RESUMEN
Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to generate superoxide, needed for the intracellular killing of microorganisms. This is caused by mutations in any one of the four subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of this enzyme (p67-phox) is missing. We here report on a patient with a mutation in the p67-phox gene that leads to expression of a nonfunctional p67-phox protein. The purified granulocytes of this patient failed to produce superoxide and contained about half of the normal amount of p67-phox. Analysis of the cDNA and genomic DNA of this patient showed that the patient is a compound heterozygote for a triplet nucleotide deletion in the p67-phox gene, predicting an in-frame deletion of lysine 58 in the p67-phox protein and a larger deletion of 11-13 kb in the other allele. Interestingly, the 58Lys deletion in p67-phox disrupts the interaction with p21-rac1, a ras-related protein involved in the activation of the NADPH oxidase. In contrast to normal neutrophils, in which p47-phox and p67-phox translocate to the plasma membrane upon cell activation, the cells of the patient did not show this translocation, indicating that an interaction between p67-phox and p21-rac1 is essential for translocation of these cytosolic proteins and activation of the NADPH oxidase. Moreover, this CGD patient represents the first case of disease caused by a disturbed binding of a ras-related protein to its target protein.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Enfermedad Granulomatosa Crónica/etiología , Enfermedad Granulomatosa Crónica/genética , Mutación , Fosfoproteínas/genética , Transporte Biológico , Compartimento Celular , Niño , Chile/etnología , Femenino , Enfermedad Granulomatosa Crónica/clasificación , Humanos , NADPH Oxidasas , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Proteínas de Unión al GTP racRESUMEN
Rho and Rac, two members of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins, regulate a variety of signal transduction pathways in eukaryotic cells. Upon stimulation of phagocytic cells, Rac enhances the activity of the enzyme nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase, resulting in the production of superoxide radicals. Activation of the NADPH oxidase requires the assembly of a multimolecular complex at the plasma membrane consisting of two integral membrane proteins, gp91phox and p21phox, and two cytosolic proteins, p67phox and p47phox. Rac1 interacted directly with p67phox in a GTP-dependent manner. Modified forms of Rac with mutations in the effector site did not stimulate oxidase activity or bind to p67phox. Thus, p67phox appears to be the Rac effector protein in the NADPH oxidase complex.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fagocitos/enzimología , Fosfoproteínas/metabolismo , Sitios de Unión , Activación Enzimática , Guanosina Trifosfato/metabolismo , Humanos , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Proteínas Recombinantes de Fusión/metabolismo , Superóxidos/metabolismo , Proteínas de Unión al GTP racRESUMEN
In keratinocytes, the beta1 integrins mediate adhesion to the extracellular matrix and also regulate the initiation of terminal differentiation. To explore the relationship between these functions, we stably infected primary human epidermal keratinocytes and an undifferentiated squamous cell carcinoma line, SCC4, with retroviruses encoding wild-type and mutant chick beta1 integrin subunits. We examined the ability of adhesion-blocking chick beta1-specific antibodies to inhibit suspension-induced terminal differentiation of primary human keratinocytes and the ability of the chick beta1 subunit to promote spontaneous differentiation of SCC4. A D154A point mutant clustered in focal adhesions but was inactive in the differentiation assays, showing that differentiation regulation required a functional ligand-binding domain. The signal transduced by beta1 integrins in normal keratinocytes was "do not differentiate" (transduced by ligand-occupied receptors) as opposed to "do differentiate" (transduced by unoccupied receptors), and the signal depended on the absolute number, rather than on the proportion, of occupied receptors. Single and double point mutations in cyto-2 and -3, the NPXY motifs, prevented focal adhesion targeting without inhibiting differentiation control. However, deletions in the proximal part of the cytoplasmic domain, affecting cyto-1, abolished the differentiation-regulatory ability of the beta1 subunit. We conclude that distinct signaling pathways are involved in beta1 integrin-mediated adhesion and differentiation control in keratinocytes.
Asunto(s)
Adhesión Celular , Integrina beta1/metabolismo , Queratinocitos/citología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Virus del Sarcoma Aviar/genética , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos , Técnica del Anticuerpo Fluorescente , Humanos , Integrina beta1/química , Integrina beta1/genética , Queratinocitos/química , Queratinocitos/metabolismo , Queratinocitos/virología , Datos de Secuencia Molecular , Mutación/genética , Precursores de Proteínas/análisis , Transducción de Señal , Suspensiones , Transfección , Células Tumorales CultivadasRESUMEN
Either radiolabeled Tc-99m- or Re-188-labeled MAG3-4-nitrophenylester or unlabeled Bz-MAG3-4-nitrophenylester was reacted with amines and peptides to follow a pre- or a postconjugate radiolabeling route, respectively. The model compounds were N'-t-butyloxycarbonyl-1,6-diaminohexane (DH-Boc) and a Lys-protected derivative of the somatostatin analog RC-160 (cyclic D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2). In the case of labeling DH-Boc, both the preconjugate labeling and the postconjugate labeling were found by using analytical HPLC to provide identical radiolabeled compounds regardless whether Re-188 or Tc-99m was used. The results are supported by infrared and mass-spectral data obtained from compounds synthesized using stable rhenium. The 188Re- or 99mTc-MAG3-RC-160 somatostatin analog were synthesized following the preconjugate labeling route and subsequent removal of the protecting group. Biodistributions of 188Re-and 99mTc-MAG3-RC-160 were evaluated in normal and tumor-bearing mice, and were similar to those of radioiodinated 131-RC-160. All radiolabeled analogs of RC-160 were rapidly cleared from the blood and were excreted through the hepatobiliary system with very little normal organ uptake. The tumor uptake (PC-3, human prostate adenocarcinoma) of systemically administered Re-188-MAG3-RC160 was very low, and it reached only 0.28% injected dose/g (%IDg) at 24 h postinjection, similar to what was obtained with I-131-RC-160. Intratumor injections resulted in significant tumor retentions (9.3% ID/g at 24 h).
Asunto(s)
Aminas/química , Oligopéptidos/química , Compuestos Organometálicos/química , Tecnecio Tc 99m Mertiatida/química , Animales , Cromatografía Líquida de Alta Presión , Diaminas/química , Femenino , Ésteres del Ácido Fórmico/química , Ratones , Ratones Desnudos , Oligopéptidos/farmacocinética , Compuestos Organometálicos/farmacocinética , Compuestos de Organotecnecio , Radiofármacos , Somatostatina/análogos & derivados , Somatostatina/química , Somatostatina/farmacocinética , Tecnecio Tc 99m Mertiatida/farmacocinética , Factores de Tiempo , Distribución TisularAsunto(s)
Proteínas de Unión al GTP/análisis , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Animales , Clonación Molecular/métodos , Escherichia coli , Proteínas de Unión al GTP/biosíntesis , Proteínas Activadoras de GTPasa , Guanosina Trifosfato/metabolismo , Indicadores y Reactivos , Cinética , Mamíferos , Mutagénesis Sitio-Dirigida , Radioisótopos de Fósforo , Mutación Puntual , Unión Proteica , Biosíntesis de Proteínas , Proteínas/análisis , Técnica de Dilución de Radioisótopos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Tritio , Proteínas de Unión al GTP racRESUMEN
BACKGROUND: An efficient medical documentation is mandatory for a trauma-oriented department in the DRG environment. Besides the continuously increasing clinical/administrative demands, the additional documentation for quality assurance, clinical studies, and research requires additional efforts. Standard solutions are only partially effective. Especially in hand surgery there is a high demand for sophisticated clinical documentation, represented by a wide variety of classifications in diagnosis and therapy. The standard documentation tools lack accuracy. The development of a software tool that defines administrative/business processes and simultaneously generates clinical and administrative information was the goal of this project. METHODS AND RESULTS: With a standard medical terminology, an innovative semantic network, and a completely new graphical user interface, it was possible to develop and introduce a software program specifically adjusted for hand surgery. This program facilitated for the first time a single-stage acquisition of clinically relevant scientific data and the simultaneous generation of DRG, quality assurance, and administrative data relevant for the hospital's revenues. CONCLUSIONS: The newly developed software tool is a step forward into a new dimension of medical software, obviating the need for multi/documentation and significantly improving the quality of clinically relevant medical data.
Asunto(s)
Grupos Diagnósticos Relacionados/clasificación , Documentación/métodos , Traumatismos de la Mano/clasificación , Administración Hospitalaria , Sistemas de Registros Médicos Computarizados/organización & administración , Garantía de la Calidad de Atención de Salud/organización & administración , Terminología como Asunto , Gráficos por Computador , Grupos Diagnósticos Relacionados/organización & administración , Alemania , Traumatismos de la Mano/diagnóstico , Traumatismos de la Mano/cirugía , Humanos , Internet , Fracturas del Radio/clasificación , Fracturas del Radio/diagnóstico , Fracturas del Radio/cirugía , Hueso Escafoides/lesiones , Hueso Escafoides/patología , Hueso Escafoides/cirugía , Programas Informáticos , Gestión de la Calidad Total/organización & administración , Interfaz Usuario-ComputadorRESUMEN
The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.
Asunto(s)
Actinas/metabolismo , Membrana Celular/fisiología , Proteínas de Unión al GTP/fisiología , Sustancias de Crecimiento/fisiología , Células 3T3 , Animales , Adhesión Celular/fisiología , Medio de Cultivo Libre de Suero , Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ratones , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/fisiología , Pinocitosis , Especificidad por Sustrato , Proteínas de Unión al GTP racRESUMEN
Rac, a small GTPase in the ras superfamily, regulates at least two biological processes in animal cells: (i) the polymerization of actin and the assembly of integrin complexes to produce lamellipodia and ruffles; and (ii) the activity of an NADPH oxidase in phagocytic cells. NADPH oxidase activation is mediated through a rac effector protein, p67phox, and using chimeras made between rac and the closely related GTPase, rho, we have identified two distinct effector sites in rac, one N-terminal and one C-terminal, both of which are required for activation of p67phox. The same two effector sites are essential for rac-induced actin polymerization in fibroblasts. p65PAK, a ubiquitous serine/threonine kinase, interacts with rac at both the N- and C-terminal effector sites, but the GTPase-activating protein, bcr interacts with rac at a different region. This makes p65PAK, but not bcr, a candidate effector of rac-induced lamellipodium formation.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Células 3T3 , Animales , Sitios de Unión , Proteínas de Unión al GTP/química , Proteínas Activadoras de GTPasa , Ratones , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rac , Proteínas Activadoras de ras GTPasaRESUMEN
A novel widely expressed type of myosin (fifth unconventional myosin from rat: myr 5) from rat tissues, defining a ninth class of myosins, was identified. The predicted amino acid sequence of myr 5 exhibits several features not found previously in myosins. The myosin head domain contains a unique N-terminal extension and an insertion of 120 amino acids at a postulated myosin-actin contact site. Nevertheless, myr 5 is able to bind actin filaments in an ATP-regulated manner. The head domain is followed by four putative light chain binding sites. The tail domain of myr 5 contains a region which coordinates two atoms of zinc followed by a region that stimulates GTP hydrolysis of members of the ras-related rho subfamily of small G-proteins. Myr 5 therefore provides the first direct link between rho GTPases which have been implicated in the regulation of actin organization and the actin cytoskeleton. It is also the first unconventional myosin for which a tail binding partner(s), namely members of the rho family, has been identified.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Miosinas/genética , Miosinas/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , GTP Fosfohidrolasas/genética , Datos de Secuencia Molecular , Miosinas/química , Miosinas/clasificación , Ésteres del Forbol/metabolismo , Unión Proteica , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Zinc/metabolismoRESUMEN
The therapeutic potential of the somatostatin analogue RC-160 radiolabeled with 188Re was evaluated in nude mice bearing xenografts of human prostate adenocarcinoma. 188Re-RC-160 was selectively retained in both DU-145 and PC-3 tumors following direct intra-tumor injection at all time points examined (2, 6 and 24 hr post-injection). Unbound 188Re-RC-160 was rapidly excreted via the hepatobiliary system and, with the exception of the gastrointestinal tract, very little normal organ uptake was found at any time point examined. Negative control compounds, 188Re-perrhenate and 188Re-mercaptoacetyl-triglycine (188Re-MAG3), were essentially washed out of the tumor by 6 hr post-injection and were rapidly excreted through the kidneys. 131I-RC-160, used as reference compound, had a biodistribution in tumor-bearing animals similar to that of 188Re-RC-160. In PC-3 xenografts, 188Re-RC-160 gave a dose-dependent therapeutic response (stasis or regression) even in animals with relatively large tumor masses (greater than 600 mm3), whereas the macro-aggregated form of 188Re-RC-160 did not. Long-term studies with 188Re-RC-160 demonstrated a protracted reduction of tumor volume and a positive effect on animal survival. Neither RC-160 by itself nor a 188Re-labeled peptide, unrelated to somatostatin (PA-22-2, a laminin peptide), demonstrated the reduction in tumor mass observed with 188Re-RC-160. 188Re-RC-160 shows potential as a new clinical agent for treatment of somatostatin-receptor-positive cancers.
Asunto(s)
Adenocarcinoma/radioterapia , Neoplasias de la Próstata/radioterapia , Receptores de Somatostatina/análisis , Renio/uso terapéutico , Somatostatina/uso terapéutico , Adenocarcinoma/metabolismo , Animales , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias de la Próstata/metabolismo , Radioisótopos/uso terapéutico , Somatostatina/análogos & derivados , Trasplante HeterólogoRESUMEN
More than thirty small guanine nucleotide-binding proteins related to the ras-encoded oncoprotein, termed Ras or p21ras, are known. They regulate many fundamental processes in all eukaryotic cells, such as growth, vesicle traffic and cytoskeletal organization. GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis of Ras-related proteins, leading to down-regulation of the active GTP-bound form. For p21ras, two GAP proteins are known, rasGAP and the neurofibromatosis (NF1) gene product. There is evidence that rasGAP may also be a target protein for regulation by Ras and be involved in downstream signalling. We have purified a GAP protein for p21rho, which is involved in the regulation of the actin cytoskeleton. Partial sequencing of rhoGAP reveals significant homology with the product of the bcr (breakpoint cluster region) gene, the translocation breakpoint in Philadelphia chromosome-positive chronic myeloid leukaemias. We show here that the carboxy-terminal domains of the bcr-encoded protein (Bcr) and of a Bcr-related protein, n-chimaerin, are both GAP proteins for the Ras-related GTP-binding protein, p21rac. This result suggest that Bcr could be a target for regulation by Rac and has important new implications for the role of bcr translocations in leukaemia.