RESUMEN
BACKGROUND: CYT997 is a novel microtubule inhibitor and vascular-disrupting agent with marked preclinical anti-tumour activity. METHODS: This phase I dose-escalation study assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of CYT997 administered by continuous intravenous infusion over 24 h every 3 weeks to patients with advanced solid tumours. RESULTS: Thirty-one patients received CYT997 over 12 dose levels (7-358 mg m(-2)). Doses up to 202 mg m(-2) were well tolerated. Dose-limiting toxicities were observed at 269 and 358 mg m(-2), consisting of grade 3 prolonged corrected QT interval in two patients and grade 3 hypoxia and grade 4 dyspnea in one patient. All toxicities were reversible. The pharmacokinetics of CYT997 were linear over the entire dose range. Dynamic contrast-enhanced magnetic resonance imaging scans showed significant changes in tumour K(trans) values consistent with vascular disruption in 7 out of 11 evaluable patients treated at CYT997 doses of >or=65 mg m(-2). Moreover, plasma levels of von Willebrand factor and caspase-cleaved cytokeratin-18 increased post-treatment at higher dose levels. Among 22 patients evaluable for response, 18 achieved stable disease for >2 cycles. CONCLUSIONS: CYT997 was well tolerated at doses that were associated with pharmacodynamic evidence of vascular disruption in tumours.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Antineoplásicos/administración & dosificación , Piridinas/administración & dosificación , Pirimidinas/administración & dosificación , Adulto , Anciano , Inhibidores de la Angiogénesis/efectos adversos , Inhibidores de la Angiogénesis/farmacocinética , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Recuento de Células , Células Endoteliales , Femenino , Humanos , Queratina-18/análisis , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Piridinas/efectos adversos , Piridinas/farmacocinética , Pirimidinas/efectos adversos , Pirimidinas/farmacocinética , Factor de von Willebrand/análisis , Factor de von Willebrand/inmunologíaRESUMEN
BACKGROUND: Primary cutaneous B-cell lymphomas (PCBCLs) are characterized by restriction to the skin and a variable but mostly favourable prognosis. Since 1997 the recombinant, chimeric anti-CD20 antibody rituximab has been used in patients suffering from non-Hodgkin's B-cell lymphomas. Different studies have shown that the effectiveness and safety in the treatment of patients with low-grade follicular lymphoma is comparable to or even higher than the standard CHOP chemotherapy. So far it has been unclear whether an extended duration of therapy leads to a benefit for the patients with PCBCL. OBJECTIVES: To evaluate the objective response rate, time to progression, remission quality and histological changes and to compare our data with the literature. PATIENTS/METHODS: Ten patients with PCBCL [eight with follicle centre cell lymphoma (FCCL), one with marginal zone lymphoma (MZL) and one with diffuse large B-cell lymphoma of the leg (DLBCL)] were treated by intravenous application of a chimeric antibody against the CD20 transmembrane antigen (rituximab) with a dosage of eight cycles, 375 mg m(-2) body surface, weekly. RESULTS: The treatment regimen resulted in clinical overall response in 9 of 10 patients, in particular there were seven complete responses (70%) plus two partial responses (20%). The median duration of remission (durable remission, DR) is 23 months (4-30 months) to date. Histological assessment of responses in four patients showed no tumour-specific infiltration. In two patients histology revealed a residual infiltration and in one patient an increasing infiltration. In two patients no histology was taken after treatment; one patient developed a new lesion. No severe side-effects occurred. Observed side-effects were two bacterial infections, two patients with shivering during infusion, one patient with sweating for months and one patient with persisting itching. As expected the B-cell count in peripheral blood was depressed in all patients after infusion. CONCLUSIONS: Intravenous therapy with eight cycles of the anti-CD20 antibody rituximab is a non-toxic and effective treatment for a subset of patients with PCBCL (relapsed, aggressive entity, old patients, multiple lesions) with a long DR.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Linfoma de Células B/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales de Origen Murino , Antígenos CD20/inmunología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Esquema de Medicación , Evaluación de Medicamentos , Humanos , Linfoma de Células B/patología , Masculino , Persona de Mediana Edad , Rituximab , Neoplasias Cutáneas/patología , Resultado del TratamientoRESUMEN
The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.
Asunto(s)
Proteínas Bacterianas/genética , Hemo Oxigenasa (Desciclizante)/fisiología , Hemo/metabolismo , Neisseria meningitidis/enzimología , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/fisiología , Biliverdina/metabolismo , Prueba de Complementación Genética , Hemo Oxigenasa (Desciclizante)/genética , Peróxido de Hidrógeno/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Mutación , Pseudomonas putida/enzimología , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
We report the crystal structure of heme oxygenase from the pathogenic bacterium Neisseria meningitidis at 1.5 A and compare and contrast it with known structures of heme oxygenase-1 from mammalian sources. Both the bacterial and mammalian enzymes share the same overall fold, with a histidine contributing a ligand to the proximal side of the heme iron and a kinked alpha-helix defining the distal pocket. The distal helix differs noticeably in both sequence and conformation, and the distal pocket of the Neisseria enzyme is substantially smaller than in the mammalian enzyme. Key glycine residues provide the flexibility for the helical kink, allow close contact of the helix backbone with the heme, and may interact directly with heme ligands.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/química , Neisseria meningitidis/enzimología , Animales , Catálisis , Cristalografía por Rayos X/métodos , Glicina , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Humanos , Mamíferos , Proteínas de la Membrana , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The ability of Shigella dysenteriae to utilize heme as an iron source is dependent on the iron-regulated expression of a number of genes including the outermembrane receptor ShuA and the cytoplasmic protein ShuS. The ShuS protein has no sequence homology with any proteins of known function and its role in heme acquisition has not been determined. In this paper we describe the purification and characterization of ShuS. The soluble oligomeric protein (650 kDa) is composed of a single type of subunit with a molecular mass of 37 kDa and binds one heme per monomer (Kd = 13 microM). In addition, the ShuS protein was shown to nonspecifically bind double-stranded DNA. It appears, therefore, that ShuS may function as both a heme storage protein, during periods of active heme transport, and as a DNA binding protein to protect the DNA from any ensuing heme mediated oxidative damage.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hemo/metabolismo , Shigella dysenteriae/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Transporte Biológico Activo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/ultraestructura , Hierro/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Shigella dysenteriae/patogenicidad , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
A full-length heme oxygenase gene from the gram-negative pathogen Neisseria meningitidis was cloned and expressed in Escherichia coli. Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the E. coli cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen Corynebacterium diphtheriae and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXalpha and carbon monoxide as the final products. Homologs of the hemO gene were identified and characterized in six commensal Neisseria isolates, Neisseria lactamica, Neisseria subflava, Neisseria flava, Neisseria polysacchareae, Neisseria kochii, and Neisseria cinerea. All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/metabolismo , Neisseria/enzimología , Secuencia de Aminoácidos , Catálisis , Clonación Molecular , Expresión Génica , Genes Bacterianos , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/genética , Hemo/química , Hemo Oxigenasa (Desciclizante)/clasificación , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/aislamiento & purificación , Humanos , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Neisseria/genética , Neisseria/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-alpha/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-beta2, Tgf-beta3 and activin-betaA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor 'related to tyrosine kinases' (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction-associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.
Asunto(s)
Anomalías Craneofaciales/genética , Receptor Cross-Talk/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Animales Recién Nacidos , Anomalías Craneofaciales/embriología , Desarrollo Embrionario y Fetal/genética , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Mutación , Fenotipo , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Receptor EphB2 , Transducción de SeñalRESUMEN
The peroxiredoxin AhpC from Mycobacterium tuberculosis has been expressed, purified, and characterized. It differs from other well characterized AhpC proteins in that it has three rather than one or two cysteine residues. Mutagenesis studies show that all three cysteine residues are important for catalytic activity. Analysis of the M. tuberculosis genome identified a second protein, AhpD, which has no sequence identity with AhpC but is under the control of the same promoter. This protein has also been cloned, expressed, purified, and characterized. AhpD, which has only been identified in the genomes of mycobacteria and Streptomyces viridosporus, is shown here to also be an alkylhydroperoxidase. The endogenous electron donor for catalytic turnover of the two proteins is not known, but both can be turned over with AhpF from Salmonella typhimurium or, particularly in the case of AhpC, with dithiothreitol. AhpC and AhpD reduce alkylhydroperoxides more effectively than H(2)O(2) but do not appear to interact with each other. These two proteins appear to be critical elements of the antioxidant defense system of M. tuberculosis and may be suitable targets for the development of novel anti-tuberculosis strategies.
Asunto(s)
Antioxidantes/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Peroxidasas/metabolismo , Proteínas Bacterianas/química , Secuencia de Bases , Catálisis , Cartilla de ADN , Ditiotreitol/metabolismo , Oxidación-Reducción , Peroxidasas/química , Peroxirredoxinas , Conformación Proteica , Especificidad por SustratoRESUMEN
The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium diphtheriae/enzimología , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/metabolismo , Histidina/metabolismo , Proteínas Bacterianas/genética , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Hemo-Oxigenasa 1 , Humanos , Hidroxilación , Ligandos , Proteínas de la Membrana , Modelos Químicos , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Espectrofotometría Atómica , Espectrometría Raman , Relación Estructura-ActividadRESUMEN
The tie2 receptor tyrosine kinase plays a key role in angiogenesis, and the remodeling and maturation of blood vessels. In this study we have used a factor-dependent cell line (Ba/F3) expressing a chimeric receptor containing the extracellular domain of mouse tie2 and the transmembrane and cytoplasmic domain of the erythropoietin receptor to identify specific binding activity associated with an adipogenic sub-line of 3T3 fibroblasts (3T3-L1). 3T3-L1 fibroblasts are capable of undergoing differentiation to adipocytes under specific culture conditions. When compared to 3T3-L1 cells, the adipocyte differentiated cultures, which contain both pre-adipocytes and adipocytes, exhibited a significantly increased ability to support the growth of Ba/F3 cells expressing the chimeric receptor. Using probes specific for two recently described ligands for tie2, Ang-1 and Ang-2, we have shown that mRNA encoding Ang-1 is upregulated when 3T3-L1 fibroblasts are differentiated to adipocytes. These results suggest that the levels of Ang-1 protein and mRNA in 3T3-L1 cells can be regulated by cellular differentiation in adipose development.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Glicoproteínas de Membrana/genética , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3 , Adipocitos/efectos de los fármacos , Angiopoyetina 1 , Angiopoyetina 2 , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Bioensayo , Diferenciación Celular/efectos de los fármacos , Cartilla de ADN/genética , Dexametasona/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Insulina/farmacología , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones , Fenotipo , Placenta/metabolismo , Embarazo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Regulación hacia ArribaRESUMEN
Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.
Asunto(s)
Permeabilidad Capilar/genética , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Técnicas Biosensibles , Western Blotting , Células CHO , Células COS , Cromatografía , Cricetinae , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/fisiología , Citometría de Flujo , Cobayas , Linfocinas/fisiología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Homología de Secuencia de Aminoácido , Factor A de Crecimiento Endotelial Vascular , Factor D de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Members of the JAK family of protein tyrosine kinase (PTK) proteins are required for the transmission of signals from a variety of cell surface receptors, particularly those of the cytokine receptor family. JAK function has been implicated in hematopoiesis and regulation of the immune system, and recent data suggest that the vertebrate JAK2 gene may play a role in leukemia. We have isolated and characterized jak cDNAs from the zebrafish Danio rerio. The zebrafish genome possesses 2 jak2 genes that occupy paralogous chromosome segments in the zebrafish genome, and these segments conserve syntenic relationships with orthologous genes in mammalian genomes, suggesting an ancient duplication in the zebrafish lineage. The jak2a gene is expressed at high levels in erythroid precursors of primitive and definitive waves and at a lower level in early central nervous system and developing fin buds. jak2b is expressed in the developing lens and nephritic ducts, but not in hematopoietic tissue. The expression of jak2a was examined in hematopoietic mutants and found to be disrupted in cloche and spadetail, suggesting an early role in hematopoiesis. Taken together with recent gene knockout data in the mouse, we suggest that jak2a may be functionally equivalent to mammalian Jak2, with a role in early erythropoiesis.
Asunto(s)
Eritropoyesis , Regulación del Desarrollo de la Expresión Génica , Genes , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas , Pez Cebra/genética , Alelos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Inducción Enzimática , Células Precursoras Eritroides/enzimología , Eritropoyesis/genética , Evolución Molecular , Hematopoyesis/genética , Células Madre Hematopoyéticas/enzimología , Humanos , Janus Quinasa 2 , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Fenotipo , Proteínas Tirosina Quinasas/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción Genética , Pez Cebra/embriologíaRESUMEN
Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the alpha-meso heme carbon is due primarily to steric influence of the distal helix.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Glicina/química , Glicina/metabolismo , Hemo/química , Hemo-Oxigenasa 1 , Histidina/química , Histidina/metabolismo , Humanos , Hierro/química , Hierro/metabolismo , Ligandos , Proteínas de la Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Oxígeno/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Docilidad , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Solventes , Electricidad Estática , Especificidad por SustratoRESUMEN
Transcription factors of the STAT family are required for cellular responses to multiple signaling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and translocate to the nucleus. Subsequent STAT binding to specific DNA elements in the promoters of signal-responsive genes alters the transcriptional activity of these loci. STAT function has been implicated in the transduction of signals for growth, reproduction, viral defense, and immune regulation. We have isolated and characterized two STAT homologs from the zebrafish Danio rerio. The stat3 gene is expressed in a tissue-restricted manner during embryogenesis, and larval development with highest levels of transcript are detected in the anterior hypoblast, eyes, cranial sensory ganglia, gut, pharyngeal arches, cranial motor nuclei, and lateral line system. In contrast, the stat1 gene is not expressed during early development. The stat3 gene maps to a chromosomal position syntenic with the mouse and human STAT3 homologs, whereas the stat1 gene does not. Despite a higher rate of evolutionary change in stat1 relative to stat3, the stat1 protein rescues interferon-signaling functions in a STAT1-deficient human cell line, indicating that cytokine-signaling mechanisms are likely to be conserved between fish and tetrapods. Dev Dyn 1999;215:352-370.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Sistema Nervioso Central/metabolismo , Mapeo Cromosómico , Cromosomas , Secuencia Conservada , Proteínas de Unión al ADN/análisis , Embrión no Mamífero/anatomía & histología , Evolución Molecular , Humanos , Janus Quinasa 1 , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Sistema Nervioso Periférico/metabolismo , Filogenia , Polimorfismo Genético , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Transactivadores/análisis , Transfección , Proteínas de Pez CebraRESUMEN
UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/química , Hemo/química , Hierro/química , Mutagénesis Sitio-Dirigida , Mioglobina/química , Oxígeno/química , Alanina/genética , Animales , Dicroismo Circular , Transporte de Electrón , Glicina/genética , Hemo Oxigenasa (Desciclizante)/genética , Histidina/genética , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Mioglobina/genética , Espectrofotometría Ultravioleta , Espectrometría Raman , Volumetría , BallenasRESUMEN
The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Histidina/metabolismo , Oxidorreductasas/metabolismo , Sustitución de Aminoácidos , Catálisis , Cisteína/genética , Cisteína/metabolismo , Transporte de Electrón , Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/química , Hemo Oxigenasa (Desciclizante)/genética , Histidina/genética , Humanos , Hierro/metabolismo , Ligandos , Mutagénesis , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Peróxidos/metabolismo , Espectrometría Raman , Tirosina/genética , Tirosina/metabolismoRESUMEN
We report the genomic organization of the mouse orphan receptor related to tyrosine kinases (Ryk), a structurally unclassified member of the growth factor receptor family. The mouse RYK protein is encoded by 15 exons distributed over a minimum of 81 kilobases. Genomic DNA sequences encoding a variant protein tyrosine kinase ATP-binding motif characteristic of RYK are unexpectedly found in two separate exons. A feature of the gene is an unmethylated CpG island spanning exon 1 and flanking sequences, including a TATA box-containing putative promoter and single transcription start site. Immunohistochemical examination of RYK protein distribution revealed widespread but developmentally regulated expression, which was spatially restricted within particular adult organs. Quantitative reduction of Southern blotting stringency for the detection of Ryk-related sequences provided evidence for a retroprocessed mouse pseudogene and a more distantly related gene paralogue. Extensive cross-species reactivity of a mouse Ryk kinase subdomain probe and the cloning of a Ryk orthologue from Caenorhabditis elegans demonstrate that Ryk and its relatives encode widely conserved members of a novel receptor tyrosine kinase subfamily.
Asunto(s)
Proteínas Tirosina Quinasas Receptoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Codón , Islas de CpG/genética , ADN , Exones , Humanos , Inmunohistoquímica , Intrones , Ratones , Datos de Secuencia Molecular , Filogenia , Proteínas Tirosina Quinasas Receptoras/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Trans effects at the sn-glycerol-3-phosphate dehydrogenase locus (Gpdh) of Drosophila melanogaster give rise to an increase in GPDH activity and mRNA from the wild-type allele in heterozygotes with some low-activity alleles. Either the low-activity alleles that induce the effect have a defective P-element inserted between the promoter and a downstream intronic enhancer element or the promoter region is deleted. The trans effect is pairing dependent, characteristic of transvection at some other loci. The defective P-elements that mediate transvection are located between the promoter and at least up to 6 bp downstream of the transcription start site. Transvection at Gpdh appears similar to the "enhancer action in trans" mode at the yellow locus.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Glicerolfosfato Deshidrogenasa/genética , Proteínas de Insectos/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Northern Blotting , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Femenino , Glicerolfosfato Deshidrogenasa/metabolismo , Heterocigoto , Proteínas de Insectos/metabolismo , Masculino , Datos de Secuencia Molecular , ARN Mensajero , Transcripción GenéticaRESUMEN
In the Drosophila embryo, a subset of muscles require expression and function of the RYK subfamily RTK gene derailed (drl) for correct attachment. We have isolated a second RYK homolog, doughnut (dnt), from Drosophila. The DNT protein exhibits 60% amino acid identity to DRL, and is structurally as similar to the mammalian RYK proteins as is DRL, indicating an ancient duplication event. dnt is expressed in dynamic patterns in the embryonic epidermis, being found at high level in epithelia adjacent to cells that are invaginating into the interior of the embryo, including ventral furrow, cephalic furrow, fore- and hindgut, optic lobe and tracheal pits. dnt is capable of a partial rescue of the muscle attachment defect of drl-/- embryos, indicating that it encodes a receptor with a related and significantly overlapping biochemical function.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas Musculares/biosíntesis , Músculos/embriología , Proteínas Tirosina Quinasas/biosíntesis , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Clonación Molecular , Drosophila melanogaster/embriología , Epidermis/embriología , Epidermis/metabolismo , Prueba de Complementación Genética , Mamíferos/genética , Datos de Secuencia Molecular , Morfogénesis/genética , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Músculos/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA. The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immuno-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor alpha-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) non-activated STAT1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.