RESUMEN
Organ donors are sources of physiologically healthy organs and tissues for life-saving transplantation, and have been recently used for human immunology studies which are typically confined to the sampling of peripheral blood. Donors comprise a diverse population with different causes of death and clinical outcomes during hospitalization, and the effects of such variations on immune parameters in blood and tissues are not known. We present here a coordinate analysis of innate and adaptive immune components in blood, lymphoid (bone marrow, spleen, lymph nodes), and mucosal (lungs, intestines) sites from a population of brain-dead organ donors (2 months-93 years; n = 291) across eight clinical parameters. Overall, the blood of donors exhibited similar monocyte and lymphocyte content and low serum levels of pro-inflammatory cytokines as healthy controls; however, donor blood had increased neutrophils and serum levels of IL-8, IL-6, and MCP-1 which varied with cause of death. In tissues, the frequency and composition of monocytes, neutrophils, B lymphocytes and T cell subsets in lymphoid or mucosal sites did not vary with clinical state, and was similar in donors independent of the extent of clinical complications. Our results reveal that organ donors maintain tissue homeostasis, and are a valuable resource for fundamental studies in human immunology.
Asunto(s)
Muerte Encefálica/inmunología , Linfocitos/inmunología , Células Mieloides/inmunología , Trasplante de Órganos , Donantes de Tejidos , Obtención de Tejidos y Órganos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Muerte Encefálica/patología , Estudios de Casos y Controles , Niño , Preescolar , Citocinas/sangre , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Pronóstico , Subgrupos de Linfocitos T/inmunología , Adulto JovenRESUMEN
The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. Here the APC gene product is shown to bind through its armadillo repeat domain to a Rac-specific guanine nucleotide exchange factor (GEF), termed Asef. Endogenous APC colocalized with Asef in mouse colon epithelial cells and neuronal cells. Furthermore, APC enhanced the GEF activity of Asef and stimulated Asef-mediated cell flattening, membrane ruffling, and lamellipodia formation in MDCK cells. These results suggest that the APC-Asef complex may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Transactivadores , Proteínas de Unión al GTP rac/metabolismo , Proteína de la Poliposis Adenomatosa del Colon , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Línea Celular , Membrana Celular/ultraestructura , Tamaño de la Célula , Colon/citología , Colon/metabolismo , Citoplasma/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Guanosina Difosfato/metabolismo , Humanos , Immunoblotting , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Transfección , Técnicas del Sistema de Dos Híbridos , beta CateninaRESUMEN
The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, and its product binds to the adherens junction protein beta-catenin. Overexpression of APC blocks cell cycle progression. The APC-beta-catenin complex was shown to bind to DLG, the human homolog of the Drosophila discs large tumor suppressor protein. This interaction required the carboxyl-terminal region of APC and the DLG homology repeat region of DLG. APC colocalized with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila , Hormonas de Insectos/metabolismo , Transactivadores , Proteínas Supresoras de Tumor , Proteína de la Poliposis Adenomatosa del Colon , Secuencia de Aminoácidos , Animales , Ciclo Celular , Células Cultivadas , Colon/química , Colon/citología , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/química , Drosophila , Células Epiteliales , Epitelio/química , Técnica del Anticuerpo Fluorescente , Hipocampo/química , Hipocampo/citología , Humanos , Hormonas de Insectos/análisis , Hormonas de Insectos/química , Ratones , Datos de Secuencia Molecular , Neuronas/química , Neuronas/citología , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Sinapsis/química , beta CateninaRESUMEN
Ovarian stromal hyperthecosis is characterized by diffuse distribution of luteinized stromal cells accompanied by varying degrees of stromal hyperplasia. We report a case of ovarian stromal hyperthecosis with particular regard to magnetic resonance (MR)-pathologic correlation. At initial MR imaging, the central areas of the bilateral ovarian masses showed hypointensity on T1-weighted images and hyperintensity on T2-weighted images, while the peripheries of the bilateral masses showed isointensity to myometrium on T1-weighted images and heterogeneous signal intensities on T2-weighted images. At 15 days after the initial MR imaging examination, a second MR imaging demonstrated shrinkage of the bilateral ovarian masses. Change in the peripheries to predominantly isointensity to myometrium on the T2-weighted images was also observed. The patient underwent bilateral oophorectomy. Microscopic examination revealed scattered nests of lutein cells on a background of densely proliferated ovarian stroma with minimal collagen production in both ovaries. Edema was occasionally seen in the outer portion but was marked in the central zone of the ovaries, particularly on the left. The final pathologic diagnosis was stromal hyperthecosis. With regard to MR-pathologic correlation, the MR findings in the peripheries of the bilateral masses (isointensity relative to myometrium on both T1- and T2-weighted imaging) showed the characteristics of stromal hyperthecosis.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Ovario/patología , Células del Estroma/patología , Medios de Contraste , Femenino , Humanos , Hiperplasia/patología , Persona de Mediana Edad , Ovariectomía , Ovario/cirugíaRESUMEN
BACKGROUND: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes. RESULTS: The crystal structure of substrate-free LigAB was solved at 2.2 A resolution. The molecule is an alpha2beta2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the beta subunit, which is covered by the alpha subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site. CONCLUSIONS: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism.
Asunto(s)
Dioxigenasas , Oxigenasas/química , Secuencia de Aminoácidos , Catálisis , Cristalografía por Rayos X , Hierro/química , Modelos Moleculares , Datos de Secuencia Molecular , Oxigenasas/metabolismo , Conformación Proteica , Pseudomonas/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Mutations in the APC gene are linked to the development of sporadic colorectal tumors as well as to familial adenomatous polyposis. Recently, the APC protein was reported to associated with catenins, proteins that bind to the cell adhesion molecule E-cadherin. In the present study, we examined the distribution and localization of the APC protein and alpha -catenin in the normal mouse intestine by light and immunoelectron microscopy using specific antibodies. The APC protein was found to be localized in microvilli and in the apical and lateral cytoplasm of the epithelial cells, whereas alpha-catenin was detected only in the lateral cytoplasm. Double-labeling immunoelectron microscopy showed colocalization of the APC protein with alpha-catenin in the lateral cytoplasm, especially along the lateral plasma membrane, although a certain portion of the APC protein in this region was distributed independently of alpha-catenin. These results suggest that a portion of the APC protein localized in the lateral cytoplasm of intestinal epithelial cells functions in cooperation with catenins, whereas the APC protein in microvilli and in the apical cytoplasm has other functions independent of catenins.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteína de la Poliposis Adenomatosa del Colon , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/ultraestructura , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Microscopía Inmunoelectrónica , Unión Proteica , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Células Tumorales Cultivadas , alfa CateninaRESUMEN
Intracellular movement of secretory granules is a proximal stage in the secretory cascade that ends in the release product from cells. We investigated mechanisms underlying the control of this movement by acetylcholine using an insulinoma cell line, MIN6, in which acetylcholine increases both insulin secretion and granule movement. The peak activation of movement was observed 3 min after an acetylcholine challenge. The effects were nullified by the muscarinic inhibitor atropine, phospholipase C (PLC) inhibitors (D 609 and compound 48/80), and pretreatment with the Ca2+ pump inhibitor, thapsigargin. Inhibitors of Ca2+-dependent phospholipase A2 (arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphate) also partially inhibited the movement caused by acetylcholine, but downregulation of protein kinase C by overnight incubation with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate failed to exert any influence. Acetylcholine stimulation of granule movement was not reproduced by membrane depolarization with high K+. Phosphorylation of the endogenous myosin light chain in MIN6 cells was increased by addition of acetylcholine and decreased by the Ca2+ chelator BAPTA (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid). The calmodulin inhibitor W-7 and the myosin light-chain kinase inhibitor ML-9 decreased the motile events in the beta-cells under both nonstimulated and acetylcholine-stimulated conditions. These findings led us to conclude that inositol trisphosphate [corrected] causes Ca2+ mobilization by muscarinic activation of PLC, leading to intracellular translocation of insulin granules to the ready-releasable pool in pancreatic beta-cells via Ca2+/calmodulin-dependent phosphorylation of myosin light chains.
Asunto(s)
Acetilcolina/farmacología , Calcio/metabolismo , Gránulos Citoplasmáticos/fisiología , Fosfatos de Inositol/farmacología , Insulina/metabolismo , Islotes Pancreáticos/ultraestructura , Animales , Atropina/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Gránulos Citoplasmáticos/ultraestructura , Inhibidores Enzimáticos/farmacología , Secreción de Insulina , Insulinoma , Islotes Pancreáticos/efectos de los fármacos , Cinética , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Antagonistas Muscarínicos/farmacología , Ácido Ocadaico/farmacología , Neoplasias Pancreáticas , Fosforilación , Ratas , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Protein phosphorylation by myosin light-chain kinase (MLCK), protein kinase A, and protein kinase C (PKC) plays a positive role in insulin secretion from the pancreatic beta-cell. To investigate the underlying mechanisms, we examined intracellular distribution of the insulin granules and MLCK by immunofluorescence and immunoelectron microscopies and also investigated intracellular traffic of the granules in cultured beta-cells (MIN6) by video microscopy. Considerable parts of MLCK immunoreactivity were colocalized with the insulin granules. Subcellular fractionation of MIN6 cell extracts revealed that myosin light chain (MLC) may be distributed with the insulin-rich fractions, and immunofluorescence staining using specific antibodies against mono- and diphosphorylated MLCs depicted presence of phosphorylated MLCs in the cytoplasm, in part, with colocalization with the insulin granules. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) caused a shift of both insulin granules and MLCK to the cell periphery, which was not reproduced by the adenylate cyclase activator, forskolin. In contrast, forskolin, but not TPA, increased the granule movement. Costimulation of the beta-cell by TPA and forskolin induced drastic translocation of insulin granules and MLCK to the cell periphery, resulting in enormous potentiation of insulin release. These findings suggest that these protein kinases increase insulin granules in the ready-releasable pool by acting on different steps in the secretory cascade.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Islotes Pancreáticos/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteína Quinasa C/metabolismo , Línea Celular , Colforsina/farmacología , Gránulos Citoplasmáticos/metabolismo , Sinergismo Farmacológico , Insulina/metabolismo , Secreción de Insulina , Membranas Intracelulares/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Fracciones Subcelulares/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Distribución TisularRESUMEN
The crystal structure of recombinant murine interferon-beta as elucidated by Senda et al. (Proc. Jap. Acad. 66B: 77-80 (1990); EMBO J. 11: 3193-3201 (1992)) appears to represent the basic structural framework of all Type I interferons including interferons-beta and all subtypes of interferons-alpha of various mammalian origin. Now the huge accumulated data on the structure-activity relationship of Type I interferons using various chemical and genetic techniques can be systematically evaluated in terms of the three-dimensional structure. Structural comparison with other cytokines, for which three-dimensional structures have been established, including interferon-gamma and considerations on the evolution of cytokines and cytokine receptors are also given.
Asunto(s)
Interferón beta/química , Secuencia de Aminoácidos , Animales , Evolución Biológica , Cristalización , Cristalografía por Rayos X , Citocinas/química , Humanos , Interferón-alfa/química , Interferón beta/genética , Interferón beta/fisiología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Homología de Secuencia de AminoácidoRESUMEN
The crystal structure of recombinant murine interferon-beta (reMuIFN-beta) has been refined at 2.15 A resolution using newly collected synchrotron data. Based on 11,228 reflections (8.0 to 2.15 A), a final R-factor of 19.1% (with a free R-factor of 25.8%) was obtained with a model obeying standard geometry within 0.013 A in bond lengths and 1.4 degrees in bond angles. Compared with the previously reported model, several amino acid residues in helix A are frame-shifted, the conformations are changed for parts of loops AB and BC, helix C is extended and a new short helix exists in loop CD. Evolutionary considerations taken together, the type I interferons appear to share common structural features with respect to the chain-folding topology and the hydrogen-bond networks between various polypeptide segments. Specifically, the disposition of the C-terminal segment of loop AB (after Arg33), known to be an important receptor-binding site, seems to be strictly maintained among the type I interferons. The exposed amino acid residues on helices A and C, which have recently been implicated as the binding site for another receptor molecule, are less well conserved. This may be responsible for varied cellular effects among the subtypes of type I interferons.
Asunto(s)
Interferón beta/química , Conformación Proteica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Enlace de Hidrógeno , Proteínas de la Membrana , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptor de Interferón alfa y beta , Receptores de Interferón/metabolismo , Proteínas Recombinantes/química , Alineación de SecuenciaRESUMEN
2-Hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (the BphD enzyme) hydrolyzes a ring-cleavage product of an aromatic compound generated in a biphenyl/polychlorinated biphenyl (PCB) degradation pathway of bacteria. The crystal structure of the BphD enzyme has been determined at 2.4 A resolution by the multiple isomorphous replacement method. The final refined model of the BphD enzyme yields an R-factor of 17.5 % at 2.4 A resolution with reasonable geometry. The BphD enzyme is an octameric enzyme with a 422 point-group symmetry. The subunit can be divided into core and lid domains. The active site of the enzyme is situated in the substrate-binding pocket, which is located between the two domains. The substrate-binding pocket can be divided into hydrophobic and hydrophilic regions. This feature of the pocket seems to be necessary for substrate binding, as the substrate is composed of hydrophilic and hydrophobic parts. The proposed orientation of the substrate seems to be consistent with the general catalytic mechanism of alpha/beta-hydrolases.
Asunto(s)
Hidrolasas/química , Hidrolasas/metabolismo , Bifenilos Policlorados/metabolismo , Rhodococcus/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Electrones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Rhodococcus/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Oxidative biodegradation of aromatic compounds by bacteria usually begins with hydroxylation of the aromatic ring by multi-component dioxygenases like benzene dioxygenase, biphenyl dioxygenase, and others. These enzymes are composed of ferredoxin reductase, ferredoxin, and terminal oxygenase. Reducing equivalents that originate from NADH are transferred from ferredoxin reductase to ferredoxin and, in turn, to the terminal oxygenase, thus resulting in the activation of a dioxygen. BphA4 is the ferredoxin reductase component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102. The amino acid sequence of BphA4 exhibits significant homology with the putidaredoxin reductase of the cytochrome P450cam system in Pseudomonas putida, as well as with various other oxygenase-coupled NADH-dependent ferredoxin reductases (ONFRs) of bacteria. To date, no structural information has been provided for the ferredoxin reductase component of the dioxygenase systems. In order to provide a structural basis for discussing the mechanism of electron transport between ferredoxin reductase and ferredoxin, crystal structures of BphA4 and its NADH complex were solved. The three-dimensional structure of BphA4 is different from those of ferredoxin reductases whose structures have already been determined, but adopts essentially the same fold as the enzymes of the glutathione reductase (GR) family. Also the three-dimensional structure of the first two domains of BphA4 adopts a fold similar to that of adrenodoxin reductase (AdR) in the mitochondrial cytochrome P450 system. Comparing the amino acid sequence with what is known of the three-dimensional structure of BphA4 strongly suggests that the other ONFRs have secondary structural features that are similar to that of BphA4. This analysis of the crystal structures of BphA4 suggests that Lys53 and Glu159 seem to be involved in the hydride transfer from NADH to FAD. Since the amino acid residues around the active site, some of which seem to be important to electron transport, are highly conserved among ONFRs, it is likely that the mechanism of electron transport of BphA4 is quite applicable to other ONFRs.
Asunto(s)
Proteínas Hierro-Azufre , Oxidorreductasas/química , Oxigenasas/química , Pseudomonas/enzimología , Secuencia de Aminoácidos , Factor Inductor de la Apoptosis , Sitios de Unión , Cristalografía por Rayos X , Transporte de Electrón , Evolución Molecular , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Flavoproteínas/química , Glutatión Reductasa/química , Humanos , Proteínas de la Membrana/química , Modelos Moleculares , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , Oxidorreductasas/metabolismo , Oxigenasas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp. strain KKS102, has been solved as a free form at 1.8 A resolution. This is the first three-dimensional structure among the extradiol-type dioxygenases. Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules. The coordination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT). These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mechanism proposed so far.
Asunto(s)
Dioxigenasas , Oxigenasas/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Pseudomonas/enzimología , Secuencia de Aminoácidos , Bacterias/enzimología , Sitios de Unión , Cristalografía por Rayos X , Análisis de los Mínimos Cuadrados , Sustancias Macromoleculares , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Oxigenasas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Interspecific crossing between L. temulentum L. and L. persicum Boiss. & Hohen. ex Boiss. was performed to clarify their interfertility based on the results of chromosome pairing, pollen fertility and seed set. Both parents were normal with a high percentage of chromosome association of ring bivalents in contrast to rod bivalents at metaphase I, pollen fertility and seed set, but F1 hybrids showed different proportions of them for each crossing combination. Chromosome affinity expressed by pairing was certainly a factor affecting the pollen fertility or seed set in F1 hybrids, but it was not the most important. The positive correlation was generally found between pollen fertility and seed set of F1 hybrids. The L. persicum accession with relatively high interfertility with L. temulentum was supposed to be derived from natural hybridization between L. temulentum and L. persicum. The degree of cytogenetic differentiation between L. temulentum and L. persicum existed because of lower chromosomal pairing, pollen fertility and seed set, but their F1 hybrids were partially fertile.
Asunto(s)
Emparejamiento Cromosómico/genética , Cromosomas de las Plantas/genética , Poaceae/genética , Cruzamientos Genéticos , Fertilidad/genética , Genoma de Planta/genética , Polen/genética , Semillas/genética , Especificidad de la EspecieRESUMEN
Midkine (MK) is a heparin-binding growth/differentiation factor implicated in the control of development and repair of various tissues. Upon fracture of the murine tibia, MK was found to be transiently expressed during bone repair. MK was immunohistochemically detected in spindle-shaped mesenchymal cells at the fracture site on day 4 after fracture and in chondrocytes in the area of endochondral ossification on day 7. MK expression was decreased on day 14 and scarcely seen on day 28 when bone repair was completed. This mode of MK expression is reminiscent of MK expression during development. MK was expressed in hypertrophic chondrocytes of the prebone cartilage rudiments on embryonic day 14 in mouse embryos. MK was also strongly expressed in the epiphyseal growth plate. MK was localized intracellularly during both bone repair and development, and this localization was confirmed by immunoelectron microscopy for embryonic chondrocytes. When MK cDNA was transfected into ATDC5 chondrogenic cells and overexpressed, the majority of transfected cells with strong MK expression showed enhanced chondrogenesis as revealed by increased synthesis of sulfated glycosaminoglycans, aggrecan, and type II collagen. These results suggest that MK plays important roles in chondrogenesis and contributes to bone formation and repair.
Asunto(s)
Proteínas Portadoras/biosíntesis , Condrogénesis/fisiología , Proteínas de la Matriz Extracelular , Curación de Fractura/fisiología , Osteogénesis/fisiología , Agrecanos , Animales , Animales Recién Nacidos , Northern Blotting , Proteínas Portadoras/genética , Línea Celular , Condrocitos/metabolismo , Condrocitos/ultraestructura , Condrogénesis/genética , Citocinas/biosíntesis , Citocinas/genética , ADN Complementario/genética , Embrión de Mamíferos , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Inmunohistoquímica , Lectinas Tipo C , Esbozos de los Miembros/metabolismo , Esbozos de los Miembros/ultraestructura , Ratones , Ratones Endogámicos ICR , Microscopía Inmunoelectrónica , Midkina , Osteogénesis/genética , Proteoglicanos/biosíntesis , TransfecciónRESUMEN
Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Bsg knock-out mice exhibit infertility of both sexes. Based on limited results, defective implantation has been considered to be the cause of the female infertility. We demonstrate here that disruption of the Bsg gene produces the failure of female reproductive processes including not only implantation but also fertilization. Bsg mRNA expression in cumulus cells and basolateral localization of the Bsg protein in the endometrial epithelium further support the importance of Bsg in these processes.
Asunto(s)
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie , Proteínas Aviares , Proteínas Sanguíneas , Infertilidad Femenina/genética , Glicoproteínas de Membrana/genética , Animales , Basigina , Copulación/fisiología , Femenino , Eliminación de Gen , Expresión Génica , Genitales Femeninos/fisiología , Humanos , Inmunoglobulinas/genética , Masculino , Ratones , Ratones Noqueados , Ovario/metabolismo , Ovulación/fisiología , Embarazo , Espermatozoides/fisiología , Útero/metabolismoRESUMEN
The pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6-dichlorohydroquinone (2,6-DCHQ). The pathway converting PCP to 2,6-DCHQ has been established previously; however, the pathway beyond 2,6-DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2, 6-DCHQ conversion. In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring-cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6-DCHQ to 2-chloromaleylacetate.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/metabolismo , Pentaclorofenol/metabolismo , Sphingomonas/enzimología , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Escherichia coli , Hidroquinonas/metabolismo , Maleatos/metabolismo , Datos de Secuencia Molecular , Oxigenasas , Homología de Secuencia de AminoácidoRESUMEN
The scientific and personal backgrounds of the crystallographic elucidation of the three-dimensional structure of murine interferon-beta (Mu-IFN-beta) are described. This structure, elucidated in 1990, is still the only experimentally determined structure for type I IFNs. Model-building studies for various type I IFNs based on the Mu-IFN-beta structure and the arguments on the receptor-binding epitopes appearing since then are reviewed. An updated set of a table and a figure demonstrating a strong correlation between the degree of amino acid sequence variation in various cytokine proteins and that in their cognate receptor proteins is given. The origin of a remarkably larger rate of evolutionary change in amino acid sequences of cytokine proteins despite their physiologic significance is discussed in view of the cytokine network and the neutral theory of evolution.
Asunto(s)
Evolución Molecular , Interferón Tipo I/química , Conformación Proteica , Animales , Sitios de Unión , Citocinas/química , Citocinas/fisiología , Historia del Siglo XX , Humanos , Interferón Tipo I/historia , Interferón Tipo I/fisiología , Interferón beta/química , Homología de Secuencia de AminoácidoRESUMEN
Crystal structures, forms 1 and 2, of recombinant native stromal cell-derived factor-1alpha (SDF-1alpha), expressed using the Sendai virus expression vector system, have been determined by x-ray crystallography at 2.0 A resolution. The crystal of form 1 is almost isomorphous with that used in the previous crystal structure analysis of the synthetic [N33A] mutant of SDF-1alpha (Dealwis, C., et al. Proc. Natl. Acad. Sci. USA 1998;95, 6941-6946). However, the present structure analysis led to considerably better refinement statistics, revealing an error in the structural assignment of N-terminal residues in the previous report. Comparison of the solution structure, as previously determined by nuclear magnetic resonance (NMR) spectroscopy, and the present structure, with two monomers in the asymmetric unit, reveals several local conformational differences. Alanine scan mutagenesis studies for each residue in the so-called RFFESH motif revealed that only the first residue, Arg12, is effective in enhancing receptor binding (and successive activation). A new notion that steric restraint between Arg8 and Arg12 is favorable (if not vital) for retaining SDF activities appears to explain more consistently the structure-activity relationship data accumulated to date. Four guiding principles are presented that may be useful for designing potent therapeutic compounds interfering with HIV-1 infection through competition at the CXCR4 coreceptor.