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1.
Am J Transplant ; 18(1): 74-88, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28719147

RESUMO

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.


Assuntos
Morte Encefálica/imunologia , Linfócitos/imunologia , Células Mieloides/imunologia , Transplante de Órgãos , Doadores de Tecidos , Obtenção de Tecidos e Órgãos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Morte Encefálica/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Citocinas/sangue , Feminino , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Prognóstico , Subpopulações de Linfócitos T/imunologia , Adulto Jovem
2.
Kaibogaku Zasshi ; 90(3): 13-4, 2015 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-26164997
3.
Science ; 289(5482): 1194-7, 2000 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-10947987

RESUMO

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.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transativadores , Proteínas rac de Ligação ao GTP/metabolismo , Proteína da Polipose Adenomatosa do Colo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Membrana Celular/ultraestrutura , Tamanho Celular , Colo/citologia , Colo/metabolismo , Citoplasma/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Guanosina Difosfato/metabolismo , Humanos , Immunoblotting , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Transfecção , Técnicas do Sistema de Duplo-Híbrido , beta Catenina
4.
Science ; 272(5264): 1020-3, 1996 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-8638125

RESUMO

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.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila , Hormônios de Inseto/metabolismo , Transativadores , Proteínas Supressoras de Tumor , Proteína da Polipose Adenomatosa do Colo , Sequência de Aminoácidos , Animais , Ciclo Celular , Células Cultivadas , Colo/química , Colo/citologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/química , Drosophila , Células Epiteliais , Epitélio/química , Imunofluorescência , Hipocampo/química , Hipocampo/citologia , Humanos , Hormônios de Inseto/análise , Hormônios de Inseto/química , Camundongos , Dados de Sequência Molecular , Neurônios/química , Neurônios/citologia , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Sinapses/química , beta Catenina
5.
Acta Radiol ; 50(8): 954-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19863423

RESUMO

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.


Assuntos
Imageamento por Ressonância Magnética/métodos , Ovário/patologia , Células Estromais/patologia , Meios de Contraste , Feminino , Humanos , Hiperplasia/patologia , Pessoa de Meia-Idade , Ovariectomia , Ovário/cirurgia
6.
Structure ; 7(8): 953-65, 1999 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10467151

RESUMO

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.


Assuntos
Dioxigenases , Oxigenases/química , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Ferro/química , Modelos Moleculares , Dados de Sequência Molecular , Oxigenases/metabolismo , Conformação Proteica , Pseudomonas/enzimologia , Homologia de Sequência de Aminoácidos
7.
Oncogene ; 11(1): 89-96, 1995 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-7624136

RESUMO

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.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteína da Polipose Adenomatosa do Colo , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/ultraestrutura , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Microscopia Imunoeletrônica , Ligação Proteica , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Células Tumorais Cultivadas , alfa Catenina
8.
Diabetes ; 47(11): 1699-706, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9792538

RESUMO

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.


Assuntos
Acetilcolina/farmacologia , Cálcio/metabolismo , Grânulos Citoplasmáticos/fisiologia , Fosfatos de Inositol/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Animais , Atropina/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Grânulos Citoplasmáticos/ultraestrutura , Inibidores Enzimáticos/farmacologia , Secreção de Insulina , Insulinoma , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Antagonistas Muscarínicos/farmacologia , Ácido Okadáico/farmacologia , Neoplasias Pancreáticas , Fosforilação , Ratos , Células Tumorais Cultivadas , Fosfolipases Tipo C/antagonistas & inibidores
9.
Diabetes ; 49(6): 945-52, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10866046

RESUMO

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.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ilhotas Pancreáticas/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Proteína Quinase C/metabolismo , Linhagem Celular , Colforsina/farmacologia , Grânulos Citoplasmáticos/metabolismo , Sinergismo Farmacológico , Insulina/metabolismo , Secreção de Insulina , Membranas Intracelulares/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Frações Subcelulares/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Distribuição Tecidual
10.
Pharmacol Ther ; 58(1): 93-132, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8415875

RESUMO

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.


Assuntos
Interferon beta/química , Sequência de Aminoácidos , Animais , Evolução Biológica , Cristalização , Cristalografia por Raios X , Citocinas/química , Humanos , Interferon-alfa/química , Interferon beta/genética , Interferon beta/fisiologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos
11.
J Mol Biol ; 253(1): 187-207, 1995 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-7473712

RESUMO

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.


Assuntos
Interferon beta/química , Conformação Proteica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Ligação de Hidrogênio , Proteínas de Membrana , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptor de Interferon alfa e beta , Receptores de Interferon/metabolismo , Proteínas Recombinantes/química , Alinhamento de Sequência
12.
J Mol Biol ; 304(3): 397-410, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11090282

RESUMO

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.


Assuntos
Proteínas Ferro-Enxofre , Oxirredutases/química , Oxigenases/química , Pseudomonas/enzimologia , Sequência de Aminoácidos , Fator de Indução de Apoptose , Sítios de Ligação , Cristalografia por Raios X , Transporte de Elétrons , Evolução Molecular , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/química , Glutationa Redutase/química , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , NAD/química , NAD/metabolismo , Oxirredutases/metabolismo , Oxigenases/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência
13.
J Mol Biol ; 309(5): 1139-51, 2001 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-11399084

RESUMO

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.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Bifenilos Policlorados/metabolismo , Rhodococcus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Elétrons , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Rhodococcus/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
14.
J Mol Biol ; 255(5): 735-52, 1996 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-8636975

RESUMO

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.


Assuntos
Dioxigenases , Oxigenases/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Pseudomonas/enzimologia , Sequência de Aminoácidos , Bactérias/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Análise dos Mínimos Quadrados , Substâncias Macromoleculares , Modelos Moleculares , Modelos Estruturais , Dados de Sequência Molecular , Oxigenases/metabolismo , Homologia de Sequência de Aminoácidos
15.
Hereditas ; 142(2005): 45-50, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16970611

RESUMO

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.


Assuntos
Pareamento Cromossômico/genética , Cromossomos de Plantas/genética , Poaceae/genética , Cruzamentos Genéticos , Fertilidade/genética , Genoma de Planta/genética , Pólen/genética , Sementes/genética , Especificidade da Espécie
16.
J Bone Miner Res ; 14(7): 1132-44, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10404013

RESUMO

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.


Assuntos
Proteínas de Transporte/biossíntese , Condrogênese/fisiologia , Proteínas da Matriz Extracelular , Consolidação da Fratura/fisiologia , Osteogênese/fisiologia , Agrecanas , Animais , Animais Recém-Nascidos , Northern Blotting , Proteínas de Transporte/genética , Linhagem Celular , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Condrogênese/genética , Citocinas/biossíntese , Citocinas/genética , DNA Complementar/genética , Embrião de Mamíferos , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Imuno-Histoquímica , Lectinas Tipo C , Botões de Extremidades/metabolismo , Botões de Extremidades/ultraestrutura , Camundongos , Camundongos Endogâmicos ICR , Microscopia Imunoeletrônica , Midkina , Osteogênese/genética , Proteoglicanas/biossíntese , Transfecção
17.
FEBS Lett ; 425(2): 191-4, 1998 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-9559645

RESUMO

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.


Assuntos
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície , Proteínas Aviárias , Proteínas Sanguíneas , Infertilidade Feminina/genética , Glicoproteínas de Membrana/genética , Animais , Basigina , Copulação/fisiologia , Feminino , Deleção de Genes , Expressão Gênica , Genitália Feminina/fisiologia , Humanos , Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Knockout , Ovário/metabolismo , Ovulação/fisiologia , Gravidez , Espermatozoides/fisiologia , Útero/metabolismo
18.
FEBS Lett ; 459(3): 395-8, 1999 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-10526172

RESUMO

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.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Pentaclorofenol/metabolismo , Sphingomonas/enzimologia , Sequência de Aminoácidos , Proteínas de Transporte/genética , Escherichia coli , Hidroquinonas/metabolismo , Maleatos/metabolismo , Dados de Sequência Molecular , Oxigenases , Homologia de Sequência de Aminoácidos
19.
J Interferon Cytokine Res ; 17(6): 319-26, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9197998

RESUMO

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.


Assuntos
Evolução Molecular , Interferon Tipo I/química , Conformação Proteica , Animais , Sítios de Ligação , Citocinas/química , Citocinas/fisiologia , História do Século XX , Humanos , Interferon Tipo I/história , Interferon Tipo I/fisiologia , Interferon beta/química , Homologia de Sequência de Aminoácidos
20.
J Interferon Cytokine Res ; 20(8): 691-700, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10954912

RESUMO

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.


Assuntos
Quimiocinas CXC/química , Quimiocinas CXC/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Embrião de Galinha , Cristalografia por Raios X , Vetores Genéticos , Humanos , Modelos Moleculares , Mutagênese , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Respirovirus/genética , Soluções , Relação Estrutura-Atividade
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