RESUMO
We have determined the DNA sequence of a gene encoding a thymus leukemia (TL) antigen in the BALB/c mouse, and have more definitively mapped the cloned BALB/c Tla-region class I gene clusters. Analysis of the sequence shows that the Tla gene is less closely related to the H-2 genes than H-2 genes are to one another or to a Qa-2,3-region genes. The Tla gene, 17.3A, contains an apparent gene conversion. Comparison of the BALB/c Tla genes with those from C57BL shows that BALB/c has more Tla-region class I genes, and that one of the genes absent in C57BL is gene 17.3A.
Assuntos
Antígenos de Neoplasias/genética , Genes , Glicoproteínas de Membrana , Camundongos Endogâmicos BALB C/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Deleção Cromossômica , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Conversão Gênica , Ligação Genética , Complexo Principal de Histocompatibilidade , Camundongos , Polimorfismo GenéticoRESUMO
We have previously described the isolation of pH-2d-37, a cDNA clone that encodes a so far unknown, poorly polymorphic, class I surface molecule. We report here the isolation of the corresponding gene, its nucleotide sequence, and its localization in the Tla region of the murine MHC. Using a RNase mapping assay, we have confirmed that the second domain coding region of the 37 gene displays very limited polymorphism, and that the gene is transcribed in a broad variety of cell types, in contrast to the genes encoding the known Qa and TL antigens. Possible functions are discussed.
Assuntos
Genes MHC da Classe II , Antígenos H-2/genética , Camundongos Endogâmicos BALB C/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Enzimas de Restrição do DNA , Camundongos , Polimorfismo Genético , RNA Mensageiro/análise , Distribuição TecidualRESUMO
DNA sequences of the fifth exon, which encodes the transmembrane domain, were determined for the BALB/c mouse class I MHC genes and used to study the relationships between them. Based on nucleotide sequence similarity, the exon 5 sequences can be divided into seven groups. Although most members within each group are at least 80% similar to each other, comparison between groups reveals that the groups share little similarity. However, in spite of the extensive variation of the fifth exon sequences, analysis of their predicted amino acid translations reveals that only four class I gene fifth exons have frameshifts or stop codons that terminate their translation and prevent them from encoding a domain that is both hydrophobic and long enough to span a lipid bilayer. Exactly 27 of the remaining fifth exons could encode a domain that is similar to those of the transplantation antigens in that it consists of a proline-rich connecting peptide, a transmembrane segment, and a cytoplasmic portion with membrane-anchoring basic residues. The conservation of this motif in the majority of the fifth exon translations in spite of extensive variation suggests that selective pressure exists for these exons to maintain their ability to encode a functional transmembrane domain, raising the possibility that many of the nonclassical class I genes encode functionally important products.
Assuntos
Éxons , Genes MHC Classe I , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Citoplasma/análise , DNA/análise , Antígenos de Histocompatibilidade Classe I/análise , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
BACKGROUND: Leukocyte activation results in a rapid increase in adhesion to the extracellular matrix due to the activation of beta 1 integrin receptors. A role for phosphatidylinositol (PI) 3-kinase in integrin activation has been proposed, as activation of integrins by many receptors can be blocked by PI 3-kinase inhibitors. One receptor that regulates integrins is the CD28 surface antigen; here, we investigated the mechanisms responsible for CD28-mediated integrin activation. RESULTS: CD28-mediated integrin activation was blocked by mutation of the binding site for the p85 catalytic subunit of PI 3-kinase in the CD28 cytoplasmic domain, and by expression of a dominant-negative form of the p85 subunit. Substitution of the Src homology 2 (SH2)-binding motif in the CD28 cytoplasmic domain for the corresponding motif in the CD28-related CTLA-4 surface antigen also blocked integrin activation but did not affect the recruitment and activation of PI 3-kinase. Mutations of the CD28 cytoplasmic domain that blocked integrin activation also impaired the tyrosine phosphorylation of the Cbl adaptor protein and the activation of the PI 3-kinase that was associated with Cbl. This Cbl-associated PI 3-kinase was distinct from the PI 3-kinase that coprecipitated with the CD28 cytoplasmic domain. CD28-mediated activation of beta 1 integrins was inhibited by expression of a mutant Cbl protein that shows reduced association with PI 3-kinase. CONCLUSIONS: Cbl is required for PI-3-kinase-dependent regulation of integrin receptors by CD28. Furthermore, CD28 is coupled to two distinct pools of PI 3-kinase, one directly associated with the CD28 cytoplasmic tail and the other associated with Cbl.
Assuntos
Antígenos CD/fisiologia , Antígenos CD2/fisiologia , Antígenos CD28/fisiologia , Adesão Celular/fisiologia , Integrina beta1/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases , Substituição de Aminoácidos , Antígenos CD/biossíntese , Sítios de Ligação , Antígenos CD2/biossíntese , Antígenos CD28/biossíntese , Citometria de Fluxo , Proteínas de Fluorescência Verde , Células HL-60 , Humanos , Cinética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotirosina/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-cbl , Proteínas Recombinantes de Fusão/biossíntese , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity. CD2-induced increases in beta1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.
Assuntos
Antígenos CD2/química , Antígenos CD2/fisiologia , Integrina beta1/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Prolina , Linfócitos T/fisiologia , Sequência de Aminoácidos , Antígenos CD/biossíntese , Antígenos CD/química , Antígenos CD/fisiologia , Antígenos CD2/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citoplasma , Ativação Enzimática , Fibronectinas/fisiologia , Glutationa Transferase/metabolismo , Células HL-60 , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Linfócitos T/imunologia , TransfecçãoRESUMO
T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in beta1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the beta1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human beta1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the beta1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the beta1 integrin with the activating beta1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the beta1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of beta1 integrin structure and function in human T cells.
Assuntos
Integrina beta1/química , Integrina beta1/fisiologia , Linfócitos T/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Adesão Celular , Primers do DNA , Fibronectinas/fisiologia , Humanos , Integrina beta1/genética , Células Jurkat , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transfecção , TirosinaRESUMO
Autoantibodies to type VII collagen are associated with the blistering diseases epidermolysis bullosa acquisita and bullous systemic lupus erythematosus. We showed previously that these autoantibodies recognize epitopes within the noncollagenous (NC1) region of type VII collagen. That region is composed of fibronectin type III homology units that may contribute to intermolecular cross-linking and basement membrane adhesion functions of type VII collagen. In this study, we defined the specific amino acid sequences recognized by these autoantibodies. By fusion protein analysis, sera from patients with epidermolysis bullosa acquisita and bullous lupus were found to react with two regions within the fourth (E-1) and eighth (E-2) fibronectin homology repeats, each consisting of approximately 100 amino acids. Affinity purification studies showed E-1 and E-2 to be independent and non-cross-reactive epitope regions. These regions were probed further by enzyme-linked immunosorbent assay analysis of overlapping octapeptide sets derived from the amino acid sequences of E-1 and E-2. The results showed two reactive, closely associated octapeptide sequences within each region, both lying in amphipathic portions of fibronectin type III homology repeats. These studies identify short peptide sequences within the NC1 domain of type VII collagen that are targeted independently by autoantibodies. These sequences may play a direct role in determining the properties of type VII collagen that influence adhesion between this molecule and other basement membrane proteins, and their alteration by antibody binding may be the immunopathogenic event underlying epidermolysis bullosa acquisita and bullous lupus.
Assuntos
Colágeno/imunologia , Epitopos/química , Fibronectinas/química , Peptídeos/análise , Sequência de Aminoácidos , Autoanticorpos/análise , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Autoantibodies to type VII collagen are characteristic of the blistering diseases epidermolysis bullosa acquisita and bullous systemic lupus erythematosus (SLE). Blisters in those diseases are due to defective adhesion of the lamina densa subregion of the epithelial basement membrane to the underlying dermis. Previous studies indicating that type VII collagen contributes to lamina densa-dermal adhesion by cross-linking lamina densa and dermal matrix proteins suggests that autoantibodies may contribute to blisters by interfering with type VII collagen function. That hypothesis is supported by previous studies showing autoantibodies from a small number of epidermolysis bullosa acquisita patients recognize proteolytic fragments containing the 145-kD noncollagenous domain of type VII collagen. In this study, we examined reactivity of autoantibodies from a large number of epidermolysis bullosa acquisita and bullous SLE patients with fusion proteins representing most of the noncollagenous domain of type VII collagen and that those regions are homologous to type III repeats of fibronectin. These results suggest autoantibodies binding to fibronectin homology regions within the 145-kD noncollagenous domain may interfere with the adhesion function of type VII collagen and contribute to lamina densa-dermal dysadhesion in epidermolysis bullous acquisita and bullous SLE.
Assuntos
Autoanticorpos/imunologia , Colágeno/genética , Colágeno/imunologia , Epitopos , Fibronectinas/genética , Western Blotting , DNA/genética , Ensaio de Imunoadsorção Enzimática , Epidermólise Bolhosa Adquirida/imunologia , Humanos , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Homologia de SequênciaRESUMO
Type VII collagen (C7) is a stratified squamous epithelial basement membrane protein composed of three identical alpha chains, each consisting of a 145-kDa amino-terminal noncollagenous (NC1) domain and a 145-kDa carboxyl-terminal collagenous domain. Morphologic and biochemical studies have shown that tissue-specific aggregates of C7 dimers called anchoring fibrils may contribute to epithelial basement membrane organization and adherence by interacting with extracellular matrix (ECM) proteins such as type IV collagen. In this study, we cloned a cDNA encoding most of the NC1 domain of C7. The deduced amino acid sequence revealed motifs characteristic of multidomain ECM proteins that contribute to the tissue-specific organization of ECM including a region of 7 1/2 sequential fibronectin type III (Fn III) homology repeats, a potential collagen-binding region homologous to the A domain of von Willebrand factor (vWf) and an RGD sequence. A purified C7 fusion protein containing these motifs specifically bound to type IV collagen in a functional assay. These results suggest that regions within the NC1 domain of C7 mediate interactions with lamina densa and dermal ECM proteins including type IV collagen. Structural mutations and autoepitopes in these regions may represent mechanisms for the development of defective basement membrane organization and adherence in genetic and autoimmune forms of epidermolysis bullosa.
Assuntos
Colágeno/química , Matriz Extracelular/química , Sequência de Aminoácidos , Sequência de Bases , Moléculas de Adesão Celular/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.
Assuntos
Antígenos CD4/fisiologia , Fusão Celular , Genes Reporter , Proteína gp160 do Envelope de HIV/fisiologia , Luciferases/genética , Proteínas Luminescentes/genética , Macrófagos/virologia , Receptores CCR5/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Fusão Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde , Humanos , Luciferases/biossíntese , Proteínas Luminescentes/biossíntese , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Sensibilidade e Especificidade , TransfecçãoRESUMO
Two different methods of achieving upper airway anesthesia for awake fiberoptic intubation were prospectively compared in patients undergoing surgery for cervical spine instability. Forty patients were randomized to either topical anesthesia or nerve block groups. Topical anesthesia patients were administered nebulized 4% lidocaine (approximately 20 ml) via the oropharynx plus a transtracheal injection of 4% lidocaine (3 ml). Nerve block patients underwent bilateral glossopharyngeal and superior laryngeal nerve blocks with 2% lidocaine (0.5-2 ml per injection site) plus a transtracheal injection of 4% lidocaine (3 ml). The quality of anesthesia for intubation was graded by observers blinded to group assignment. Mean arterial pressure, heart rate, Pao2, Paco2, pHa, SpO2, and plasma lidocaine concentrations were measured during the intubation sequence. Patient recall of intubation and discomfort were assessed during the postoperative period with visual analog scales. Time required for successful intubation and quality of intubation were not different between groups. Physiologic values for the two groups were similar. The mean total dose of lidocaine in the topical anesthesia group was approximately 2 times greater than that in the nerve block group (815 versus 349 mg; p < 0.0001). In contrast, mean plasma lidocaine concentration at initiation of intubation in the topical anesthesia group was half that of nerve block group (2.16 versus 4.23 micrograms/ml; p < 0.0001). Ten minutes later there was no difference for plasma lidocaine concentration between groups. No patients had evidence of seizures or neurologic change during the procedure. There was no difference in patient perception of discomfort during the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anestesia Local , Vértebras Cervicais/cirurgia , Intubação Intratraqueal , Lidocaína/administração & dosagem , Bloqueio Nervoso , Vigília , Adulto , Pressão Sanguínea , Dióxido de Carbono/sangue , Feminino , Nervo Glossofaríngeo , Frequência Cardíaca , Humanos , Intubação Intratraqueal/efeitos adversos , Intubação Intratraqueal/métodos , Nervos Laríngeos , Lidocaína/sangue , Masculino , Orofaringe , Oxigênio/sangue , Dor/etiologia , Estudos Prospectivos , Doenças da Coluna Vertebral/cirurgia , TraqueiaAssuntos
Adenosina Desaminase/genética , Coformicina/toxicidade , Nucleosídeo Desaminases/genética , Ribonucleosídeos/toxicidade , Inibidores de Adenosina Desaminase , Animais , Linhagem Celular , Bandeamento Cromossômico , Coformicina/análogos & derivados , Cricetinae , Cricetulus , Resistência a Medicamentos , Feminino , Cariotipagem , Cinética , Células L/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Camundongos , Ovário , Pentostatina , RatosRESUMO
Several approaches aimed at detecting and cloning interactive transcriptional regulatory proteins have been presented. All of the techniques can effectively identify specific interactions between two transcription proteins. However, interaction cloning and the two hybrid system have the added advantage of yielding a cDNA expression clone directly. The other methods, EMSA-mediated cloning, co-immunoprecipitation, oligonucleotide/PCR-facilitated cloning, Southwestern, and Farwestern, require additional manipulations to obtain a cDNA clone. Clearly, the interactive cloning system of choice will depend on the proteins under investigation.
Assuntos
Clonagem Molecular/métodos , DNA/genética , Fatores de Transcrição/genéticaRESUMO
Deoxycoformycin-resistant rat hepatoma cells exhibit up to a 2000-fold increase in adenosine deaminase activity compared to the sensitive parental cells. The increased enzyme activity in these cells is accompanied by similar increases in 1) the amount of adenosine deaminase protein, 2) the relative rate of adenosine deaminase synthesis in vivo, and 3) adenosine deaminase mRNA activity. To further investigate the mechanism(s) responsible for the overproduction of adenosine deaminase in these cells, we have isolated a recombinant plasmid containing a 1.4-kilobase insert complementary to at least part of the adenosine deaminase mRNA. Using this cDNA as a specific hybridization probe, all deoxycoformycin-resistant variants were shown to have increased amounts of adenosine deaminase mRNA and gene sequences. The relative increase in the level of mRNA and gene copy number was similar to the relative increase in enzyme activity for most resistant cell lines. However, the degree of adenosine deaminase gene amplification in one deoxycoformycin-resistant cell line (6-10-200) was 3-4-fold less than the relative increase in adenosine deaminase mRNA. These results indicate that the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells is due in large part, but not exclusively, to gene amplification.
Assuntos
Adenosina Desaminase/genética , Clonagem Molecular , Coformicina/farmacologia , Amplificação de Genes , Genes , Neoplasias Hepáticas Experimentais/enzimologia , Nucleosídeo Desaminases/genética , Ribonucleosídeos/farmacologia , Adenosina Desaminase/isolamento & purificação , Animais , Sequência de Bases , Linhagem Celular , Coformicina/análogos & derivados , DNA/metabolismo , Resistência a Medicamentos , Hibridização de Ácido Nucleico , Pentostatina , RatosRESUMO
Deoxycoformycin-resistant rat hepatoma cells exhibit up to 300-fold increase in adenosine deaminase activity compared to the sensitive parental cells. In order to determine the basis of the increased enzyme activity in deoxycoformycin-resistant cells, adenosine deaminase was purified from rat liver and deoxycoformycin-sensitive and -resistant cells. Physical, kinetic, and immunological properties of the purified enzymes were compared. Purified adenosine deaminase from all sources was found to be a monomer with an Mr approximately 45,000. In addition, the purified enzymes had a similar isozyme pattern in nondenaturing polyacrylamide gels. Km values for adenosine and Ki values for deoxycoformycin did not differ among the purified enzymes. By double diffusion analysis and quantitative immunoprecipitation, the purified enzymes were found to be immunologically indistinguishable. These data indicate that deoxycoformycin-resistant rat hepatoma cells produce increased amounts of adenosine deaminase protein which results in increased enzymatic activity.
Assuntos
Adenosina Desaminase/metabolismo , Coformicina/farmacologia , Neoplasias Hepáticas Experimentais/enzimologia , Nucleosídeo Desaminases/metabolismo , Ribonucleosídeos/farmacologia , Adenosina Desaminase/isolamento & purificação , Inibidores de Adenosina Desaminase , Animais , Coformicina/análogos & derivados , Resistência a Medicamentos , Imunodifusão , Cinética , Fígado/enzimologia , Peso Molecular , Pentostatina , RatosRESUMO
The basis for the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells was investigated. Three variant cell lines with different levels of adenosine deaminase activity showed increases in the relative rate of synthesis of the enzyme in vivo. No difference in the rate of degradation of the enzyme was seen between the parental cell line and one variant cell line which exhibits a 180-fold increase in adenosine deaminase activity. Polysomal RNA isolated from this variant exhibited a 175-fold increase in the ability to direct the synthesis of adenosine deaminase in vitro.
Assuntos
Adenosina Desaminase/genética , Coformicina/farmacologia , Neoplasias Hepáticas Experimentais/enzimologia , Nucleosídeo Desaminases/genética , RNA Mensageiro/genética , Ribonucleosídeos/farmacologia , Animais , Linhagem Celular , Coformicina/análogos & derivados , Resistência a Medicamentos , Cinética , Pentostatina , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , RNA Ribossômico/genética , RatosRESUMO
Deoxycoformycin (dCF) is a specific inhibitor of adenosine deaminase (ADA). Rat hepatoma cells deficient in adenosine kinase and growing on adenosine as the sole carbon source are sensitive to the lethal action of dCF. Mutants resistant to dCF arise spontaneously with a frequency of 1.7 x 10(-6). This frequency is increased to 2.6 x 10(-5) by prior mutagenesis with ethyl methane sulfonate. Initially, dCF-resistant cell lines have 3-10 times the level of adenosine deaminase when compared to sensitive parental cells. Subsequent selection of mutants resistant to increased concentrations of dCF results in cells with a 15- to 30-fold increase in ADA levels. Quantitative immunoprecipitation tests indicate that the increase in enzyme activity in one line tested is due to an increase in the number of ADA molecules. These dCF' cell lines may serve as a model system to study the human disease state, hereditary hemolytic anemia, which is associated with increased levels of ADA.
Assuntos
Adenosina Desaminase/genética , Separação Celular/métodos , Coformicina/farmacologia , Resistência a Medicamentos , Nucleosídeo Desaminases/genética , Ribonucleosídeos/farmacologia , Adenosina Desaminase/metabolismo , Animais , Linhagem Celular , Coformicina/análogos & derivados , Cinética , Neoplasias Hepáticas Experimentais , Mutação , Pentostatina , RatosRESUMO
T-lymphocyte movement out of the bloodstream and into tissue is critical to the success of these cells in their role in immunosurveillance. This process involves interactions of the T-cell with endothelium as well as with extracellular matrix. Central to these interactions are a number of T-cell adhesion molecules and their endothelial and extracellular matrix ligands. The identification and functional characterization of adhesion molecules have been the subject of intensive research in recent years. We highlight here the latest developments in this rapidly expanding field as they pertain to T-cell interactions with endothelial cells and extracellular matrix components, including: (1) identification of adhesion molecule families, including the selectins, mucins, integrins, immunoglobulin superfamily members, and cadherins; (2) elucidation of the multi-step adhesion cascade that mediates the rolling, arrest, and eventual diapedesis of T-cells through the vascular endothelium into the surrounding tissue; (3) the changes in adhesion molecule expression that accompany T-cell maturation and activation, and the impact of those changes on T-cell migration; (4) the functional relevance of the extracellular matrix for T-cell function; and (5) the clinical relevance of adhesion molecules and the potential for targeting these molecules for the amelioration of immune-mediated diseases.
Assuntos
Moléculas de Adesão Celular/imunologia , Adesão Celular/imunologia , Endotélio Vascular/imunologia , Matriz Extracelular/imunologia , Linfócitos T/imunologia , Animais , Doenças Autoimunes/imunologia , Quimiotaxia de Leucócito , Endotélio Vascular/citologia , Matriz Extracelular/enzimologia , Humanos , Memória Imunológica , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T , Transdução de SinaisRESUMO
The beta1 integrin adhesion receptors activate signal transduction pathways that induce tyrosine phosphorylation of a variety of substrates. Increased tyrosine phosphorylation is mediated by the beta1 subunit cytoplasmic domain, which consists of 46 amino acids and contains no intrinsic kinase activity. In the H9 T cell line, beta1 integrin engagement leads to the increased tyrosine phosphorylation of three 105 to 115-kDa substrates that are distinct from focal adhesion kinase (FAK): HEF1 (human enhancer of filamentation 1), a protein with structural homology to p130Cas, and two novel substrates, pp105 and pp115. DNA-mediated gene transfer was used to explore the role of the beta1 cytoplasmic domain in integrin-mediated tyrosine phosphorylation of HEF1, pp105, and pp115 in human T cells. Using a chimeric receptor composed of the cytoplasmic domain of the beta1 integrin subunit and the extracellular and transmembrane domains of the CD2 Ag, we demonstrate that the beta1 cytoplasmic domain is necessary and sufficient for inducing tyrosine phosphorylation of each of these three substrates in H9 T cells. Analysis of a series of beta1 cytoplasmic domain truncations reveals that a truncation of only five amino acids from the carboxyl-terminal end of the beta1 cytoplasmic domain abrogates the ability of the CD2/beta1 chimera to activate tyrosine phosphorylation of HEF1, pp105, or pp115. Thus, the carboxyl-terminal five amino acids, Lys-Tyr-Glu-Gly-Lys (KYEGK), of the beta1 integrin cytoplasmic domain are critical for the coordinate tyrosine phosphorylation of three non-FAK substrates in human T cells.