RESUMO
Mannheimiosis is the major respiratory disease among some ruminants, whereas it is not pathogenic for other mammals, an observation that has been attributed to a specific interaction between Mannheimia haemolytica leukotoxin (Lkt) and bovine or ovine CD18 subunit of lymphocyte function-associated antigen-1 (LFA-1) and Mac-1. We therefore hypothesized that Lkt utilizes CD18 as its receptor on caprine leukocytes as well. We have transiently transfected the beta2-integrins-deficient K-562 cell line with cDNAs encoding caprine CD11a and caprine CD18 to determine the susceptibility of the transfectants to Lkt-induced cytolysis. Flow cytometric analysis of the transfectants revealed surface expression of caprine LFA-1 and lysis by Lkt in a concentration-dependent manner whereas the parent cells were not. Moreover, K562 cells expressing caprine CD18 and human or bovine CD11a were also sensitive to Lkt whereas K-562 cells expressing caprine CD11a and human CD18 were not. Taken together, these results indicate that CD18 on caprine leukocytes serves as a receptor for Lkt.
Assuntos
Antígenos CD18/imunologia , Citotoxinas/imunologia , Exotoxinas/imunologia , Leucócitos/imunologia , Mannheimia haemolytica/imunologia , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/isolamento & purificação , Antígenos CD11/genética , Antígenos CD11/imunologia , Antígenos CD18/genética , Bovinos , Linhagem Celular , Citotoxinas/genética , Citotoxinas/isolamento & purificação , Exotoxinas/genética , Exotoxinas/isolamento & purificação , Doenças das Cabras/imunologia , Doenças das Cabras/microbiologia , Cabras , Humanos , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/imunologia , Mannheimia haemolytica/genética , Mannheimia haemolytica/patogenicidade , Infecções por Pasteurellaceae/imunologia , Infecções por Pasteurellaceae/microbiologia , Infecções por Pasteurellaceae/veterinária , Ligação ProteicaRESUMO
The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. The ovine cDNA encoding CD11a, the predominant alpha subunit of the beta2-integrin family, was sequenced and compared with the human, bovine and murine sequences. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues. Along with the ovine CD18-encoding cDNA, which is available for a few months, the sequence data provided here will allow the Ovis aries beta2-integrin CD11a/CD18 (LFA-1, alpha(L)beta2) expression in vitro as a tool to examine the specificities of inflammation in the ovine species.
Assuntos
Antígeno CD11a/genética , Antígeno-1 Associado à Função Linfocitária/genética , Carneiro Doméstico/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígeno CD11a/isolamento & purificação , Bovinos , Clonagem Molecular , DNA Complementar/genética , Humanos , Antígeno-1 Associado à Função Linfocitária/química , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The leukocyte integrins play a critical role in a great number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the caprine beta(2) (CD18) sub-unit, common to the leukocyte beta(2)-integrin family. The deduced 770-amino-acid sequence reveals a transmembrane protein with 80, 81, 83, 96 and 99% identity with its canine, murine, human, bovine and ovine homologues respectively. Analysis of CD18 sequences emphasizes the functional importance of the beta(2) sub-unit I-like domain, and included metal ion-dependent adhesion site-like motif and confirms that of the cytoplasmic tail. Moreover, comparisons of ruminant versus non-ruminant CD18 sequences allowed the identification of 16 potential mutation sites that could be held responsible for the unique virulence of Mannheimia haemolytica for ruminants. Mannheimiosis is known to be the major respiratory disease among ruminants, whereas it is not pathogenic for other mammals, an observation that has been attributed to a specific interaction between M. haemolytica leukotoxin and ruminants' CD18. Therefore, the data provided here offer the possibility to explore new avenues in studies based on the caprine model and provide key information for future studies aimed at elucidating the molecular mechanisms underlying the ruminant-specific virulence of M. haemolytica.
Assuntos
Antígenos CD18/genética , Cabras/genética , Mannheimia haemolytica/patogenicidade , Mutação Puntual/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , DNA Complementar/genética , Doenças das Cabras/microbiologia , Cabras/microbiologia , Dados de Sequência Molecular , Infecções por Pasteurellaceae/microbiologia , Infecções por Pasteurellaceae/veterinária , Subunidades Proteicas/genéticaRESUMO
The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the ovine beta(2) (CD18) subunit, common to the leukocyte beta(2)-integrin family. The deduced 770-amino-acid sequence reveals a transmembrane protein with 81%, 83% and 95% identity with its murine, human and bovine homologues, respectively. Comparisons of CD18 sequences emphasize the functional importance of the beta(2) subunit I-like domain and included metal ion-dependent adhesion site (MIDAS)-like motif and confirm that of the cytoplasmic tail. The data provided here will offer the possibility to explore new avenues in studies based on the ovine model.
Assuntos
Antígenos CD18/genética , Carneiro Doméstico/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The bovine cDNA encoding CD11a, a cell-surface glycoprotein involved in multiple leukocyte functions, was sequenced and compared with the human and murine sequences. Despite some focal differences, it shares all the main characteristics of its known mammalian homologs. Along with the bovine CD18-encoding cDNA, which is available for a long time, the sequence data provided here will allow the successful expression of bovine CD11a, thus giving the first opportunity to express the Bos taurus beta(2)-integrin CD11a/CD18 (LFA-1, alpha(L)beta(2)) in vitro as a tool to examine the specificities of inflammation in the bovine species.
Assuntos
Antígeno CD11a/genética , Bovinos/genética , DNA Complementar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Glicoproteínas/genética , Humanos , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Organisms living in permanently cold environments, which actually represent the greatest proportion of our planet, display at low temperatures metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. They produce cold-evolved enzymes partially able to cope with the reduction in chemical reaction rates and the increased viscosity of the medium induced by low temperatures. In most cases, the adaptation is achieved through a reduction in the activation energy, leading to a high catalytic efficiency, which possibly originates from an increased flexibility of either a selected area of or the overall protein structure. This enhanced plasticity seems in return to be responsible for the weak thermal stability of cold enzymes. These particular properties render cold enzymes particularly useful in investigating the possible relationships existing between stability, flexibility, and specific activity and make them potentially unrivaled for numerous biotechnological tasks. In most cases, however, the adaptation appears to be far from being fully achieved.
Assuntos
Clima Frio , Enzimas/química , Enzimas/metabolismo , Adaptação Fisiológica , Biotecnologia , Evolução Molecular Direcionada , Estabilidade Enzimática , Enzimas/genética , Cinética , Conformação ProteicaRESUMO
Double-stimulation was used to demonstrate that, in a T lymphocytic cell line (CEM), phorbol myristate acetate (PMA) rapidly induced NF-kappa B through a signaling pathway which did not involve reactive oxygen species (ROS) and was different from the activation triggered by either H2O2 or tumor necrosis factor-alpha (TNF-alpha). Since these latter compounds were known to activate NF-kappa B translocation in a redox-sensitive way, we have demonstrated that NF-kappa B activation by PMA was resistant to antioxidant N-acetyl-L-cysteine (NAC) and sensitive to kinase inhibitors staurosporine and H7 while activation by H2O2 or TNF-alpha were not.
