Asunto(s)
Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/cirugía , Escisión del Ganglio Linfático , Linfadenitis/etiología , Linfadenitis/cirugía , Disección del Cuello , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Cuello , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: The efficacy of granulocyte transfusions in patients with HLA alloimmunization is uncertain. A flow cytometric assay using dihydrorhodamine 123 (DHR), a marker for cellular NADPH oxidase activity, was used to monitor the differential survival of transfused oxidase-positive granulocytes in alloimmunized patients with chronic granulomatous disease (CGD). STUDY DESIGN AND METHODS: Ten patients with CGD and serious infections were treated with daily granulocyte transfusions derived from steroid and granulocyte-colony-stimulating factor-stimulated donors. The proportion of neutrophils with intact oxidase activity was quantitated by DHR fluorescence on samples drawn before and 1 hour after transfusion. The incidence of acute transfusion reactions was correlated with the results of DHR fluorescence and biweekly HLA serologic screening assays. RESULTS: Eight of 10 patients experienced acute adverse reactions in association with granulocyte transfusions. Four had only chills and/or fever, and four experienced respiratory compromise; all eight exhibited HLA alloimmunization. Mean (± SD) oxidase-positive cell recovery was 19.7 ± 17.4% (n = 15 transfusions) versus 0.95 ± 1.59% (n = 16) in the absence and presence of HLA allosensitization, respectively (p < 0.01). Greater than 1% in vivo recovery of DHR-enhancing donor granulocytes was strongly correlated with lack of HLA alloimmunization. CONCLUSION: The ability to detect DHR-positive donor granulocytes by flow cytometry is strongly correlated with absence of HLA alloimmunization and lack of acute reactions to granulocyte transfusions in patients with CGD. If HLA antibodies are present and the survival of donor granulocytes is low by DHR analysis, transfusions should be discontinued, avoiding a therapy associated with high risk and unclear benefit.
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Granulocitos/trasplante , Enfermedad Granulomatosa Crónica/terapia , Transfusión de Leucocitos/métodos , Adolescente , Niño , Preescolar , Femenino , Citometría de Flujo , Humanos , Masculino , Neutrófilos/citología , Adulto JovenRESUMEN
The occurrence and structure of microfilaments in epidermal cancers induced in mice by treatment with 3,4-benzpyrene were investigated with the electron microscope. With malignant change, pleomorphic, undifferentiated cells with a cortical zone of microfilaments became increasingly abundant. The microfilaments were 40 A in diameter and occupied the cortex of the cells beneath the plasma membrane, extended into cell processes, and were situated in the cores of microvilli. At high magnification, the filamentous areas were formed by an interconnected meshwork of filaments which in favorable planes had a polygonal arrangement. When exposed to high concentrations of cytochalasin B, the microfilaments became clumped and moderately disrupted. At the same time, the processes and microvilli of the cells were blunted. The structure of these filaments and their sensitivity to cytochalasin B place them in a class of microfilaments believed to be related to cell motility. Their presence in malignant cells may be correlated with the motile, invasive properties of these cells.
Asunto(s)
Carcinoma de Células Escamosas/patología , Carcinoma/patología , Neoplasias Cutáneas/patología , Piel/citología , Acetona/farmacología , Animales , Benzo(a)Antracenos/farmacología , Benzopirenos , Carcinoma de Células Escamosas/inducido químicamente , Movimiento Celular , Citocalasina B/farmacología , Femenino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Piel/efectos de los fármacos , Piel/patología , Neoplasias Cutáneas/inducido químicamenteRESUMEN
Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 mum) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 mug/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.
Asunto(s)
Quimiotaxis , Citoplasma/fisiología , Citoesqueleto/fisiología , Microtúbulos/fisiología , Neutrófilos/fisiología , Sangre , Núcleo Celular , Quimiotaxis/efectos de los fármacos , Colchicina/farmacología , Citocalasina B/farmacología , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Organoides/fisiología , SeudópodosRESUMEN
Chronic granulomatous diseases of childhood (CGD) are a group of disorders of phagocytic cell superoxide (O2.-) production (respiratory burst). Anion exchange chromatography separated from normal neutrophil cytosol a 47-kilodalton neutrophil cytosol factor, NCF-1, that restored activity to defective neutrophil cytosol from most patients with autosomally inherited CGD in a cell-free O2.--generating system. A 65-kilodalton factor, NCF-2, restored activity to defective neutrophil cytosol from one patient with autosomal CGD. NCF-1, NCF-2, and a third cytosol fraction, NCF-3, were inactive alone or in pairs, but together replaced unfractionated cytosol in cell-free O2.- generation. Neutrophils deficient in NCF-1, but not NCF-2, did not phosphorylate the 47-kilodalton protein. It is proposed that NCF-1, NCF-2, and NCF-3 are essential for generation of O2.- by phagocytic cells and that genetic abnormalities of these cytosol components can result in the CGD phenotype.
Asunto(s)
Enfermedad Granulomatosa Crónica/metabolismo , Neutrófilos/metabolismo , Superóxidos/biosíntesis , Western Blotting , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Técnicas In Vitro , Peso Molecular , Fosfoproteínas/metabolismoRESUMEN
A 47-kilodalton neutrophil cytosol factor (NCF-47k), required for activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase superoxide (O2-.) production, is absent in most patients with autosomal recessive chronic granulomatous disease (AR-CGD). NCF-47k cDNAs were cloned from an expression library. The largest clone predicted a 41.9-kD protein that contained an arginine and serine-rich COOH-terminal domain with potential protein kinase C phosphorylation sites. A 33-amino acid segment of NCF-47k shared 49% identity with ras p21 guanosine triphosphatase activating protein. Recombinant NCF-47k restored O2-. -producing activity to AR-CGD neutrophil cytosol in a cell-free assay. Production of active recombinant NCF-47k will enable functional regions of this molecule to be mapped.
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ADN/genética , Enfermedad Granulomatosa Crónica/genética , NADH NADPH Oxidorreductasas/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Enfermedad Granulomatosa Crónica/enzimología , Humanos , Immunoblotting , Datos de Secuencia Molecular , NADPH Oxidasas , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxidos/metabolismoRESUMEN
The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.
Asunto(s)
Grupo Citocromo b/sangre , NADH NADPH Oxidorreductasas/sangre , Neutrófilos/enzimología , Fagocitos/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Sistema Libre de Células , Grupo Citocromo b/genética , Grupo Citocromo b/aislamiento & purificación , Ferredoxina-NADP Reductasa/genética , Ferredoxina-NADP Reductasa/metabolismo , Humanos , Insectos , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/aislamiento & purificación , NADP/metabolismo , NADPH Oxidasas , Plantas/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Superóxidos/sangre , TransfecciónRESUMEN
Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.
Asunto(s)
NADH NADPH Oxidorreductasas/genética , Neutrófilos/enzimología , Proteínas Proto-Oncogénicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Enfermedad Granulomatosa Crónica/sangre , Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/genética , Humanos , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/sangre , NADPH Oxidasas , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas pp60(c-src) , Homología de Secuencia de Ácido NucleicoRESUMEN
Myeloperoxidase (MPO), a heme enzyme present in the azurophilic granules of human polymorphonuclear neutrophils (PMN), is important in the oxygen-dependent microbicidal activity of PMN. MPO deficiency, defined as the lack of PMN peroxidative activity, is a common genetic defect of human PMN. The purpose of our study was to characterize the structural basis for this loss of enzymatic activity, using protein biochemical and immunochemical techniques to examine PMN from three subjects with partial MPO deficiency and from five subjects with complete MPO deficiency.We purified MPO from normal PMN and defined its electrophoretic mobility after two-dimensional electrophoretic separation, using nondenaturing acidic polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecyl sulfate (SDS) denaturation and SDS-PAGE separation of MPO subunit peptides. In agreement with previous studies, we found that normal MPO had subunits of 59,000 and 13,500 mol wt when subjected to SDS-PAGE under reducing conditions. Granule protein extracts of normal PMN, partially MPO-deficient PMN, and completely MPO-deficient PMN were analyzed with two-dimensional PAGE. Partially MPO-deficient PMN granules contained electrophoretically normal MPO in less than normal amounts, whereas completely MPO-deficient PMN granules contain no protein with the electrophoretic mobility of normal MPO. Using rabbit antiserum against purified MPO, we used immunoautoradiographic analysis to examine whole PMN for peptides immunochemically related to MPO. PMN from normal, partially MPO-deficient, and completely MPO-deficient subjects were solubilized in SDS and component peptides separated by SDS-PAGE. The peptides were electroblotted onto nitrocellulose paper that was exposed sequentially to rabbit anti-MPO and (125)I-protein A before autoradiography. Radiolabeled bands were identical when partially purified MPO or normal PMN were compared except that whole PMN contained a small amount of an immunologically cross-reactive membrane associated material of 75,000-90,000 mol wt. Using a modification of this immunoautoradiographic analysis, we quantitated the relative amounts of MPO peptides in PMN. PMN from MPO-deficient subjects contain 41.0-52.3% the amount of MPO peptides present in normal PMN. Similar analysis showed that completely MPO-deficient PMN lacked any peptides corresponding to MPO peptides.We conclude that partial MPO deficiency is characterized by the presence of electrophoretically and immunologically normal MPO in amounts approximately one-half that seen in PMN from normal subjects. Completely MPO-deficient PMN lack any normal MPO peptides. No MPO-deficient subject studied had an immunologically cross-reacting variant of MPO. Since this deficiency is associated with the absence of more than one peptide, it is possible that the underlying genetic defect may involve: (a) failure to synthesize a single precursor peptide; (b) abnormal regulation of the synthesis of two separate peptides; or (c) an aberration in postsynthetic processing or packaging into azurophilic granules.
Asunto(s)
Neutrófilos/enzimología , Peroxidasa/deficiencia , Peroxidasas/deficiencia , Adolescente , Adulto , Autorradiografía , Gránulos Citoplasmáticos/enzimología , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Femenino , Humanos , Técnicas Inmunológicas , Masculino , Persona de Mediana Edad , Peso Molecular , Peroxidasa/análisis , Peroxidasa/inmunologíaRESUMEN
Normal and antibiotic-pretreated staphylococci were incubated with human neutrophils to determine the interactions between cells and antimicrobials in the killing of the organisms. Staphylococcus aureus 502A pretreated during log-phase growth with subinhibitory ((1/4) minimum inhibiting concentration) (MIC) concentrations of penicillin G were more susceptible to killing by normal neutrophils than untreated bacteria (intracellular survival 0.17+/-0.04 vs. 1.5+/-0.38%, mean+/-SEM, respectively, at 35 min in 14 experiments; P < 0.01 by t test). Furthermore, this enhanced susceptibility to killing was observed even when phagosome formation was inhibited by cytochalasin B (65.6+/-4.6% pencillintreated vs. 30.5+/-4.5% untreated killed at 30 min in 14 experiments, P < 0.001). Pretreatment of S. aureus with vancomycin similarly enhanced susceptibility to killing by cytochalasin B-treated polymorphonuclear leukocytes (PMN), whereas pretreatment with gentamicin did not. The enchancement of killing by pretreatment with cell wall-active antibiotics was present in a dose-response fashion to 1/16th the MIC. It required specific antimicrobial activity; i.e., penicillin activity was inhibited by penicillinase or by incubation with bacteria at 4 degrees C. It also required active cellular metabolism and intact neutrophils. For antibiotic-pretreated bacteria to be killed by normal and cytochalasin B-treated cells, phagocytosis or binding to the cells was essential via a serum opsonindependent mechanism. In experiments with the cytochalasin B-treated cells, all bound penicillin-treated bacteria were killed vs. only a fraction (70%) of the bound untreated bacteria. Penicillin in 10 times the MIC had no direct effects on PMN phagocytic, metabolic, or microbicidal functions against a nonsusceptible organism, Candida albicans. The results indicate a cooperative effect between cell wall-active antibiotics at low concentrations and human PMN in the killing of staphylococci. The model establishes conditions for the study of the mechanisms involved in the cooperation of these bactericidal systems.
Asunto(s)
Neutrófilos/fisiología , Penicilina G/farmacología , Fagocitosis , Staphylococcus aureus/fisiología , Citocalasina B/farmacología , Relación Dosis-Respuesta a Droga , Gentamicinas/farmacología , Humanos , Lisostafina/farmacología , Neutrófilos/microbiología , Temperatura , Vancomicina/farmacologíaRESUMEN
Neutrophil specific granule deficiency (SGD) is a congenital disorder associated with an impaired inflammatory response and a deficiency of several granule proteins. The underlying abnormality causing the deficiencies is unknown. We examined mRNA transcription and protein synthesis of two neutrophil granule proteins, lactoferrin and myeloperoxidase in SGD. Metabolically labeled SGD nucleated marrow cells produced normal amounts of myeloperoxidase, but there was no detectable synthesis of lactoferrin. Transcripts of the expected size for lactoferrin were detectable in the nucleated marrow cells of two SGD patients, but were markedly diminished in abundance when compared with normal nucleated marrow cell RNA. Because lactoferrin is secreted by the glandular epithelia of several tissues, we also assessed lactoferrin in the nasal secretions of one SGD patient by ELISA and immunoblotting. Nasal secretory lactoferrin was the same molecular weight as neutrophil lactoferrin and was secreted in normal amounts. From these data, we conclude that lactoferrin deficiency in SGD neutrophils is tissue specific and is secondary to an abnormality of RNA production. We speculate that the deficiency of several granule proteins is due to a common defect in regulation of transcription that is responsible for the abnormal myeloid differentiation seen in SGD patients.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Regulación de la Expresión Génica , Enfermedades Hematológicas/congénito , Lactoferrina/genética , Lactoglobulinas/genética , Neutrófilos/ultraestructura , Northern Blotting , Células de la Médula Ósea , Sondas de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactoferrina/deficiencia , Mucosa Nasal/metabolismo , Peroxidasa/genética , Peroxidasa/metabolismo , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
The phagocyte NADPH oxidase is a complex system consisting of membrane and cytosolic components that must assemble at the membrane for proper activation. Studies of patients with chronic granulomatous diseases of childhood have enabled the molecular characterization of these components, which has led to studies defining their interaction during NADPH complex assembly. Understanding NADPH oxidase assembly provides an opportunity to develop therapeutics for the regulation of this important reaction of inflammation.
Asunto(s)
Enfermedad Granulomatosa Crónica/enzimología , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fagocitos/enzimología , Niño , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Citosol/metabolismo , Inducción Enzimática , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/patología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasas , Neutrófilos/enzimología , Procesamiento Proteico-Postraduccional , Estallido Respiratorio , Superóxidos/metabolismoRESUMEN
We have produced a murine IgM monoclonal antibody (Y201) that recognizes a cell surface antigen present on HL60 cells. Seventy percent of uninduced HL60 cells expressed Y201 antigen, while the remainder did not. There were no morphological differences between HL60 cells that expressed Y201 antigen and cells that did not express Y201 antigen. Cells with the greatest number of antigenic sites were found to have greater proliferative capacity in liquid culture and in soft agar than did HL60 cells deficient in this marker. Expression or lack of expression of the Y201 antigen is not constant over a prolonged period in that both subpopulations ultimately reproduced the original pattern of antigenic expression when grown in liquid culture. The antigen identified by Y201 was lost with terminal differentiation of HL60 cells using a variety of inducers. Loss of Y201 antigen during differentiation was associated with a decrease in proliferative capacity in soft agar. Loss of Y201 antigen by greater than 95% of differentiated HL60 cells was associated with loss of proliferative capacity. These data suggest that HL60 cells are heterogeneous in regard to proliferative capacity and that this heterogeneity is associated with expression of the cell surface antigen identified by Y201.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Diferenciación Celular , Leucemia Mieloide Aguda/inmunología , Especificidad de Anticuerpos , Diferenciación Celular/efectos de los fármacos , División Celular , Línea Celular , Dimetilsulfóxido/farmacología , Citometría de Flujo , Granulocitos/citología , Humanos , Monocitos/citología , Tretinoina/farmacologíaRESUMEN
A "Hollow Cylinder Protein' (HCP) similar to the protein originally isolated by Harris from human erythrocyte membranes (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534--537) is present in the cytosol of erythrocytes at a concentration of more than 15 micrograms/ml packed erythrocytes. When negatively stained and examined in the electron microscope, cytosol HCP is similar in morphology to the HCP associated with erythrocyte ghost membranes. Cytosol HCP can be purified by isoelectric precipitation at pH 5.2 followed by repeated sucrose gradient centrifugation at alkaline pH. Negatively stained purified cytosol HCP appears as a hollow cylinder with apparent dimensions of 18.0 nm in length by 11.8 nm in diameter and contains a hollow core. Purified cytosol HCP migrates as a single band by non-denaturing polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis shows that it is composed of five peptides having apparent molecular weights 21 500, 23 500, 26 000, 27 500 and 29 000. Chymotryptic peptide maps of each of these bands indicate that each is a unique polypeptide chain. These results indicate that erythrocyte cytosol HCP is a macromolecular complex composed of multiple copies of five non-identical subunits arranged as a hollow cylinder.
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Proteínas Sanguíneas , Eritrocitos/análisis , Proteínas de la Membrana/aislamiento & purificación , Citosol/análisis , Electroforesis en Gel de Poliacrilamida , Membrana Eritrocítica/análisis , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Peso Molecular , Fragmentos de Péptidos/análisis , Conformación ProteicaRESUMEN
Protein kinase C (PKC) inhibitors, staurosporine or 1,5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited NADPH oxidase activity and phosphorylation of 47 kDa protein (p47) in PMA-stimulated neutrophils in a dose-dependent manner. These PKC inhibitors, at the same doses, did not affect oxidase activity and caused only partial inhibition of p47 phosphorylation in OZ-stimulated neutrophils. There was residual (20%) phosphorylated p47 in the membranes of OZ-stimulated cells in the presence of PKC inhibitors, at concentrations which caused total inhibition of oxidase activity and p47 phosphorylation in PMA-stimulated neutrophils. In the presence of ionomycin, which increased intracellular calcium ion concentrations, staurosporine was less effective in inhibiting both superoxide generation and p47 phosphorylation stimulated by PMA, similar to its effect in OZ-stimulated cells. The results indicate that some phosphorylation of p47 always accompanied oxidase activation induced by PMA or OZ, though the degree of phosphorylation of membrane-bound p47 does not directly correlate with rates of superoxide production.
Asunto(s)
Alcaloides/farmacología , Proteínas Sanguíneas/metabolismo , Isoquinolinas/farmacología , NADH NADPH Oxidorreductasas/sangre , Neutrófilos/enzimología , Piperazinas/farmacología , Proteína Quinasa C/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Proteínas Sanguíneas/aislamiento & purificación , Membrana Celular/metabolismo , Citosol/metabolismo , Activación Enzimática , Humanos , Ionomicina/farmacología , Cinética , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADPH Oxidasas , Neutrófilos/efectos de los fármacos , Fosfatos/sangre , Fosfoproteínas/sangre , Fosfoproteínas/aislamiento & purificación , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina , Superóxidos/sangre , Acetato de Tetradecanoilforbol/farmacología , Zimosan/farmacologíaRESUMEN
BACKGROUND: Previous investigations provide evidence that an enzyme related to the phagocyte NADPH oxidase produces superoxide in the blood vessel wall. These data, however, are confounded by observations that both NADPH and NADH serve as substrates for superoxide production in vascular cells. To clarify this issue, we compared the superoxide-generating capabilities of vascular smooth muscle cells (VSMCs) derived from wild-type (p47phox(+/+); phagocyte oxidase) mice with those from mice that lack p47phox (p47phox(-/-); "knockout"), an essential component of the phagocyte NADPH oxidase. METHODS AND RESULTS: VSMCs were derived from aortic explants harvested from p47phox(+/+) or p47phox(-/-) mice. VSMCs from p47phox(+/+) but not those from p47phox(-/-) mice produced superoxide after stimulation by phorbol myristate acetate. Consistent with this, p47phox was detected only in p47phox(+/+) VSMCs. p47phox-transduced p47phox(-/-) but not enhanced green fluorescent protein-transduced p47phox(-/-) VSMCs generated significant levels of superoxide after stimulation by angiotensin II or platelet-derived growth factor-BB (PDGF-BB). Enhanced expression of recombinant p47phox in p47phox-transduced p47phox(-/-) cells correlated with superoxide production in these cells. CONCLUSIONS: These data provide direct functional proof that an oxidase requiring the p47phox component mediates superoxide release from VSMCs in the blood vessel wall in response to angiotensin II or PDGF-BB.
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , Músculo Liso Vascular/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Superóxidos/metabolismo , Actinas/biosíntesis , Angiotensina II/farmacología , Animales , Aorta , Becaplermina , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Genes Reporteros , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía de Contraste de Fase , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , NADPH Oxidasas/metabolismo , Fosfoproteínas/deficiencia , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Acetato de Tetradecanoilforbol/farmacología , Transducción GenéticaRESUMEN
OBJECTIVE: The CD34 cell surface marker is used widely for stem/progenitor cell isolation. Since several recent studies reported that CD34(-) cells also have in vivo engrafting capacity, we quantitatively compared the engraftment potential of CD34(+) vs CD34(-) cell preparations from normal human placental/umbilical cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PBSC) specimens, using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. METHODS: CD34(+) and CD34(-) cell preparations were purified by four different approaches in 14 individual experiments involving 293 transplanted NOD/SCID mice. In most experiments, CD34(+) cells were depleted twice (CD34(=)) in order to obtain efficient depletion of CD34(+) cells from the CD34(-) cell preparations. RESULTS: Dose-dependent levels of human hematopoietic cells were observed after transplantation of CD34(+) cell preparations. To rigorously assess the complementary CD34(-) cell preparations, cell doses 10- to 1000-fold higher than the minimum dose of the CD34(+) cell preparations necessary for engraftment were transplanted. Nevertheless, of 125 NOD/SCID mice transplanted with CD34(-) cell preparations purified from the same starting cells, only six mice had detectable human hematopoiesis, by flow cytometric or PCR assay. CONCLUSIONS: CD34(-) cells provide only a minor contribution to hematopoietic engraftment in this in vivo model system, as compared to CD34(+) cells from the same samples of noncultured human cells. Hematopoiesis derived from actual CD34(-) cells is difficult to distinguish from that due to CD34(+) cells potentially contaminating the preparations.
Asunto(s)
Antígenos CD34/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Células Madre Hematopoyéticas , Animales , Antígenos CD34/biosíntesis , Biomarcadores , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas , Humanos , Ratones , Ratones SCID , Trasplante HomólogoRESUMEN
We compared the cell cycle status and expression of mRNA for the amphotropic retroviral receptor in hematopoietic stem cells isolated from bone marrow and cytokine mobilized peripheral blood. CD34+ cells from six normal volunteers were enriched by immune selection from steady-state bone marrow and granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (10 microg/kg/day for 5 days). Cell cycle status of the phenotypically primitive CD34+CD38- hematopoietic stem cell population was analyzed using a four-color flow cytometry technique that distinguished the G0, G1, and S/IG2/M phases of the cell cycle. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to measure mRNA expression of the amphotropic retroviral receptor. Peripheral blood hematopoietic stem cells had 2.6-fold more cells in the G1 phase of the cell cycle compared to steady-state bone marrow. Furthermore, lineage CD34+CD38- cells from G-CSF mobilized peripheral blood had a fourfold higher level of amphotropic retrovirus receptor mRNA. In conclusion, we found that CD34+ CD38- hematopoietic stem cells isolated from G-CSF mobilized peripheral blood differ from those isolated from steady-state bone marrow in that a significant proportion have entered the G1 phase of the cell cycle and express higher levels of amphotropic receptor mRNA. These biologic properties are consistent with the reported rapid recovery of hematopoietic function following transplantation with peripheral blood hematopoietic stem cells and make these cells a preferred target for retroviral-based gene transfer.
Asunto(s)
Fase G1 , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , ARN Mensajero/biosíntesis , Receptores Virales/biosíntesis , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células Cultivadas , Citometría de Flujo , Terapia Genética , Vectores Genéticos/fisiología , Células Madre Hematopoyéticas/metabolismo , Humanos , Especificidad de Órganos , ARN Mensajero/genética , Receptores Virales/genética , Retroviridae/fisiologíaRESUMEN
Advances in molecular genetic understanding of disease processes has been extended to a number of phagocytic disorders. Most of these disorders were extensively characterized at the functional and protein level prior to cloning of the relevant genes. Nucleotide sequence data has been essential for establishing the mechanism and mode of inheritance of genetically transmitted phagocyte disorders. Such data provides insights into the functionally important regions of affected proteins and information regarding regulation of these genes and homologies to other known proteins. From such data it is also possible to determine the evolutionary history of these genes. Chronic granulomatous disease, a phenotypic classification of a heterogeneous group of defects in oxidative metabolism, has now been defined in terms of specific molecular defects. Cloning of the two subunits of cytochrome b558 has led the way to characterization of the X-linked form and one of the autosomal recessive forms of this disease and confirmed the importance of this protein in the phagocyte oxidative burst. The absence of lactoferrin associated with hereditary specific granule deficiency is a result of decreased transcription of the lactoferrin gene in myeloid cells. Myeloperoxidase deficiency is likely a result of a mutation of the gene coding for myeloperoxidase. More precise understanding of expression of the lactoferrin and myeloperoxidase genes may be important in elucidating some of the underlying mechanisms in the pathogenesis of myeloid malignancies. A rare disorder, leukocyte adhesion deficiency, has provided a model for establishing the relationship between the several distinct alpha subunits and the shared common beta subunit of leukocyte adhesion proteins. These proteins have been shown to be genetically related to the superfamily of extracellular matrix receptors termed 'integrins'. Because these proteins have been highly conserved at the genetic level across a variety of species, comparison of nucleotide sequence data has illuminated some of the evolutionary history of these genes as they arose from ancestral genes. Studies of these adhesion protein genes may contribute new information in the broader context of how the functions of these genes evolved.
Asunto(s)
Enfermedades del Sistema Inmune/genética , Fagocitos/metabolismo , Humanos , Enfermedades del Sistema Inmune/inmunología , Enfermedades del Sistema Inmune/metabolismo , Fagocitos/inmunologíaRESUMEN
Comparison of cDNA sequences from multiple sources predicts a genus of highly homologous but structurally distinct G protein alpha-subunits, designated as Gi alpha, that may include the alpha-subunit of the functionally defined adenylate cyclase inhibitory G protein. Using specific oligonucleotide probes on Northern blots, we show that Gi alpha-2 and Gi alpha-3, but not Gi alpha-1, are expressed in HL60 cells. Antisera raised against synthetic peptides from regions predicted to be conserved (AS6) and divergent (LE3) among Gi alpha subtypes bind to a 40 kDa protein in Western blots of HL60 membranes. AS6 identifies an additional protein at 41 kDa. Thus, Northern blot and immunoblot results show that at least two Gi alpha subtypes, a 40 kDa protein likely to correspond to Gi alpha-2 and a 41 kDa protein possibly representing Gi alpha-3, may be expressed in a single cell type.