RESUMO
A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Antígenos Ly/isolamento & purificação , Proteínas do Sistema Complemento/fisiologia , Linfócitos/imunologia , Anticorpos Monoclonais , Antígenos Ly/genética , Antígenos Ly/fisiologia , Sequência de Bases , Antígenos CD59 , Proteínas Inativadoras do Complemento , Complexo de Ataque à Membrana do Sistema Complemento , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , DNA/análise , Epitopos/análise , Humanos , Dados de Sequência MolecularRESUMO
Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.
Assuntos
Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Antígenos de Diferenciação/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Extratos Celulares , Escherichia coli/genética , Expressão Gênica , Humanos , Proteína Fosfatase 1 , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
The yolk sac of the fetal rat and the proximal small intestine of the neonatal rat selectively transport maternal IgG. IgG-Fc receptors are thought to mediate transport across the epithelium of both tissues. We used a mouse mAb (MC-39) against the 45-54-kD component of the Fc receptor of the neonatal intestine to find an antigenically related protein that might function as an Fc receptor in fetal yolk sac. In immunoblots of yolk sac, MC-39 recognized a protein band with apparent molecular mass of 54-58 kD. MC-39 bound to the endoderm of yolk sac in immunofluorescence studies. In immunogold-labeling experiments MC-39 was associated mainly with small vesicles in the apical cytoplasm and in the region near the basolateral membrane of endodermal cells. The MC-39 cross-reactive protein and beta 2-microglobulin, a component of the intestinal Fc receptor, were copurified from detergent-solubilized yolk sac by an affinity purification that selected for proteins which, like the intestinal receptor, bound to IgG at pH 6.0 and eluted at pH 8.0. In summary, the data suggest that we have isolated the Fc receptor of the yolk sac and that this receptor is structurally and functionally related to the Fc receptor of the neonatal intestine. An unexpected finding is that, unlike the intestinal receptor which binds maternal IgG on the apical cell surface, the yolk sac receptor appears to bind IgG only within apical compartments which we suggest represent the endosomal complex.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Intestinos/embriologia , Receptores Fc/isolamento & purificação , Saco Vitelino/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais , Transporte Biológico , Cromatografia de Afinidade , Reações Cruzadas , Endoderma/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Intestinos/imunologia , Peso Molecular , Ratos , Ratos Endogâmicos , Receptores de IgG , Útero/metabolismo , Saco Vitelino/química , Saco Vitelino/citologia , Microglobulina beta-2/isolamento & purificaçãoRESUMO
The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.
Assuntos
Antígenos de Diferenciação/genética , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Antígenos de Diferenciação/isolamento & purificação , Sequência de Bases , Evolução Biológica , Clonagem Molecular , DNA/genética , Enzimas de Restrição do DNA , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Integrinas , Antígeno-1 Associado à Função Linfocitária , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
HNK-1 carbohydrate antigen in an epitope expressed commonly in many cell surface adhesion and recognition molecules in the nervous system. We purified and characterized from rat brain a novel phosphatidylinositol (PI)-anchored 150-kD glycoprotein belonging to the HNK-1 family. The molecule (PI-GP150) was detected by combination of PI-specific phospholipase C treatment of brain membranes and Western blot analysis with mAb HNK-1. HNK-1-positive PI-GP150 was purified from the PI-PLC-released materials with three successive chromatographies (Sephacryl S-300, mAb HNK-1-Sepharose 4B, and Mono Q) and proven to be a novel molecule by immunoblot and structural analyses. Polyclonal antibody was raised against PI-GP150 and used to show that (a) PI-GP150 is expressed on the surface of neuronal cell bodies and their processes in culture, and (b) PI-GP150 appears during embryonic development and is present throughout all postnatal life in all brain regions. However, the expression of the HNK-1 epitope on PI-GP150 is regulated in both developmental stage-specific and region-specific manners. In newborn rats, the HNK-1 epitope is expressed on PI-GP150 throughout the brain. The level of HNK-1 epitope on PI-GP150 decreases after postnatal day 7 in hindbrain and becomes completely absent in adult myelencephalon and metencephalon. In contrast, HNK-1 epitope on PI-GP150 was constitutively expressed in telencephalon. Thus, while the HNK-1 carbohydrate epitope is strongly coupled to PI-GP150, its expression can be regulated independently of that of PI-GP150. The differential expression of the HNK-1 epitope at different rostro-caudal axial levels was observed also in other HNK-1 family molecules in brain membranes. These results suggest that the HNK-1 epitope plays an important role in adding region-specific and developmental stage-specific modifications on the function of the cell surface molecules.
Assuntos
Antígenos de Diferenciação/genética , Encéfalo/crescimento & desenvolvimento , Glicolipídeos/fisiologia , Glicoproteínas/fisiologia , Neurônios/fisiologia , Fosfatidilinositóis/fisiologia , Envelhecimento , Animais , Anticorpos , Antígenos de Diferenciação/isolamento & purificação , Encéfalo/embriologia , Antígenos CD57 , Embrião de Mamíferos , Epitopos/análise , Imunofluorescência , Expressão Gênica , Idade Gestacional , Glicoproteínas/isolamento & purificação , Glicosilfosfatidilinositóis , Membranas Intracelulares/fisiologia , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Sinaptossomos/fisiologia , Fosfolipases Tipo C/metabolismoRESUMO
CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Antígenos CD , Antígenos de Diferenciação/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adenosina Difosfato Ribose/metabolismo , Adenosina Difosfato Ribose/farmacologia , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/isolamento & purificação , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Cálcio/metabolismo , ADP-Ribose Cíclica , Ativação Linfocitária , Glicoproteínas de Membrana , Camundongos , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , NAD/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Plasmodium falciparum-infected erythrocytes (IE) specifically adhere to vascular endothelium in vivo and to human endothelial cells, some human melanoma cell lines, and human monocytes in vitro. The tissue cell receptor for a ligand on the surface of the infected erythrocytes is an Mr 88,000 glycoprotein (GP88) recognized by the MAb OKM5, which also blocks cytoadherence of IE. Isolated, affinity-purified GP88 (CD36) competitively blocks cytoadherence and when absorbed to plastic surfaces, specifically binds P. falciparum IE. Additionally, monoclonal and polyclonal antibodies to GP88 block cytoadherence to both target cells and immobilized GP88. Binding to GP88 by IE is unaffected by the absence of calcium or the absence of thrombospondin, a putative mediator for cytoadherence of P. falciparum IE. Thus, GP88 (CD36), which has been demonstrated to be the same as platelet glycoprotein IV, interacts directly with P. falciparum IE, presumably via a parasite-induced ligand exposed on the surface of the infected erythrocytes. CD36 is shown to be present on brain endothelium in both individuals without malaria and individuals with cerebral malaria. This would suggest that factors other than just cerebral sequestration of IE play an initiating role in the genesis of cerebral malaria.
Assuntos
Antígenos de Diferenciação/imunologia , Eritrócitos/metabolismo , Glicoproteínas de Membrana/imunologia , Plasmodium falciparum/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas , Receptores Imunológicos/fisiologia , Formação de Roseta , Animais , Anticorpos Monoclonais/fisiologia , Antígenos de Diferenciação/isolamento & purificação , Ligação Competitiva , Encéfalo/irrigação sanguínea , Antígenos CD36 , Linhagem Celular , Endotélio Vascular/análise , Eritrócitos/imunologia , Humanos , Melanoma/imunologia , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/farmacologia , Peso Molecular , Plasmodium falciparum/imunologia , TrombospondinasRESUMO
We characterized Fc receptor III (FcR III) on human neutrophils and found it to be heavily glycosylated and polymorphic. In some individuals, FcR III that had been digested with N-glycanase appeared after SDS-PAGE under reducing conditions as two bands with apparent molecular masses of 33 and 29 kD. In other individuals, N-glycanase-treated FcR III appeared as a single band with an Mr of either 33 or 29 kD. After SDS-PAGE of N-glycanase-treated FcR III under nonreducing conditions, the apparent Mr of each structural type was decreased, suggesting the presence of intramolecular disulfide bonds. Digestion of the 33-kD band and the 29-kD band with Staphylococcus aureus V8 protease yielded similar, but not identical, peptide maps. Thus, at least two polymorphic forms of FcR III are expressed on human neutrophils. The structural polymorphism of neutrophil FcR III correlated with previously described antigenic polymorphisms detected by monoclonal antibody Gran 11 and by alloantisera which recognize epitopes of the biallelic, neutrophil antigen (NA) system. Individuals whose neutrophils expressed the two-band structural type of FcR III were NA1NA2 heterozygotes. Individuals whose neutrophils expressed the single 33-kD band structural type were NA2NA2 homozygotes, and individuals whose neutrophils expressed the single 29-kD band structural type were NA1NA1 homozygotes. These findings indicate that antigenic and structural polymorphisms of human neutrophil FcR III are related and can be accounted for by differences at the level of primary protein structure.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Imunoglobulina G/metabolismo , Polimorfismo Genético , Receptores Fc/isolamento & purificação , Anticorpos Monoclonais , Reações Antígeno-Anticorpo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos Heterófilos/imunologia , Glicosilação , Humanos , Isoanticorpos , Peso Molecular , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores Fc/genética , Receptores Fc/imunologia , Receptores de IgG , Relação Estrutura-AtividadeRESUMO
Whole human and bovine pancreases were extracted in 20 mM Tris-HCl buffer without detergents and fractionated by high-speed centrifugation. The 80,000 x g supernatant was used to coat microtiter plates at a concentration of 5 micrograms protein/ml in phosphate-buffered saline. This solid-phase ELISA system was used for the detection of islet cell antigens defined by a series of monoclonal islet cell antibodies (HISL-1, -4, -5, -8, -14, and -19 and 4F2, 3G5, and A2B5). Both glycoprotein and glycolipid islet cell antigens in the total pancreatic extracts were detected by the monoclonal islet cell antibody in the ELISA system, indicating that epitope preservation had occurred during the extraction procedure. There was a good correlation between islet cell antigen quantitated by the ELISA system and the corresponding islet immunohistochemical reaction. Studies along these lines have the potential to facilitate the design of large-scale protocols for the purification of diabetes-related islet cell antigens to homogeneity.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Ilhotas Pancreáticas/imunologia , Animais , Anticorpos Monoclonais , Bovinos , Humanos , Ilhotas Pancreáticas/análise , MétodosRESUMO
Using tumour cell lines expressing specific isoforms of murine Ly-5 (molecular weights of 180,000, 200,000 and 240,000) we find that all forms of Ly-5 and immuno-affinity purified forms of Ly-5 contain tyrosyl phosphatase activity. These results demonstrate that these isoforms of Ly-5 belong to the same family of functional receptor-linked tyrosine phosphatases as the human leukocyte common antigen. CD45.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígenos Ly/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Antígenos de Diferenciação/isolamento & purificação , Antígenos Ly/isolamento & purificação , Galinhas , Antígenos de Histocompatibilidade/isolamento & purificação , Humanos , Antígenos Comuns de Leucócito , Camundongos , Testes de Precipitina , Proteínas Tirosina Fosfatases , Células Tumorais CultivadasRESUMO
Granuloma initiation factor (GIF), which elicits a granulomatous reaction in naive murine skin, contains low-molecular-weight proteins partially purified from organized granulomas developed in livers of mice with schistosomiasis. In this study, we found that 10- and 14-kDa proteins in the GIF are highly homologous to mouse migration inhibitory factor-related protein (MRP) 8 and MRP 14. Compared with the N-terminal amino acid sequence deduced from each corresponding cDNA, the 10-kDa protein from the granuloma lacks the first methionine, whereas the 14 kDa misses methionine, alanine, and asparagine. Immunohistochemically, cells expressing MRP 8 and MRP 14 considerably increased in different murine tissues after Schistosoma mansoni infection and concentrated in liver around the dilated blood vessels and at the edge of granulomas. The staining of differentiated macrophages and epithelioid cells located in the center of the granulomas was negative. Immunoreactivity of peritoneal exudate cells also was found to gradually disappear with time in cell culture. Furthermore, in vivo effects of the recombinant proteins in murine skin were described histologically. Both MRPs caused severe infiltration of neutrophils and monocytes during 7-14 days. The reaction resulting from individual MRP implantation became minimal after 50 days but inoculation of the Ca2+-dependent heterodimers showed an extensive eosinophil accumulation.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Transporte/química , Granuloma , Esquistossomose mansoni , Sequência de Aminoácidos , Animais , Calgranulina A , Calgranulina B , Células Cultivadas , Feminino , Inflamação , Isomerases , Macrófagos/química , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Schistosoma mansoniRESUMO
OBJECTIVES: To identify a substance found in female genital tract secretions that enhances HIV expression in infected cells. DESIGN: Cervico-vaginal lavages (CVL), collected in sterile normal saline, were fractionated and tested for HIV-inducing activity using HIV-infected monocytes. METHODS: To purify the component(s) of CVL that enhance HIV production, Mono-Q ion exchange chromatography followed by Superose-12 molecular sieve analysis, and SDS--PAGE were performed. The purified protein was identified by amino acid sequence analysis. RESULTS: SDS--PAGE of bioactive fractions showed a 14 kDa polypeptide band. Amino acid sequence analysis of selected peptides from the 14 kDa band showed 100% homology with the myeloid-related protein (MRP)-8, an inflammatory protein found in mucosal secretions. Western blot analysis revealed that bioactive CVL contained more immunoreactive MRP-8 than samples without bioactivity. The HIV-inducing activity of MRP-8 was further confirmed by showing that human recombinant MRP-8 increased HIV expression by up to 40-fold. CONCLUSIONS: MRP-8 in cervico-vaginal secretions stimulates HIV production. Strategies aimed at blocking MRP-8 activity in the genital tract could reduce risk of sexual as well as maternal--infant transmission of HIV.
Assuntos
Antígenos de Diferenciação/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , Colo do Útero/metabolismo , HIV-1/genética , Vagina/metabolismo , Sequência de Aminoácidos , Antígenos de Diferenciação/química , Antígenos de Diferenciação/isolamento & purificação , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/isolamento & purificação , Calgranulina A , Linhagem Celular , Colo do Útero/química , Feminino , Humanos , Proteínas Recombinantes/farmacologia , Vagina/química , Ativação ViralRESUMO
CD38 is an ectoenzyme, which can produce metabolites with intracellular Ca(2+) mobilizing properties and has multiple immunological functions. However, we have recently shown that CD38 is also localized to the nucleus of rat hepatocyte whereby its metabolite cADPR, is able to mobilize nuclear Ca(2+) stores. In this study, we further characterize the localization of nuclear CD38 in the spleen, an important immune organ. We managed to detect the presence of ADP-ribosyl cyclase activity in the nuclear fraction. With Western blotting, we managed to characterize a 42-45 kDa protein band that is typical of CD38 under reducing and non-reducing conditions. However, as a comparison, other nuclear fractions from tissues like thymus, cardiac muscle and cerebellum yielded an additional 85 kDa protein band under non-reducing conditions. Both protein bands could be blocked with a CD38 blocking peptide. Immunohistochemical studies revealed the expression of CD38 in the marginal zone and in the red pulp. In contrast, the germinal center remained largely immunonegative for CD38. This is the first report of a functionally active ADP-ribosyl cyclase/CD38 in the spleen nuclear fraction. The results here suggest that the presence of CD38 in the nuclear environment might have a corollary to functional and regulatory roles in the nucleus.
Assuntos
Antígenos CD , Antígenos de Diferenciação/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/imunologia , NAD+ Nucleosidase/metabolismo , Baço/enzimologia , Baço/imunologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos de Diferenciação/química , Antígenos de Diferenciação/isolamento & purificação , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana , Microssomos/enzimologia , Microssomos/imunologia , Peso Molecular , NAD+ Nucleosidase/química , NAD+ Nucleosidase/isolamento & purificação , Ratos , Ratos Wistar , Baço/anatomia & histologia , Distribuição TecidualRESUMO
To identify the differentiation antigen of ovarian cells, we raised a murine monoclonal antibody (POG-1 antibody) reactive to porcine granulosa and thecal cells in the ovary. Immunofluorescence staining showed that expression of the POG-1 antigen on granulosa and theca interna cells increased gradually in accordance with follicular development. The thecal cells just outside the basal lamina surrounding the follicles did not express the antigen, whereas some stromal cells around the theca externa layer in the large follicles did express it. These expression profiles indicated the heterogeneity of thecal-stromal cells and that the POG-1 antigen was a differentiation-related antigen of granulosa and thecal cells. Luteal cells also expressed the antigen. In organs other than the ovary, some endocrine and exocrine cells, such as Leydig cells and secretory cells of the breast, expressed the antigen. The POG-1 antigen was purified from granulosa cells by immunoaffinity chromatography. Polyacrylamide gel electrophoresis profiles showed that the antigen consisted of two specific proteins; the major one had a molecular mass of 77, and the other had a molecular mass of 81 kilodaltons. Analysis of the purified POG-1 antigen may contribute to understanding the differentiation mechanism of granulosa and thecal cells.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação/análise , Células da Granulosa/imunologia , Células Tecais/imunologia , Animais , Antígenos de Diferenciação/isolamento & purificação , Feminino , Masculino , Células Estromais/fisiologia , Suínos , Testículo/imunologia , Células Tecais/citologiaRESUMO
CD9 antigen (p24) was purified from human platelets and partially characterized. The yield was 75 micrograms from 10 units of platelet concentrates. p24 (38,000 copies/platelet) has intramolecular disulfide bond(s) and, in SDS-PAGE, consists of major 24-kDa molecule and minor 26- to 31-kDa molecules. The N-terminal sequence of p24, PVKGGTKXIKYLLFGFNFIF, indicates that the protein has not previously been characterized and amino terminus (position 12-20) is hydrophobic.
Assuntos
Antígenos CD/isolamento & purificação , Antígenos de Diferenciação/isolamento & purificação , Plaquetas/imunologia , Glicoproteínas de Membrana , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Tetraspanina 29RESUMO
Normal human fibroblasts have a limited replicative potential in culture and eventually reach a state of irreversible growth arrest, termed senescence. In a previous study aiming to identify genes that are differentially regulated during cellular senescence we have cloned clusterin/apolipoprotein J (Apo J), a 80 kDa secreted glycoprotein. In the current report we pursue our studies and show that senescence of human diploid fibroblasts is accompanied by up-regulation of both Apo J mRNA and protein levels, but with no altered biogenesis, binding partner profile or intracellular distribution of the two Apo J forms detected. To analyze the causal relationship between senescence and Apo J protein accumulation, we stably overexpressed the Apo J gene in primary as well as in SV40 T antigen-immortalized human fibroblasts and we showed no alteration of the proliferative capacity of the transduced cells. Despite previous reports on tumor-derived cell lines, overexpression of Apo J in human fibroblasts did not provide protection against apoptosis or growth arrest induced by hydrogen peroxide. Overall, our results suggest that Apo J overexpression does not induce senescence but it is rather a secondary consequence of the senescence phenotype. To our knowledge this is the first report that provides a functional analysis of human Apo J during replicative senescence.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Senescência Celular/fisiologia , Fibroblastos/fisiologia , Glicoproteínas/isolamento & purificação , Chaperonas Moleculares/isolamento & purificação , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Clusterina , Diploide , Fibroblastos/citologia , Glicoproteínas/biossíntese , Glicoproteínas/genética , Humanos , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Estresse Oxidativo/fisiologia , Proteínas Recombinantes/biossíntese , Regulação para CimaRESUMO
Class II HLA-DR genes play an important role in the immune response to viral antigens. The effect of measles vaccine virus (MVV) infection on the induction of self-peptides presented by HLA-DR molecules during the immune response to viral infection is poorly known. Here, we describe a strategy for isolation and rapid sequence determination of an MVV-inducible class II bound peptide from a membrane protein (Leu-13). Peptides bound to HLA-DR4 (DRB1*0401 peptide complex) were eluted from immunoaffinity-purified HLA-DR4, peptides were differentially screened by MALDI-TOF-MS and subsequently sequenced by post source decay (PSD)-MALDI-TOF-MS. Human B-cells infected with MVV demonstrated an enhanced pattern of self-peptide production after MVV infection. This relatively simple analytical protocol provides a sensitive method for the direct identification of peptides associated with MHC class II DR molecules. More broadly, this same approach can be used to identify sequences of specific MVV processed peptides presented by any class II MHC DR molecule.
Assuntos
Alelos , Antígenos HLA-DR/isolamento & purificação , Vacina contra Sarampo/imunologia , Vírus do Sarampo/imunologia , Oligopeptídeos/isolamento & purificação , Sequência de Aminoácidos , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/isolamento & purificação , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Transformada , Regulação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Cadeias HLA-DRB1 , Humanos , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
CD66a, also known as biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and the human homologue of the rat cell-CAM. There is evidence that aberrant expression or loss of CD66a in tumor tissue is of biological significance. No data about its expression in breast carcinoma cells and only sparse information about the expression of CD66a in normal breast are available thus far. In this study we used monoclonal antibodies to analyze the expression of CD66a and CEA in normal tissue, benign lesions, and in noninvasive and invasive carcinomas of the mammary gland. In normal tissue and benign lesions, CD66a was consistently expressed at the apical sites of epithelial cells and in myoepithelia, whereas CEA was absent or was restricted only to some apical membranes within the ductal tree. The specific staining of myoepithelia was most evident in pseudoinfiltrative radial scars and sclerosing adenosis. However, the apical expression of CD66a disappeared with the development of the malignant phenotype in noninvasive and invasive carcinomas, and changed gradually from low- to high-grade noninvasive carcinomas into a predominant uniform membrane staining all around the atypical cells. CEA expression was irregular in intensity and distribution. The native apical CD66a staining was partially preserved in some highly differentiated invasive carcinomas with a better prognosis, such as tubular and papillary carcinomas. These findings indicate that loss of CD66a expression rather than a change in staining patterns coincides with the development of the malignant phenotype.
Assuntos
Antígenos CD/isolamento & purificação , Antígenos de Diferenciação/isolamento & purificação , Neoplasias da Mama/patologia , Carcinoma/patologia , Moléculas de Adesão Celular/isolamento & purificação , Lesões Pré-Cancerosas/patologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Neoplasias da Mama/química , Antígeno Carcinoembrionário , Carcinoma/química , Humanos , Hiperplasia , Imuno-Histoquímica , Invasividade Neoplásica , Lesões Pré-Cancerosas/químicaRESUMO
PURPOSE: Previous studies have suggested that galectins may be involved in retinal adhesion and photoreceptor cell survival. To elucidate the underlying mechanisms, the authors isolated retinal galectins, determined their types and distributions, and investigated the validity of the hypothesis, using rat models. METHODS: An antibody was prepared against a bovine retinal lectin that was isolated by use of a lactose-agarose column. cDNA of the lectin was isolated by screening of a bovine retinal cDNA library, using the antibody, and then was sequenced. The cDNAs of rat retinal galectins were also isolated by means of polymerase chain reaction and used to produce an antibody against recombinant galectin-3. Using the described antibodies, the authors examined the distributions of galectins in bovine and rat retinas, morphologic changes of rat retinas induced by the antibodies, and distributional changes of galectins in constant-light-exposed rat retinas. RESULTS: The cDNAs of bovine galectin-1, rat galectin-1, and rat galectin-3 were isolated. Galectin-1 was found in various regions, including the retinal pigment epithelium, outer limiting membrane, and outer plexiform layer in bovine and rat retinas. Galectin-3 was increasingly detected in the cytoplasm of Müller cells after constant light exposure after an increase in its transcript. Retinal detachment and vacuolation of the outer plexiform layer were induced in rat eyes by intravitreous injection of the anti-galectin-1 antibody. CONCLUSIONS: Galectin-1 may be involved in adhesion of the photoreceptor and outer plexiform layers by interacting with glycoconjugates with beta-galactoside residues in the interphotoreceptor matrix and synaptic cleft matrix. Galectin-3 may increase in Müller cells of a degenerative rat retina, probably through endogenous anti-apoptosis.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Proteínas do Olho/isolamento & purificação , Hemaglutininas/isolamento & purificação , Retina/química , Animais , Anticorpos/farmacologia , Antígenos de Diferenciação/fisiologia , Western Blotting , Bovinos , Adesão Celular/fisiologia , Cromatografia de Afinidade , Primers do DNA/química , DNA Complementar/análise , Proteínas do Olho/fisiologia , Galectina 1 , Galectina 3 , Hemaglutininas/fisiologia , Técnicas Imunoenzimáticas , Injeções , Masculino , Coelhos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Retina/metabolismo , Descolamento Retiniano/induzido quimicamente , Descolamento Retiniano/metabolismo , Descolamento Retiniano/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Corpo VítreoRESUMO
The terms glycoprotein IV (GPIV) and glycoprotein IIIb (GPIIIb) have been used interchangeably and reports in the literature have indicated this glycoprotein as having a molecular weight variously described as either 88,000 or 97,000, a fast anodal mobility on crossed electrophoresis and either 13 or less than 1 methionine residues on amino acid analysis of the purified glycoprotein. To resolve these discrepancies, we have evaluated the characteristics of GPIV both in whole platelets and after isolation. These studies have shown that the term GPIV defines a protease-resistant platelet surface glycoprotein with Mr 88,330 +/- 2,240 which is immunologically identical with the CD36 differentiation antigen, which migrates with a relatively slow anodal mobility on crossed immunoelectrophoresis and which contains approximately 13 methionine residues per mole.