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STUDY QUESTION: Is expectant management (EM) of tubal ectopic pregnancy (EP) an effective and safe treatment strategy when compared to alternative interventions? SUMMARY ANSWER: There is insufficient evidence to conclude EM yields a difference in the resolution of tubal EP, the avoidance of surgery or time to resolution of tubal EP when compared to intramuscular methotrexate in stable patients with ß-hCG <1500 IU/l. WHAT IS ALREADY KNOWN: The utilisation of medical and surgical management for EP is well established. EM aims to allow spontaneous resolution of the EP without intervention. STUDY DESIGN SIZE AND DURATION: We performed a systematic review and meta-analysis, searching Ovid MEDLINE, Embase, PsycINFO, CINAHL, Web of Science, OpenGrey.eu, Google Scholar, cross-referencing citations and trial registries to 15 December 2019. There were no limitations placed on language or publication date. Search terms included tubal EP and EM as well as variations of these terms. PARTICIPANTS/MATERIALS SETTING AND METHOD: We considered studies that included patients with tubal EP, EM as a comparator, and that were randomised controlled trials (RCTs). The primary outcome was resolution of tubal EP. Secondary outcomes included avoidance of surgery and the time to resolution of EP. Two reviewers independently selected the studies, assessed bias and extracted data. Relative risk (RR) and mean difference with 95% CI were assessed using a random effects model. The certainty of evidence was scored according to Grading of Recommendations Assessment, Development and Evaluation guidelines. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 920 studies were screened. Five studies were eligible for inclusion in the systematic review. Two RCTs comparing methotrexate to EM were identified as being eligible for inclusion in meta-analysis. No RCTs comparing surgery to EM were identified. Compared with EM, there was insufficient evidence that methotrexate yields a difference on resolution of tubal EP (RR 1.04, 95% CI 0.88-1.23, P = 0.67; two RCTs, moderate-certainty evidence), avoiding surgery (RR 1.10, 95% CI 0.94-1.29, P = 0.25; two RCTs, low-certainty evidence) or the time to resolution of tubal EP (-2.56 days (favouring EM), 95% CI -7.93-2.80, P = 0.35; two RCTs, low-certainty evidence). LIMITATIONS REASONS FOR CAUTION: Only two RCTs with a total of 103 patients were eligible for inclusion in this meta-analysis. Further RCTs comparing EM to medical and surgical management are needed and these should also report adverse events. Patient preference should also be evaluated. WIDER IMPLICATIONS OF THE FINDINGS: We found insufficient evidence of differences in terms of resolution, avoidance of surgery and time to resolution between expectant and medical management. Given the imprecision in the effect estimates as demonstrated by the wide CIs, resulting in the downgrading of certainty of evidence for all outcomes in this meta-analysis, larger RCTs comparing interventions for tubal EP are needed. Caution should be exercised when trying to decide between EM and methotrexate to treat tubal EP. STUDY FUNDING/COMPETING INTERESTS: There was no funding for this study. NICM receives funding from various sources; none specifically supported this research. M.L. reports grants from Australian Women and Children's Research Foundation, outside the submitted work. M.A.: As a medical research institute, NICM Health Research Institute receives research grants and donations from foundations, universities, government agencies and industry. Sponsors and donors provide untied and tied funding for work to advance the vision and mission of the Institute. This systematic review was not specifically supported by donor or sponsor funding to NICM. M.A. reports a partnership grant with Metagenetics outside the submitted work. G.C. reports grants from Australian Women and Children's Research Foundation, personal fees from Roche and GE Healthcare, outside the submitted work. The remaining authors report no conflicts of interest. PROSPERO REGISTRATION NUMBER: CRD42020142736.
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Replacement of pancreatic ß-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional ß-cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress-induced ß-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated ß-cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin+ /urocortin-3- cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate ß-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative ß-cell sources, graft sites, and ultimately fully vascularized bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature ß-cell phenotype has been maintained.
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Diferenciación Celular , Fibrosis Quística/terapia , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/patología , Trasplante de Islotes Pancreáticos/métodos , Adulto , Femenino , Humanos , Fenotipo , PronósticoRESUMEN
Estimated human inhalation toxicity values for Sarin (GB) were calculated using a new two independent (concentration, exposure time), one dependent (toxic response), non-linear dose response (toxicity) model combined with re-evaluated allometric equations relating to animal and human respiration. Historical animal studies of GB toxicity containing both exposure and fractional animal response data were used to test the new process. The final data set contained 6621 animals, 762 groups, 37 studies and 7 species. The toxicity of GB for each species was empirically related to exposure concentration (C; mg m(-3)) and exposure time (T; min) through the surface function Y = b0 + b1 Log10C + b2 Log10T or Y = b0 + b2 Log10C(n)T where Y is the Normit, b0, b1 and b2 are constants and n is the 'toxic load exponent' (Normit is PROBIT - 5). Between exposure times of 0.17 and 30 min, the average value for n in seven species was 1.35 +/- 0.15. The near parallel toxic load equations for each species and the linear relationship between minute volume/body weight ratio and the inhalation toxicity (LCt50) for GB were used to create a pseudo-human data set and then an exposure time/toxicity surface for the human. The calculated n for the human was 1.40. The pseudo-human data had much more variability at low exposure times. Raising the lower exposure limit to 1 min, did not change the LCt50 but did result in lower variability. Raising the lower value to 2 min was counterproductive. Based on the toxic load model for 1-30 min exposures, the human GB toxicities (LCt01, LCt05, LCt50 and LCt95) for 70 kg humans breathing 15 l min(-1) were estimated to be 11, 16, 36 and 83; 18, 25, 57 and 132 and 24, 34, 79 and 182 mg x min m(-3) for 2, 10 and 30 min exposures, respectively. These values are recommended for general use for the total human population. The empirical relationships employed in the calculations may not be valid for exposure times >30 min.
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Sustancias para la Guerra Química/toxicidad , Sarín/toxicidad , Algoritmos , Animales , Aves , Gatos , Interpretación Estadística de Datos , Perros , Drosophila , Cobayas , Haplorrinos/fisiología , Humanos , Exposición por Inhalación , Dosificación Letal Mediana , Ratones , Dinámicas no Lineales , Valor Predictivo de las Pruebas , Conejos , Ratas , Sarín/administración & dosificación , Ovinos , Especificidad de la Especie , PorcinosRESUMEN
During apoptosis, the mitochondrial membrane potential (MMP) decreases, but it is not known how this relates to the apoptotic process. It was recently suggested that cytochrome c is compartmentalized in closed cristal regions and therefore, matrix remodeling is required to attain complete cytochrome c release from the mitochondria. In this work we show that, at the onset of apoptosis, changes in MMP control matrix remodeling prior to cytochrome c release. Early after growth factor withdrawal the MMP declines and the matrix condenses. Both phenomena are reversed by adding oxidizable substrates. In mitochondria isolated from healthy cells, matrix condensation can be induced by either denying oxidizable substrates or by protonophores that dissipate the membrane potential. Matrix remodeling to the condensed state results in cristal unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria during apoptosis. In contrast, when a transmembrane potential is generated due to either electron transport or a pH gradient formed by acidifying the medium, mitochondria maintain an orthodox configuration in which most cytochrome c is sequestered in the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane.
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Apoptosis/fisiología , Carbonil Cianuro m-Clorofenil Hidrazona/análogos & derivados , Citocromos c/metabolismo , Citosol/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Animales , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Línea Celular , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/fisiología , Citosol/efectos de los fármacos , Citosol/ultraestructura , Inmunohistoquímica , Interleucina-3/deficiencia , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/ultraestructura , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Péptidos/farmacología , Rotenona/farmacología , Succinatos/farmacologíaRESUMEN
INTRODUCTION: Change must begin with education. Theme 8 explored issues that need attention in Disaster Medicine education. METHODS: Details of the methods used are provided in the introductory paper. The chairs moderated all presentations and produced a summary that was presented to an assembly of all of the delegates. The chairs then presided over a workshop that resulted in the generation of a set of action plans that then were reported to the collective group of all delegates. RESULTS: Main points developed during the presentations and discussion included: (1) formal education, (2) standardized definitions, (3) integration, (4) evaluation of programs and interventions, (5) international cooperation, (6) identifying the psychosocial consequences of disaster, (7) meaningful research, and (8) hazard, impact, risk and vulnerability analysis. DISCUSSION: Three main components of the action plans were identified as evaluation, research, and education. The action plans recommended that: (1) education on disasters should be formalized, (2) evaluation of education and interventions must be improved, and (3) meaningful research should be promulgated and published for use at multiple levels and that applied research techniques be the subject of future conferences. CONCLUSIONS: The one unanimous conclusion was that we need more and better education on the disaster phenomenon, both in its impacts and in our response to them. Such education must be increasingly evidence-based.
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Planificación en Desastres/organización & administración , Medicina de Emergencia/educación , Evaluación de Necesidades/organización & administración , Competencia Clínica/normas , Salud Global , Planificación en Salud/organización & administración , Investigación sobre Servicios de Salud , Humanos , Relaciones Interinstitucionales , Cooperación Internacional , Desarrollo de Programa , Evaluación de Programas y Proyectos de Salud , Investigación , Medición de RiesgoRESUMEN
The relationship between body weight (BW) and respiratory minute volume (V(m)) was reviewed by collecting a database from the literature. The data were separated into anaesthetized and non-anaesthetized groups. Only young adult terrestrial mammals were included in the final data set. This database is the largest to be reported to date, is the first to separate the anaesthetized and non-anaesthetized groups and is matched to the target population of young, fit adult humans. The data set of non-anaesthetized animals contained 142 studies representing 2616 animals and 18 species from mice at 12 g body weight to horses and a giraffe at ca. 500 kg body weight. Analysis of the data indicated a power law (allometric) relationship between the minute volume and body weight. The resulting allometric equations for the empirical relationship between minute volume and body weight are: log(10)V()(m)= -0.302 + 0.809 log(10)BW and V(m) = 0.499 BW(0.809)where V(m) is the minute volume (l min(-1)) and BW is the body weight (kg). From these equations, a minute volume of 15.5 lmin(-1)was obtained for a 70 kg human in the same physiological and/or emotional state as the animals. The results of the analyses were compared to other empirical studies in the literature, the more recent of which also indicated a scaling factor of 0.8. The relationship between minute volume and body weight is recommended for use in estimating the inhalation toxicity to young adult humans (military personnel), because this is the first study to use a large database focused exclusively upon non-anaesthetized young adult terrestrial mammals.
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Peso Corporal , Exposición por Inhalación/efectos adversos , Respiración , Pruebas de Toxicidad/normas , Adulto , Anestesia , Animales , Animales de Laboratorio , Femenino , Humanos , Masculino , Valores de Referencia , Estudios Retrospectivos , Especificidad de la Especie , Estadística como AsuntoRESUMEN
This article describes the behavior of transiently transfected human receptors into melanophores and the potential use of constitutive receptor activity to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably leads to increased levels of receptor expression. This leads to an increased response to agonists (both maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated receptor to cause elevated basal cellular responses. This was observed as a decrease in the transmittance of light through melanophores (consistent with G(s) protein activation) and increased response to human calcitonin. The receptor-mediated nature of this response was confirmed by its reversal with the hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with G(i)-protein activation). NPY1 cDNA produced little constitutive response on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potency) with increasing cDNA transfection. The receptor/G(i)-protein nature of both the constitutive and agonist-mediated responses was confirmed by elimination with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applicability of this approach for the general screening of G protein-coupled orphan receptors.
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Evaluación Preclínica de Medicamentos , Proteínas de Unión al GTP/metabolismo , Receptores de Calcitonina/metabolismo , Secuencia de Aminoácidos , Animales , Calcitonina/análogos & derivados , Calcitonina/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Ligandos , Melanóforos/efectos de los fármacos , Melanóforos/metabolismo , Melanosomas/efectos de los fármacos , Melanosomas/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Calcitonina/agonistas , Receptores de Calcitonina/química , Receptores de Calcitonina/genética , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Xenopus laevisRESUMEN
This paper discusses the use of constitutively active G-protein-coupled receptor systems for drug discovery. Specifically, the ternary complex model is used to define the two major theoretical advantages of constitutive receptor screening-namely, the ability to detect antagonists as well as agonists directly and the fact that constitutive systems are more sensitive to agonists. In experimental studies, transient transfection of Chinese hamster ovary cyclic AMP response element (CRE) luciferase reporter cells with cDNA for human parathyroid hormone receptor, glucagon receptor, and glucagon-like peptide (GLP-1) receptor showed cDNA concentration-dependent constitutive activity with parathyroid hormone (PTH-1) and glucagon. In contrast, no constitutive activity was observed for GLP-1 receptor, yet responses to GLP-1 indicated that receptor expression had taken place. In another functional system, Xenopus laevi melanophores transfected with cDNA for human calcitonin receptor showed constitutive activity. Nine ligands for the calcitonin receptor either increased or decreased constitutive activity in this assay. The sensitivity of the system to human calcitonin increased with increasing constitutive activity. These data indicate that, for those receptors which naturally produce constitutive activity, screening in this mode could be advantageous over other methods.
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Evaluación Preclínica de Medicamentos , Modelos Químicos , Modelos Moleculares , Receptores de Droga/química , Animales , Células CHO , Calcitonina/farmacología , Cricetinae , ADN Complementario/genética , Evaluación Preclínica de Medicamentos/métodos , Humanos , Melanóforos/efectos de los fármacos , Melanóforos/fisiología , Receptores de Calcitonina/efectos de los fármacos , Receptores de Calcitonina/genética , TransfecciónRESUMEN
Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.
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Amiloide/farmacología , Antiulcerosos/farmacología , Calcitonina/farmacología , Proteínas de la Membrana/farmacología , Receptores de Calcitonina/efectos de los fármacos , Animales , Humanos , Péptidos y Proteínas de Señalización Intracelular , Polipéptido Amiloide de los Islotes Pancreáticos , Proteínas de la Membrana/metabolismo , Ratas , Proteína 1 Modificadora de la Actividad de Receptores , Proteína 2 Modificadora de la Actividad de Receptores , Proteína 3 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Calcitonina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , XenopusRESUMEN
This article describes the transient expression of the CXC chemokine receptor-4 in Xenopus laevis melanophores and the resulting functional assay for the endogenous ligand for this receptor stromal cell-derived factor (SDF)-1alpha. Specifically, it will be shown that SDF-1alpha produces increased light transmittance in transfected cells that is consistent with the activation of Gi protein. This stimulus pathway is further implicated by the abolition of this response after pretreatment of the cells with pertussis toxin, a known method for the inactivation of Gi protein. The fact that SDF-1alpha does not produce responses in nontransfected cells and that treatment of the cells with 12G5, an antibody specific for the CXC chemokine receptor-4, eliminates this response indicates that this ligand produces responses by activation of this receptor in these cells. The possible relevance to human immunodeficiency virus (HIV) entry into cells was explored by observing the effects of SDF-1alpha on HIV-mediated cell fusion. It was found that SDF-1alpha blocked cell-to-cell fusion (as has been previously reported) at concentrations 1200-fold greater than those required to produce Gi protein mediated responses. The implications of the functional assay to screening for new drugs to block HIV-mediated fusion is discussed.
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Quimiocinas CXC/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Receptores CXCR4/fisiología , Secuencia de Aminoácidos , Animales , Quimiocina CXCL12 , Ligandos , Melanóforos , Datos de Secuencia Molecular , Toxina del Pertussis , Proteínas Recombinantes , Transducción de Señal , Factores de Virulencia de Bordetella/farmacología , Xenopus laevisRESUMEN
Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493-497 (1993); J. Pharmacol. Exp. Ther. 270:779-787 (1994); Eur. J. Pharmacol. 262:133-141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound 125I-AC512 and 125I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125I-human calcitonin (hCAL) or 125I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125I-AC512, 125I-hCAL, and 125I-rAmylin with high affinity. The agonists 125I-hCAL and 125I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand 125I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (125I-hCAL = 24.8% Bmax, 125I-rAmylin = 8% Bmax). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand 125I-AC512. For example, the pKi value for displacement of 125I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125I-AC512 and agonist 125I-hCAL was observed; no binding to 125I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.
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Receptores de Calcitonina/efectos de los fármacos , Receptores de Calcitonina/genética , Receptores de Péptidos/metabolismo , Secuencia de Aminoácidos , Amiloide/metabolismo , Amiloide/farmacología , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Humanos , Radioisótopos de Yodo , Polipéptido Amiloide de los Islotes Pancreáticos , Datos de Secuencia Molecular , Núcleo Accumbens/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Calcitonina/metabolismo , Receptores de Polipéptido Amiloide de Islotes Pancreáticos , Receptores de Péptidos/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
We have selected a tobacco cell line, SU-27D5, that is highly resistant to sulfonylurea and imidazolinone herbicides. This line was developed by selection first on a lethal concentration of cinosulfuron and then on increasing concentrations of primisulfuron, both sulfonylurea herbicides. SU-27D5 was tested against five sulfonylureas and one imidazolinone herbicide and was shown, in every case, to be two to three orders of magnitude more resistant than wild-type cells. The acetohydroxyacid synthase (AHAS) of SU-27D5 was 50- to 780-fold less sensitive than that of wild-type cells to herbicide inhibition. The specific activity of AHAS in the SU-27D5 cell lysate was 6 to 7 times greater than that in wild-type cells. Using Southern analysis, we showed that cell line SU-27D5 had amplified its SuRB AHAS gene about 20-fold while maintaining a normal diploid complement of the SuRA AHAS gene. Genomic clones of both AHAS genes were isolated and used to transform wild-type tobacco protoplasts. SuRB clones gave rise to herbicide-resistant transformants, whereas SuRA clones did not. DNA sequencing showed that all SuRB clones contained a point mutation at nucleotide 588 that converted amino acid 196 of AHAS from proline to serine. In contrast, no mutations were found in the SuRA clones. The stability of SuRB gene amplification was variable in the absence of selection. In one experiment, the withdrawal of selection reduced the copy number of the amplified SuRB gene to the normal level within 30 days. In another experiment, amplification remained stable after extended cultivation on herbicide-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)
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Acetolactato Sintasa/genética , Amplificación de Genes/genética , Herbicidas/farmacología , Nicotiana/enzimología , Plantas Tóxicas , Compuestos de Sulfonilurea , Southern Blotting , Línea Celular , Clonación Molecular , Resistencia a Medicamentos/genética , Familia de Multigenes/genética , Mutación/genética , Nicotiana/efectos de los fármacos , Nicotiana/genéticaRESUMEN
The complete nucleotide sequence of RNA alpha from the Type strain of barley stripe mosaic virus has been determined. The RNA is 3768 nucleotides long and contains a single open reading frame which codes for a polypeptide of 1139 amino acids (mw 129,634). The open reading frame is flanked by a 5'-terminal sequence of 91 nucleotides and a 3'-nontranslated region composed of a short poly(A) tract followed by a 238-nucleotide tRNA-like structure. The amino acid sequence of the polypeptide (alpha a) encoded by the open reading frame has homology with the TMV 126K protein and with related polypeptides from other viruses. The carboxy-terminal portion of the alpha a polypeptide also has limited homology with the 58K (beta b) protein encoded by BSMV RNA beta and includes a consensus sequence found in mononucleotide-binding polypeptides.
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Virus del Mosaico/genética , Péptidos/genética , ARN Viral/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Autorradiografía , Secuencia de Bases , Hordeum , Datos de Secuencia Molecular , Virus ARN/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The complete nucleotide sequences of RNA gamma from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5'-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3'-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNA gamma ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNA gamma ORF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNA gamma ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3'-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984, Virology 139, 375-383). In the genomic RNA gamma, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.
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Genes Virales , Virus del Mosaico/genética , ARN Viral/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Virus del Mosaico/clasificación , Conformación de Ácido Nucleico , Proteínas Virales/genéticaRESUMEN
The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.
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Virus del Mosaico/genética , ARN Viral/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Clonación Molecular , Codón , ADN/genética , Genes , Genes Virales , Biosíntesis de Proteínas , Proteínas Virales/genéticaAsunto(s)
Nivel de Alerta , Ciclo Menstrual , Adolescente , Adulto , Femenino , Actividad Nerviosa Superior , Humanos , Pruebas PsicológicasRESUMEN
A bacterial gene encoding hygromycin phosphotransferase has been modified for expression in tobacco cells. The aphIV gene from Escherichia coli was inserted between the 5' sequence of an octopine synthase gene and the 3' sequence from a nopaline synthase gene. The new gene was incorporated between T-DNA border fragments in the broad-host-range vector pKT210 to form a micro-Ti plasmid. Agrobacterium tumefaciens containing this plasmid and a Ti plasmid as helper was used to incite crown gall tumors on aseptic tobacco plants. Samples of these galls could grow in the presence of hygromycin B, provided that the aph gene had been fused with the ocs gene to maintain the sense of the coding sequences. When the genes had been fused in the reverse 'antisense' orientation none of the gall samples could grow on hygromycin. Unlike wild-type galls the hygromycin-resistant tissue contained DNA sequences homologous to the aphIV gene. Thus the modified gene can be introduced into tobacco cells and confer on them the ability to grow in the presence of hygromycin B.
RESUMEN
A deletion mutant of cauliflower mosaic virus (CaMV) isolate NY8153 deficient in aphid transmissibility was constructed by BAL-31 exonuclease treatment of XhoI linearized pCMS31 (a plasmid containing the entire CaMV genome cloned in the SalI site of pBR322), followed by ligation. The resulting mutant, pSA103, lacked about 100 by from the putative protein-coding region II of CaMV DNA. In turnips there was no difference in the number and appearance of starch lesions or hybridization lesions, or in the nature of symptoms in systemically infected leaves induced by pSA103 DNA, pCMS31 DNA, and NY8153 DNA. Systemic symptoms appeared later in plants infected with pSA103 (27 +/- 2 days) than in those infected with the parental pCMS31 DNA (19 +/- 2 days). Aphids fed on virus SA103-infected mustard plants were unable to transmit the virus to healthy plants while 30-70% transmission was observed from plants infected by the parent virus. Virions extracted from turnips infected with the mutant had DNA still containing the deletion. In addition, the single-stranded discontinuity in region II of NY8153 DNA was missing in this DNA. The results suggest that region II codes for a "helper component" required for aphid transmission of CaMV.
RESUMEN
Low levels of adenosine 3',5'-monophosphate (cyclic AMP) were detected in the cyanobacterium Anabaena variabilis using a protein binding assay and two radioisotopic labelling methods. The basal concentration of intracellular cyclic AMP ranged from 0.27 pmol/mg protein in A. variabilis Kutz grown under heterotrophic conditions to 1.0--2.7 pmol/mg protein in A. variabilis strain 377 grown autotrophically. Extracellular cyclic AMP was found to comprise as much as 90% of the total cyclic AMP in rapidly growing cultures. When A. variabilis strain 377 was starved of nitrogen, a 3--4-fold increase in intracellular cyclic AMP was observed during the 24 h period coincident with early heterocyst development.