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Intraosseous vascular malformations are rare vascular anomalies that present unique treatment challenges due to structural instability, embolization and sclerotherapy resistance, and tendency to recur. Patients may have clinical manifestations including pain, functional impairment, increased fracture risk, and decreased quality of life. Image-guided ablation techniques are emerging interventional treatment options for soft tissue tumors and complicated vascular anomalies. Percutaneous image-guided cryoablation offers a potential alternative as an isolated or adjunct therapy for intraosseous vascular malformations.
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Transjugular intrahepatic portosystemic shunt (TIPS) creation treats complications of portal hypertension in appropriately selected patients by lowering the portal venous pressure. While this can be a lifesaving intervention, portal venous flow diversion is not without potential consequences. Overshunting can lead to hepatic decompensation and encephalopathy. TIPS reduction and TIPS occlusion are therapeutic options used to mitigate overshunting, with reduction being the initial alternative due to retained shunt patency and lower potential for venous thrombosis. Patient selection, techniques for TIPS reduction, and patient outcomes are reviewed in this article.
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Intercostal schwannomas can present incidentally and lead to compressive thoracic symptoms. These slow-growing and benign tumors typically arise from intercostal nerves and are supplied by intercostal arteries, which may increase the risk of hemorrhagic complications with surgical resection. Due to the rarity of intercostal schwannomas, there exists no standardized management algorithms. Pre-operative angiography and embolization can supplement surgical thoracotomy and resection by decreasing intra-operative hemorrhage and minimizing the risk of anterior spinal cord hypoperfusion.
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Embolización Terapéutica , Neurilemoma , Angiografía , Estudios de Seguimiento , Humanos , Neurilemoma/diagnóstico por imagen , Neurilemoma/cirugía , ToracotomíaRESUMEN
Background: To detect concentrations of subtherapeutic doses of the CD80-Fc fusion protein FPT155 in serum in Phase I studies, a highly sensitive assay was developed. Materials & methods: FPT155 was purified from human serum using magnetic beads coupled to cytotoxic T-lymphocyte-associated antigen-4. After washing away the serum components, FTP155 was released by acid dissociation and neutralization. The eluted drug was quantified in an ELISA using cytotoxic T-lymphocyte-associated antigen-4 as a capture reagent and biotinylated anti-human Fc for detection. The assay was validated with a calibration range of 5-40 ng/ml and a dilutional integrity of up to 100,000 ng/ml. Conclusion: A highly sensitive assay to determine serum concentrations of FPT155 using readily available reagents was developed. The results were in conformity with theoretical calculations.
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Antígeno B7-1/sangre , Ensayo de Inmunoadsorción Enzimática , Fragmentos Fc de Inmunoglobulinas/sangre , Proteínas Recombinantes de Fusión/sangre , Antígeno B7-1/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Fenómenos Magnéticos , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Neonatal cases of systemic artery to portal venous system arteriovenous malformations (AVMs) can present unique challenges in terms of diagnosis, management, and treatment. Prompt identification of these AVMs is necessary for minimizing long-term sequelae and optimizing prognosis. Our report describes the diagnosis and successful endovascular coil embolization of a congenital right internal mammary artery (IMA) to portal vein AVM in a young infant initially presenting during routine fetal screening with an incidentally discovered congenital thoracic vascular abnormality.
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Malformaciones Arteriovenosas , Embolización Terapéutica , Arterias Mamarias , Malformaciones Arteriovenosas/diagnóstico por imagen , Malformaciones Arteriovenosas/terapia , Humanos , Lactante , Arterias Mamarias/diagnóstico por imagen , Vena Porta/diagnóstico por imagen , PronósticoRESUMEN
Endovascular transshepatic access has limitations that can be exacerbated in the posttransplantation setting. Although several techniques are available for portal venous system catheterization, the transsplenic approach offers a direct pathway for accessing the portal venous system, as well as associated varices or shunts, while avoiding potential injury to the liver transplant. The purpose of this report is to present the diagnostic and interventional management of main portal vein occlusion in a 56-year-old female after liver transplantation. Endovascular transsplenic recanalization with stenting and shunt embolization is a viable method for treatment of main portal vein thrombosis in an adult liver transplant recipient.
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PURPOSE: Venous thromboembolism (VTE) refers to both deep venous thrombosis (DVT) and pulmonary embolism (PE). The risk of VTE in adult neurosurgical patients is thoroughly studied. However, the incidence and risk of VTE in a comprehensive pediatric neurosurgical population is not well-defined. The available pediatric data consists of reviews of specific high-risk groups, such as trauma, critical care, or cancer patients. This may not be reflective of the entire spectrum of a high-volume pediatric neurosurgery practice. This study was undertaken to analyze the incidence and risk factors of VTE in all hospitalizations evaluated by a pediatric neurosurgery service over a 25-year period. METHODS: A retrospective review of electronic medical records was performed for 9149 hospitalizations in 6374 unique patients evaluated by the pediatric neurosurgery service at Riley Hospital for Children (Indianapolis, IN, USA) from 1990-2014. During this time period, there was no standardized VTE prevention protocol. The study group included all patients less than 18 years of age. Patients with a known pre-existing VTE or pregnancy were excluded. RESULTS: VTE was diagnosed in 20 of the 9149 (0.22%) hospitalizations, in 18 unique patients. All DVTs were diagnosed via Doppler ultrasound and/or computed tomography. Anatomic clot locations included 9 in the upper extremity (0.098% of hospitalizations), 8 in the lower extremity (0.087%), and 4 (0.044%) pulmonary emboli. Ten of the 20 occurred in hospitalizations where the patient underwent surgery, although the need for surgery was not a statistically significant risk factor. Sixteen of the 20 (80%) occurred in patients with at least one form of central venous line (p < 0.00001). There was one VTE-related death (0.01%). CONCLUSIONS: In all pediatric neurosurgical patients, a VTE was found in 0.22% of hospitalizations over a 25-year span. Statistically significant risk factors for VTE included central venous line placement, paralysis, malignancy, intubation greater than 48 h, and hypercoagulable state.
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Embolia Pulmonar , Tromboembolia Venosa , Trombosis de la Vena , Adulto , Niño , Femenino , Humanos , Incidencia , Embarazo , Embolia Pulmonar/diagnóstico por imagen , Embolia Pulmonar/epidemiología , Embolia Pulmonar/etiología , Estudios Retrospectivos , Factores de Riesgo , Tromboembolia Venosa/epidemiología , Tromboembolia Venosa/etiologíaRESUMEN
OBJECTIVE: The purpose of this report is to discuss the presentation and the progressive magnetic resonance imaging (MRI) findings in a single patient with clinically-diagnosed Raynaud's phenomenon (RP). CONCLUSION: RP can present as non-specific toe pain and manifest as progressive abnormal MRI bone marrow signal in the toes. In addition to patient presentation and clinical assessment, this information could contribute to earlier diagnosis and treatment of RP and other coexisting rheumatologic disorders.
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BACKGROUND: Further investigation is needed to look at the impact of vestibular schwannoma (VS) on the health-related quality of life (QOL) of participants who undergo Gamma Knife® radiosurgery (GKRS). OBJECTIVES: Investigators compared the QOL for VS participants to reported US population norms in order to evaluate disease burden and long-term QOL several years after GKRS. METHODS: This cross-sectional study surveyed participants to assess hearing status, tinnitus, imbalance, vertigo, as well as the Short-Form 36-item Health Questionnaire (SF-36). The data were normalized, age adjusted, and functional status was correlated to determine clinically significant differences. RESULTS: A total of 353 participants who underwent GKRS between 1997 and 2007 were included in this study with a median postoperative period of 5 years. SF-36 scores were very similar to population norms, and age-adjusted scores for participants followed the US population curve. Frequent vertigo and balance problems had the largest statistically and clinically significant effect on physical and mental component summary scores followed by nonuseful hearing in the tumor ear. CONCLUSIONS: Participants reported a good long-term QOL that was very similar to the QOL of US population norms. Of the common VS symptoms, vertigo had the greatest impact on QOL followed by imbalance and then hearing loss.
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Neuroma Acústico/radioterapia , Neuroma Acústico/cirugía , Calidad de Vida , Radiocirugia , Adulto , Anciano , Costo de Enfermedad , Estudios Transversales , Humanos , Persona de Mediana Edad , Neuroma Acústico/complicaciones , Encuestas y Cuestionarios , Resultado del TratamientoRESUMEN
Microglia are resident inflammatory cells of the CNS and have important roles in development, homeostasis and a variety of neurologic and psychiatric diseases. Difficulties in procuring human microglia have limited their study and hampered the clinical translation of microglia-based treatments shown to be effective in animal disease models. Here we report the differentiation of human induced pluripotent stem cells (iPSC) into microglia-like cells by exposure to defined factors and co-culture with astrocytes. These iPSC-derived microglia have the phenotype, gene expression profile and functional properties of brain-isolated microglia. Murine iPSC-derived microglia generated using a similar protocol have equivalent efficacy to primary brain-isolated microglia in treatment of murine syngeneic intracranial malignant gliomas. The ability to generate human microglia facilitates the further study of this important CNS cell type and raises the possibility of their use in personalized medicine applications.
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Diferenciación Celular/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Microglía/metabolismo , Microglía/fisiología , Animales , Astrocitos/citología , Movimiento Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Glioma/terapia , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Microglía/trasplante , Fagocitosis/fisiología , Especies Reactivas de Oxígeno/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Blister aneurysms at non-branching sites of the dorsal internal carotid artery (dICA) are fragile, rare, and often difficult to treat. The purpose of this study is to address the demographics, treatment modalities, and long-term outcome of patients treated for dICA blister aneurysms. A retrospective review of medical records identified all consecutive patients who presented with a blister aneurysm from 2002 to 2011 at our institution. Eighteen patients (M=7, F=11; mean age: 48.4±15.1years; range: 15-65years) harbored a total of 43 aneurysms, 25 of which were dorsal wall blister aneurysms of the ICA. Eleven (61.1%) patients presented with aneurysmal subarachnoid hemorrhage (aSAH), and 10 (55.6%) patients had multiple aneurysms at admission. Twelve patients had 18 aneurysms that were treated microsurgically. Five (41.7%) of these patients had a single recurrence that was retreated with subsequent repeat clip ligation. Six patients had 7 blister aneurysms that were treated with endovascularly. One (16.7%) of these patients had a single recurrence that was retreated with subsequent coil embolization. Postoperative vasospasm occurred in 8 (44.4%) patients, one of whom suffered from a stroke. This is one of the largest single-institution dICA blister aneurysm studies to date. There was no detected significant difference between microsurgical clip ligation and endovascular coil embolization in terms of surgical outcome. These blister aneurysms demonstrate a propensity to be associated with multiple cerebral aneurysms. Strict clinical and angiographic long-term follow-up may be warranted. STATEMENT OF SIGNIFICANCE: Blister aneurysms are focal wall defects covered by a thin layer of fibrous tissue and adventitia, lacking the usual collagenous layer. Due to their pathologically thin vessel wall, blister aneurysms are prone to rupture. The management of these rare and fragile aneurysms presents a number of challenges. Here, we address the long-term outcome of patients treated for blister aneurysms at non-branching sites of the dICA. The presented data and analysis is imperative to determine the necessary strict long-term clinical and angiographic follow-up.
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Enfermedades de las Arterias Carótidas/cirugía , Arteria Carótida Interna/cirugía , Embolización Terapéutica/métodos , Procedimientos Endovasculares/métodos , Aneurisma Intracraneal/cirugía , Instrumentos Quirúrgicos , Adolescente , Adulto , Anciano , Vesícula/diagnóstico por imagen , Vesícula/cirugía , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Arteria Carótida Interna/diagnóstico por imagen , Embolización Terapéutica/instrumentación , Procedimientos Endovasculares/instrumentación , Femenino , Estudios de Seguimiento , Humanos , Aneurisma Intracraneal/diagnóstico por imagen , Ligadura , Masculino , Persona de Mediana Edad , Recurrencia , Retratamiento/métodos , Estudios Retrospectivos , Instrumentos Quirúrgicos/estadística & datos numéricos , Adulto JovenRESUMEN
OBJECTIVE With the recent evolution of endovascular therapies, objective evaluation of the efficacy of clip ligation for cerebral aneurysms should be performed. This study was undertaken to evaluate the durability of microsurgical clip ligation, identify risk factors for recurrence, and assess the need for long-term follow-up imaging. METHODS A retrospective review of medical records identified 616 consecutive patients (156 male and 460 female patients; mean age 48.4 ± 12.4 years; range 6-90 years) who underwent microsurgical clip ligation and follow-up imaging at least 1 year after discharge between 1990 and 2010 at our institution. Of a total of 926 aneurysms in 616 patients, 758 aneurysms were microsurgically clip-ligated. At presentation, 431 of these aneurysms were ruptured and 327 aneurysms were unruptured. All patients underwent postoperative baseline imaging within the 1st month of their operation. A logistic regression analysis was performed to identify which variables are more likely to predict recurrence. RESULTS Late follow-up angiographic imaging was obtained at a mean of 7.2 ± 4.7 years postdischarge (median 5.7 years; range 1-23 years). Of the 699 clipped aneurysms without residua, late follow-up angiography revealed only 1 (0.14%) recurrent aneurysm. Of the 59 residual aneurysms that remained after initial clip ligation on early postoperative imaging, 8 (13.6%) demonstrated growth. All of these aneurysms required treatment. None of the recurrences were due to broken or delayed displacement of clips. A total of 111 patients presented with multiple aneurysms. De novo aneurysm formation occurred in 8 (0.97%) patients, all of whom initially presented with multiple aneurysms. CONCLUSIONS This study provides additional evidence to support the long-term efficacy of aneurysm clip ligation. The chance of aneurysm recurrence after complete clip ligation is very small. However, there is a regrowth risk of 1.83% per year for aneurysm remnants after incomplete clip ligation. These findings support the necessity for continued followup, late angiographic imaging, and the potential need for further intervention of incompletely ligated aneurysms. Furthermore, completely clip-ligated aneurysms may not require additional surveillance imaging unless multiple aneurysms were evident at presentation.
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Aneurisma Intracraneal/epidemiología , Aneurisma Intracraneal/cirugía , Ligadura , Microcirugia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Estudios de Seguimiento , Humanos , Aneurisma Intracraneal/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Recurrencia , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: This review aims to identify and assess evidence on interventions to communicate risk and promote disease mitigation measures in epidemics and emerging disease outbreak settings. The study focuses on data that are relevant to low and middle-income country (LMIC) settings. METHODS: We conducted a comprehensive literature search using five major electronic databases (Pubmed Medline, Biomed Central, EMBASE, Science of Citation Index, and Cochrane Library) and other sources to identify relevant studies published from January 2002 to July 2013. The review was guided by the socio-ecological model/perspective of public health and the ideation theory and focused on interventions at the community, healthcare, and multi-sectoral settings, which also reflect key intervention levels of the Ottawa Charter for Health Promotion. Eligible quantitative studies were selected according to specific study criteria and assessed using the Critical Appraisal Skills Program (CASP) framework. Conversely, qualitative studies, reviews, case studies, and editorials were not included. Studies were selected by two independent reviewers. RESULTS: Twenty-nine relevant studies from 16 countries were included. Most studies focused on a single intervention or intervention level, rather than multi-sectoral interventions. The majority of the evidence relates to programs aimed at behavioral and social results (or relevant intermediate steps) within a specific population group. Two studies included implications for improvements in health service delivery, two studies examined the intervention's impact on health systems-related outcomes, and three had also implications for environmental health outcomes. Cost- and health equity-related implications for select evidence were also discussed. CONCLUSIONS: The paucity of well-designed quantitative evaluations of interventions to communicate health risk and promote disease control measures in LMICs does not allow for any definitive conclusions. Yet, the review identified several promising interventions and areas for future investigation. Among them, community-based and participatory interventions seemed to be central within epidemic and emerging disease settings, particularly in low-resource settings. Yet, evidence on their effectiveness is not conclusive and needs to be explored by future studies. Other promising areas for future investigation include multi-component and multi-sectoral approaches to intervention design. Major research gaps referred to any evaluation of the impact of these kinds of interventions on health policy adoption and/or implementation, and social determinants of health. Research on cost-effectiveness also needs to be strengthened. This review identified several research gaps and questions, and discusses potential future directions for increasing capacity for future and more rigorous assessments.
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Enfermedades Transmisibles Emergentes/prevención & control , Comunicación , Epidemias , Salud Pública , Enfermedades Transmisibles Emergentes/epidemiología , Países en Desarrollo , Femenino , Promoción de la Salud , Humanos , Masculino , Conducta de Reducción del RiesgoRESUMEN
In the present paper, gel-filtration studies of diferric-ovotransferrin (Fe2OTf), the individual half-molecules of ovotransferrin (OTf) and equimolar mixtures of half-molecules have been interpreted according to the Gilbert theory as developed by Ackers & Thompson [(1965) Proc. Natl. Acad. Sci. U.S.A. 53, 342-349]. The data indicate that the half-molecules associate reversibly in solution and allow determination of a dissociation constant, Kd' = 8.0 (+/- 2.7) microM. Equilibrium binding studies have been performed using NH4Cl to block removal of iron from equimolar differentially iodine-labelled half-molecules (125I and 131I), in order to evaluate the binding of each to chick-embryo red blood cells under identical conditions. The amount of associated half-molecules over a range of concentrations has been calculated using the constant derived from the gel-filtration experiments described above. A computerized non-linear least-squares regression analysis of the data leads to determination of Kd* (the apparent dissociation constant for the interaction between OTf or half-molecules and the transferrin (Tf) receptors of chick-embryo red blood cells) and Bmax (binding at infinite free-ligand concentration) for the half-molecules similar to those found for Fe2OTf. Recent reports confirm that the two iron-binding domains of both OTf and human lactotransferrin associate non-covalently in solution. Our work shows that the isolated half-molecules of OTf are able to reassociate in solution and that this reassociation has functional significance by allowing the complex to be recognized by the Tf receptor.
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Conalbúmina/metabolismo , Proteínas del Huevo/metabolismo , Reticulocitos/metabolismo , Cloruro de Amonio/farmacología , Animales , Embrión de Pollo , Cromatografía en Gel , Técnicas In Vitro , Hierro/metabolismo , Cinética , Ligandos , Sustancias Macromoleculares , Unión Proteica/efectos de los fármacos , SolucionesRESUMEN
Separation of ovotransferrin into C-terminal (OTf/2C) and N-terminal (OTf/2N) half-molecules has made possible the resolution of all expected histidinyl C(2)H resonances by proton nuclear magnetic resonance at 250 MHz. The chemical shift of many of the resonances decreases with increasing pH, allowing construction of titration curves, whereas a few resonances fail to titrate. On formation of the GaIIIOTf/2(C2O4) ternary complexes, two of the low-field C(2)H resonances in each half-molecule fail to titrate. This behavior implicates the imidazole groups giving rise to these resonances as ligands to the bound metal ion. A third C(2)H resonance in each half-molecule undergoes a marked reduction in pK'a on formation of the ternary complex. The imidazole group displaying this resonance is implicated in a proton-relay scheme involved in binding the synergistic anion, oxalate, and a water of hydration on the bound metal ion. The titration curves for the various imidazole resonances have been fit to a four-parameter equation involving estimation of the pK'a, the limiting chemical shift values, and a Hill constant n. Hill constants of less than 1 can be rationalized by correcting the titration curve for the charge Z on the protein as a function of pH and the work function w. The titration curve for the imidazole group in OTf/2C involved in the proton-relay scheme shows a value for n greater than 1, which suggests positive cooperativity in the titration of this residue. The basis for this behavior cannot be rationalized at this time.(ABSTRACT TRUNCATED AT 250 WORDS)
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Conalbúmina/metabolismo , Proteínas del Huevo/metabolismo , Histidina , Aniones , Sitios de Unión , Concentración de Iones de Hidrógeno , Cinética , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética/métodos , Metales/metabolismo , Oxalatos/metabolismo , Unión Proteica , Conformación Proteica , Urea/metabolismoRESUMEN
The ability of human-derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first-order association rate constants of 0.43 min-1 for human Tf and 0.15 min-1 for equine Tf. Labeled chicken ovo-Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37 degrees C in the presence of 2,4-dinitrophenol to prevent iron removal indicate 1 X 10(6) Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0 degrees C to prevent endocytosis reveals 4.1-6.7 X 10(5) Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast-like MRC-5 cells indicate expression of 0.82-2.78 X 10(5) Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500-fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human greater than horse greater than calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum-free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf.
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Receptores de Superficie Celular/metabolismo , Transferrina/metabolismo , Animales , Unión Competitiva , Bovinos/sangre , División Celular , Línea Celular , Células HeLa , Caballos/sangre , Humanos , Hierro/metabolismo , Radioinmunoensayo , Receptores de Transferrina , Especificidad de la Especie , Transferrina/farmacologíaRESUMEN
Two fragments, each corresponding to approximately half of the ovotransferrin (OTf) molecule and containing an iron-binding site were produced by digestion with affinity bound trypsin and were purified by isoelectric focusing and gel filtration chromatography. The immunologically distinct "half-molecules" individually have little ability to bind to transferrin receptors on chick embryo red blood cells or to donate iron to them. Combining them, however, leads to both binding and iron donation approaching that found for holo-OTf. Furthermore, similar amounts of radiolabeled iron can be extracted into the putative heme fraction from Fe2OTf and from the various combined half-molecules. These findings conflict with those reported by Keung and Azari ( (1982) J. Biol. Chem. 257, 1184-1188) for subtilisin-derived half-molecules of OTf examined in a similar system. They found that each half-molecule appeared to bind at a level of approximately one-third that of Fe2OTf and that the half-molecules competed with each other for binding sites. In contrast, our equilibrium binding studies, in the presence of 2,4-dinitrophenol to prevent iron removal, led to the determination of 4.79 X 10(4) binding sites/cell for Fe2OTf, 4.44 X 10(4) for the NH2-terminal half-molecules in the presence of excess COOH-terminal half-molecules and 4.17 X 10(4) for COOH-terminal half-molecules in the presence of NH2-terminal half-molecules; apparent binding constants were estimated to be 3.29 X 10(6), 1.19 X 10(6), and 0.67 X 10(6) M-1 for these same samples. Problems associated with equilibrium binding studies in which a narrow range of concentrations of ligand is used and/or iron is being removed are discussed. Labeled combined half-molecules were half as effective as labeled Fe2OTf in competition with unlabeled Fe2OTf. These findings are consistent with the lower apparent binding constant found in the equilibrium binding studies. Equimolar apo-OTf had no effect on binding of either Fe2OTf or the combined half-molecules. It seems apparent from our studies that the NH2- and COOH-terminal half-molecules each contain a recognition region both of which are necessary for binding to the transferrin receptor and iron donation to the chick embryo red blood cell.
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Conalbúmina/metabolismo , Proteínas del Huevo/metabolismo , Hierro/sangre , Receptores de Superficie Celular/metabolismo , Reticulocitos/metabolismo , Transferrina/metabolismo , 2,4-Dinitrofenol , Animales , Embrión de Pollo , Dinitrofenoles/farmacología , Inmunodifusión , Cinética , Unión Proteica , Receptores de TransferrinaRESUMEN
The biphasic binding of diferric transferrin to reticulocytes has been reevaluated with a series of kinetic and equilibrium studies. Identical binding progress profiles were observed for reticulocytes in the presence or absence of oxygen. The relative size of the rapid initial adsorption step could be increased to ca. 65% of the total binding by stripping the cells of endogenous transferrin or reduced to 0% by preloading the cells with nonradiolabeled diferric transferrin. Preloading the cells with 125I-labeled diferric transferrin and chasing with 131I-labeled diferric transferrin revealed identical rate constants for release and binding. Scatchard plots of equilibrium binding of diferric transferrin to reticulocytes showed no significant effects of anaerobiasis or 2,4-dinitrophenol on the equilibrium binding constant or the maximum number of binding sites. The potent microtubule inhibitor nocodazole had no effect on the progress curves for transferrin binding or iron uptake by reticulocytes. It was concluded that the rapid adsorption step in the binding profile represents binding to open receptors and that the slow first-order binding phase represents binding of radiolabeled transferrin to receptors already occupied by nonlabeled endogenous transferrin as this endogenous transferrin leaves the receptors. Furthermore, this first-order binding phase, unlike iron uptake, does not require the presence of active oxidative phosphorylation. These findings are consistent with a specific desorption-adsorption model for the interaction of diferric transferrin with reticulocytes.
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Reticulocitos/metabolismo , Transferrina/metabolismo , 2,4-Dinitrofenol , Adsorción , Animales , Embrión de Pollo , Dinitrofenoles/farmacología , Hierro/metabolismo , Cinética , Modelos Biológicos , Reticulocitos/efectos de los fármacos , Vinblastina/farmacologíaRESUMEN
The complete amino acid sequence of the beta subunit of phycocyanin from the unicellular rhodophyte Cyanidium caldarium, has been determined by automated sequential degradation of cyanogen bromide, tryptic, and Staphylococcus aureus V8 protease peptides. The beta subunit contains 172 amino acids with methionine and glutamine the NH2- and carboxyl-terminal amino acids, respectively. The calculated molecular weight of the protein, based on the sequence, is 19,572. Two phycocyanobilin chromophores are covalently attached by cysteinyl thioether linkages to residues 82 and 153. A third cystine (residue 109) occurs in the beta subunit, but it is not attached to phycocyanobilin. Comparison of the complete amino acid sequence of the beta subunit of C. caldarium phycocyanin with the sequences of the phycocyanin beta subunits from two cyanobacteria, shows that the sequence homology previously noted at the NH2 terminus of phycobiliproteins from distantly related organisms extends along the entire polypeptide chain. The amino acid sequences of the alpha and beta subunits of C. caldarium phycocyanin are also similar, and by proper alignment of the sequences it can be shown that the beta subunit contains a 12-residue insertion where the second phycocyanobilin chromophore is covalently attached. A matrix comparing the alpha and beta subunits of phycobiliproteins for which the complete sequences are known has been determined, and based on these data, a scheme is proposed for evolution of the family of phycobiliproteins in living cyanobacteria and red algae from a protein precursor which gave rise initially to a beta-type allophycocyanin subunit.
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Ficocianina , Pigmentos Biológicos , Rhodophyta/análisis , Secuencia de Aminoácidos , Bromuro de Cianógeno , Sustancias Macromoleculares , Fragmentos de Péptidos/análisis , Péptido Hidrolasas , TripsinaRESUMEN
The complete amino acid sequence of the alpha subunit of phycocyanin from the unicellular rhodophyte Cyanidium caldarium has been determined by automated sequential degradation of cyanogen bromide peptides, tryptic peptides derived from protein chemically modified with 1,2-cyclohexanedione or citraconic anhydride, and a peptide obtained after cleavage of the protein at the single tryptophan residue. The alpha subunit contains 162 amino acids and methionine and serine are the NH2- and carboxyl-terminal amino acids, respectively. The calculated molecular weight of the protein, based on the amino acid sequence, is 18,303, in good agreement with the value of 17,500 +/- 500, obtained by electrophoresis on calibrated sodium dodecyl sulfate-polyacrylamide gels. One phycocyanobilin chromophore is attached to the alpha subunit at residue 84 by a cysteinyl thioether linkage. A second cysteine (residue 98) is present but is not linked to phycocyanobilin. The amino acid sequence of the alpha subunit of phycocyanin from C. caldarium is the first complete amino acid sequence of a phycobiliprotein from a eukaryotic alga. Extensive homology occurs between the alpha subunit of phycocyanin from C. caldarium and from two prokaryotic cyanobacteria, and the significance of this is discussed.