , Animales , Encéfalo/virología , Línea Celular , Clonación Molecular , Progresión de la Enfermedad , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/mortalidad , Femenino , Ganglios Linfáticos/virología , Ratones , Fenotipo , ARN Viral/genética , ARN Viral/metabolismo , Bazo/virología , Relación Estructura-Actividad , Vacunas Atenuadas/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Vacunas Virales/genética , Viremia , Virulencia/genética , Replicación ViralRESUMEN
Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array (MEA). This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and characterize the MEA. Simultaneous extracellular recording of local field potentials (LFPs) and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy.
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Hipocampo/fisiología , Red Nerviosa , Potenciales de Acción/fisiología , Animales , Estimulación Eléctrica , Histocitoquímica , Microelectrodos , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.
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Cianuros/química , ADN/análisis , Electroforesis Capilar/métodos , Colorantes Fluorescentes/química , Carbocianinas/química , Estructura MolecularAsunto(s)
Isoenzimas/biosíntesis , Isoenzimas/genética , Schizosaccharomyces/enzimología , Fosfolipasas de Tipo C/biosíntesis , Fosfolipasas de Tipo C/genética , Genes Fúngicos , Mutagénesis , Sistemas de Lectura Abierta , Fosfolipasa C delta , Mapeo Restrictivo , Schizosaccharomyces/genética , TemperaturaRESUMEN
Malondialdehyde, a product of lipid peroxidation, produces threshold conversion of low density lipoprotein (LDL) to a form recognized by type I and type II scavenger receptors of monocyte-macrophages. To investigate whether localized domains of human apoB-100 protein provide recognition determinants, we tested the ability of several different apoB-bearing particles to interact with the scavenger receptor of human monocyte-macrophages. Genetically engineered, carboxyl-terminally truncated apoB proteins assembled into lipoprotein form were labeled by fluorescent dye. Fluorescence microscopy and quantitative fluorescent spectrophotometry showed that purified particles containing as little as 23% of the apoB amino-terminus were internalized by the scavenger receptor after, but not before, malondialdehyde modification. There was no recognition of the particles by the LDL receptor. Similar results were obtained with human plasma LDL homozygous for carboxyl-terminally truncated apoB-45.2. Liposome-incorporated fusion protein containing apoB residues 547-735 displayed specific uptake by the scavenger receptor without modification by malondialdehyde. In contrast, fusion proteins containing apoB residues 3,029-3,133 or a short amino terminal segment failed to interact. Thus, primary sequence presented by residues 1-1,084 sufficed to produce recognition of modified LDL by the scavenger receptor. These receptor-combining domains were sequestered when secreted in lipoprotein form and were expressed upon malondialdehyde modification. When packaged exogenously in liposome form, fusion protein containing apoB residues 547-735, containing approximately 4% of the primary sequence, mediated scavenger receptor-dependent uptake and hydrolysis. Our findings provide an additional function or the amino-terminal region of apoB and demonstrate that primary sequence presented by the first 2% of apoB-100 protein suffices to produce recognition on malondialdehyde-modified LDL by the scavenger receptor of human monocyte-macrophages.
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Apolipoproteínas B/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteínas B/análisis , Apolipoproteínas B/química , Apolipoproteínas B/aislamiento & purificación , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Emulsiones , Humanos , Immunoblotting , Radioisótopos de Yodo , Lipoproteínas LDL/química , Macrófagos/citología , Malondialdehído/química , Datos de Secuencia Molecular , Monocitos/citología , Ratas , Receptores Inmunológicos/química , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
A bidirectional promoter can be defined operationally as a short segment of DNA that regulates divergent transcription. In an attempt to investigate whether the intergenic region between the oleosin and a second open reading frame (ORFII) in Brassica napus (L.) is a divergent promoter, and also to characterize the ORFII, cDNA clones homologous to ORFII were isolated from a leaf cDNA library. A representative cDNA (clone D) of one of the two classes identified was identical, in DNA sequence, to the genomic ORFII. The second representative cDNA (clone O) was 97% identical at the nucleotide level to the genomic ORFII. The predicted amino acid sequence of the cDNA clones each exhibit homology with the peptide methionine sulphoxide reductase (PMSR) of Escherichia coli. The gene structure of ORFII was elucidated and the relative positions of the oleosin, ORFII, and the intergenic promoter region were determined. This confirms that the B. napus oleosin-ORFII intergenic region has divergent promoter activity. Consequently this is the first such plant nuclear divergent promoter identified. RFLP-mapping results showed that all four ORFII genes are linked to four of the six copies of the oleosin genes. This suggested that the bidirectional promoter locus is conserved within the B. napus genome. The ORFII gene product is targeted to the chloroplast, which is consistent with previous data indicating the presence of PMSR activity in the chloroplast. The over-expressed recombinant fusion protein (minus the transitpeptide) showed the capability to reduce peptide methionine sulphoxide residues in vitro, indicating PMSR activity. This study demonstrates that ORFII is transcribed and encodes a plant PMSR, and is the first example of the isolation of a eukaryotic PMSR gene.
Asunto(s)
Brassica/enzimología , Brassica/genética , Proteínas de Unión al ADN/genética , Genes de Plantas , Oxidorreductasas/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cloroplastos/enzimología , Cloroplastos/genética , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Ligamiento Genético , Metionina Sulfóxido Reductasas , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimologíaRESUMEN
The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced. THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range. A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence. HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes. In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate. At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses. Selection in cell culture often results in attenuation of virulence in animals. Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice. Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene. These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes. A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation.
Asunto(s)
Secuencia de Consenso , Virus Sindbis/genética , Infecciones por Alphavirus/virología , Animales , Arginina/metabolismo , Secuencia de Bases , Línea Celular , Células Cultivadas , Cricetinae , ADN Viral , Ratones , Datos de Secuencia Molecular , Mutación , Fenotipo , Virus Sindbis/metabolismo , Virus Sindbis/patogenicidad , Proteínas del Envoltorio Viral/genética , Virulencia/genéticaRESUMEN
A step in the maturation of Sindbis virus glycoproteins is the cleavage of the precursor glycoprotein PE2 into E3 and E2 by furin or a furin-like host cell protease. The results presented here suggest that PE2 cleavage is an obligatory event for Sindbis virus maturation in C6/36 cells and demonstrate that certain mutants display a cell-specific PE2 cleavage phenotype. We previously have described Sindbis virus variants which fail to cleave PE2 because of incorporation of a signal for N-linked glycosylation immediately adjacent to the PE2 cleavage site but are viable in BHK-21 cells by virtue of an additional mutation at E2 216 or E2 191 (TRSB-NE2G216 and TRSB-NE2T191, respectively) (H. W. Heidner, K. L. McKnight, N. L. Davis, and R. E. Johnston, J. Virol. 68:2683-2692, 1994). Other viable PE2 cleavage-defective mutants were constructed by substituting the parental residue at E2 position 1 (Arg), with Leu or Val (TRSB-E2L1 and TRSB-E2V1, respectively) (H.W. Heidner and R. E. Johnston, J. Virol. 68:8064-8070, 1994). When grown in BHK-21 cells, all four of these viruses replicated normally and incorporated PE2 in place of E2 in released virions. However, growth of TRSB-NE2G216 and TRSB-NE2T191 was severely restricted in cultured arthropod cells (C6/36 cells). Analysis of infected C6/36 cells by flow cytometry demonstrated that the restricted growth of TRSB-NE2G216 and TRSB-NE2T191 was not due to an impaired ability to initiate infection. In addition, TRSB-NE2G216 and TRSB-NE2T191 remained growth restricted in C6/36 cells following introduction of in vitro transcriptions by electroporation. In contrast, the PE2 cleavage defect of TRSB-E2L1 and TRSB-E2V1 was cell type specific. In C6/36 cells, the majority of PE2 was converted to E2, and these viruses replicated normally in C6/36 cells. These results demonstrated a consistent link between expression of a PE2 cleavage defect and restricted growth in C6/36 cells and suggest that cleavage of PE2 is required for maturation of Sindbis virus late in infection of C6/36 cells.