RESUMO
The establishment of a stable endothelial layer on a biomaterial suture is a well known strategy to achieve hemocompatibility. The endothelialisation is supported by factors as e.g. vascular endothelial growth factor (VEGF), which can be secreted by monocytes/macrophages (mo/mΦ) in an angiogenic milieu. In order to avoid detrimental inflammation triggered by these mo/mΦ, we established a protocol for the generation of alternatively activated macrophages and studied their response towards VEGF-A165. We could generate sufficient amounts of these CD14+ CD163+ IL-10+ mo/mΦ from buffy coats(8.6 ± 4.7 × 105 cells/mlbuffy coat). Furthermore, we achieved a VEGF-A165 secretion in the nanogram range. The VEGF-A165 secretion increased 2.1-fold within 14 days from 7.6 ± 2.2 to 16.1 ± 2.5ng/ml when the cells were grown with a VEGF-A165 supplemented (10ng/ml) cell culture medium. Within this time period the secretion levels of other pro-angiogenic growth factors (bFGF, PDGF-BB) and immunomodulatory cytokines (IL-10, IL-12, IL-1ra, TNFα, IFNγ) reached only the picogram range. These results suggest that angiogenically stimulated CD14+ CD163+ IL-10+ mo/mΦ might be useful as a cellular cytokine delivery system supporting endothelialisation of biomaterials without inducing pro-inflammatory effects.
Assuntos
Indutores da Angiogênese/farmacologia , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Interleucina-10/imunologia , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Fragmentos de Peptídeos/farmacologia , Receptores de Superfície Celular/imunologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Indutores da Angiogênese/imunologia , Separação Celular , Células Cultivadas , Humanos , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Fragmentos de Peptídeos/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Interleukin (IL)-10 is one of the most crucial immunoregulatory cytokines. Its short-term effects have been analysed extensively, but little is known about its long-term effects. This is of considerable importance, as high systemic IL-10 levels are present for long periods in patients with persistent viral infections, certain cancers and in critical care patients. Our study investigated the effects of the long-term presence of IL-10 on human peripheral blood monocytes. In vitro, IL-10 treatment of these cells for 7 days induced the development of a novel cell type characterized by unique phenotypical and functional characteristics. These cells showed high HLA-DR expression and low expression of CD86 and other co-stimulatory molecules on their surface. The mRNA levels of both HLA-DR and CD86 were high, but no intracellular accumulation of CD86 protein was observed. With respect to its function, these cells showed strongly diminished tumour necrosis factor-alpha production following lipopolysaccharide stimulation, strongly diminished allogenic CD4(+) T cell stimulatory capacity, and even induced a hyporesponsive state in CD4(+) T cells. The phenotype remained stable despite the removal of IL-10. In vivo, we found monocytic cells from patients exhibiting this phenotype after long-term IL-10 exposure. These results complement our knowledge further about the biological effects of IL-10 and may provide an explanation for the sustained immunodeficiency in cases of the persistent presence of systemic IL-10.
Assuntos
Interleucina-10/imunologia , Monócitos/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Imunofenotipagem , Interleucina-10/uso terapêutico , Isoantígenos/imunologia , Ativação Linfocitária/imunologia , Psoríase/tratamento farmacológico , Psoríase/imunologia , RNA Mensageiro/genética , Fator de Necrose Tumoral alfaRESUMO
The exposition of monocytes to lipopolysaccharide (LPS) primarily causes a massive inflammatory response that is then followed by a hyporesponsive state of these cells. This latter state is called endotoxin tolerance and is characterized by (i) the attenuated production of proinflammatory mediators after repeated LPS treatment, and (ii) the diminished antigen presentation and T-cell stimulation capacity. The data presented here indicate that LPS priming causes a specific decrease in the expression of legumain (the asparaginyl endopeptidase responsible for the key step in antigen processing) in monocytes. In these cells, the fraction of major histocompatibility complex (MHC) class II loaded with CLIP was increased. In contrast to monocytes, LPS priming provoked an increase of legumain expression in B cells. Reduced monocytic expression of legumain was also found in critically ill patients supporting the suitability of endotoxin tolerance as an experimental model of clinical postinflammatory immunodeficiency.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Cisteína Endopeptidases/biossíntese , Endotoxinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Monócitos/enzimologia , Apresentação de Antígeno/genética , Apresentação de Antígeno/fisiologia , Linfócitos B/enzimologia , Células Cultivadas , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica/fisiologia , Genes MHC da Classe II/fisiologia , HumanosRESUMO
The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.
Assuntos
Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Morfogênese , Mutação , Proteínas Oncogênicas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Marcação de Genes , Genótipo , Humanos , Proteínas com Domínio LIM , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Alinhamento de SequênciaRESUMO
LIM-only (LMO) proteins are transcription regulators that function by mediating protein-protein interaction and include the T cell oncogenes encoding LMO1 and LMO2. The oncogenic functions of LMO1 and LMO2 are thought to be mediated by interaction with LDB1 since they form a multimeric protein complex(es). A new member of the Lmo family, Lmo4, has also recently been identified via its interaction with Ldb1. Sequence analysis of the mouse Lmo4 gene shows that it spans about 18 kb and consists of at least six exons, including two alternatively spliced 5' exons. Unlike Lmo1, the two 5' exons of Lmo4 do not encode protein. Comparison of the Lmo4 gene structure with the other LMO family members shows the exon structure of Lmo4 differs in the position of exon junctions encoding the second LIM domain and in a novel exon-intron junction at the penultimate codon of the gene. Lmo4 is thus the least conserved known member of the LIM-only family in both nucleotide sequence and exon structure. Physical mapping of the Lmo4/LMO4 genes has shown mouse Lmo4 is located on Chromosome (Chr) 3 and human LMO4 on Chr 1p22.3. This chromosome location is of interest as it occurs in a region that is deleted in a number of human cancers, indicating a possible role of LMO4 in tumorigenesis, like its relatives LMO1 and LMO2.
Assuntos
Cromossomos Humanos Par 1 , Proteínas de Homeodomínio/genética , Proteínas Oncogênicas , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Éxons/genética , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Íntrons/genética , Proteínas com Domínio LIM , Metaloproteínas/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Muridae , Proteínas Nucleares , Proteínas Proto-Oncogênicas , Alinhamento de SequênciaRESUMO
Two general features have emerged about genes that are activated after chromosomal translocations in acute forms of cancer. The protein products of these genes are transcription regulators and are involved in developmental processes, and it seems that the subversion of these normal functions accounts for their role in tumorigenesis. The features of the LMO family of genes, which encode LIM-domain proteins involved in T-cell acute leukemia through chromosomal translocations, typify these abnormal functions in tumorigenesis. For example, the LMO2 protein is involved in the formation of multimeric DNA-binding complexes, which may vary in composition at different stages of hematopoiesis and function to control differentiation of specific lineages. In T cells, enforced expression of Lmo2 causes aberrant protein complex formation that primarily seems to hinder the T-cell differentiation program. These observations underscore the conclusion that protein-protein interaction (in this case, through the LIM domain) is a key determinant in tumorigenesis. Furthermore, the study of chromosomal translocations as naturally occurring mutations has been informative about mechanisms in hematopoiesis as well as in tumor etiology.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Leucemia Experimental/genética , Metaloproteínas/genética , Transcrição Gênica , Translocação Genética , Doença Aguda , Proteínas Adaptadoras de Transdução de Sinal , Animais , Hematopoese/fisiologia , Proteínas com Domínio LIM , Leucemia-Linfoma de Células T do Adulto/genética , Camundongos , Família MultigênicaRESUMO
The T cell oncogenes LMO1 and LMO2 are activated by distinct chromosomal translocations in childhood T cell acute leukaemias. Transgenic mouse models of this disease demonstrate that enforced expression of Lmo1 and Lmo2 cause T cell leukaemias with long latency and that Lmo2 expression leads to an inhibition of the T cell differentiation programme, prior to overt disease. These functions appear to be partly mediated by interaction of LMO1 or LMO2 with the LIM-binding protein LDB1/ NLI1. We have now identified a new member of the Lmo family, designated Lmo4, via its interaction with Ldb1. Lmo4 is widely expressed in mouse tissues, including adult thymus (mainly CD4, CD8-double positive T cells) and embryonic thymus (mainly CD4, CD8-double negative T cells). These characteristics imply that Ldb1-Lmo4 interaction may function in the T cell developmental programme and that enforced expression of LMO1 or LMO2 by chromosomal translocations or transgenesis may displace Lmo4 from this complex and thereby influence T cell differentiation prior to T cell tumour occurrence.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Metaloproteínas/fisiologia , Família Multigênica , Proteínas Oncogênicas , Subpopulações de Linfócitos T/metabolismo , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes , Proteínas de Homeodomínio/fisiologia , Proteínas com Domínio LIM , Metaloproteínas/genética , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares , Especificidade de Órgãos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Timo/embriologia , Timo/crescimento & desenvolvimento , Timo/metabolismo , Fatores de Transcrição/fisiologia , Translocação GenéticaRESUMO
The LIM-only protein LMO2 is expressed aberrantly in acute T-cell leukaemias as a result of the chromosomal translocations t(11;14) (p13;q11) or t(7;11) (q35;p13). In a transgenic model of tumorigenesis by Lmo2, T-cell acute leukaemias arise after an asymptomatic phase in which an accumulation of immature CD4(-) CD8(-) double negative thymocytes occurs. Possible molecular mechanisms underlying these effects have been investigated in T cells from Lmo2 transgenic mice. Isolation of DNA-binding sites by CASTing and band shift assays demonstrates the presence of an oligomeric complex involving Lmo2 which can bind to a bipartite DNA motif comprising two E-box sequences approximately 10 bp apart, which is distinct from that found in erythroid cells. This complex occurs in T-cell tumours and it is restricted to the immature CD4(- )CD8(-) thymocyte subset in asymptomatic transgenic mice. Thus, ectopic expression of Lmo2 by transgenesis, or by chromosomal translocations in humans, may result in the aberrant protein interactions causing abnormal regulation of gene expression, resulting in a blockage of T-cell differentiation and providing precursor cells for overt tumour formation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Metaloproteínas/metabolismo , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/imunologia , Soros Imunes , Proteínas com Domínio LIM , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Metaloproteínas/imunologia , Camundongos , Camundongos Transgênicos , Oligodesoxirribonucleotídeos , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukaemias, is normally expressed in haematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the interaction is unexplained. We now show that in erythroid cells Lmo2 forms a novel DNA-binding complex, with GATA-1, TAL1 and E2A, and the recently identified LIM-binding protein Ldb1/NLI. This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box, CAGGTG, followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding complex which may play a role in haematopoiesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Hematopoese/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Células COS , Sequência Consenso , Fatores de Ligação de DNA Eritroide Específicos , Genes Reporter , Proteínas com Domínio LIM , Metaloproteínas/metabolismo , Modelos Genéticos , Proteínas Nucleares/metabolismo , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , TransfecçãoRESUMO
The LMO2 gene associated with T cell acute leukaemia has been used as an example of a gene activated by association with the T cell receptor genes after chromosomal translocations. The gene is shown to encode a LIM protein which is involved in protein interactions and during normal haematopoiesis is necessary for erythroid development. LMO2 has been shown to cause tumours when aberrantly expressed and to be able to heterodimerise with TAL1 to facilitate tumour development.
Assuntos
Proteínas de Ligação a DNA/genética , Eritropoese/genética , Leucemia-Linfoma de Células T do Adulto/genética , Metaloproteínas/genética , Proto-Oncogenes , Transcrição Gênica , Translocação Genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cromossomos Humanos Par 11 , Proteínas de Ligação a DNA/biossíntese , Hematopoese/genética , Humanos , Proteínas com Domínio LIM , Leucemia-Linfoma de Células T do Adulto/fisiopatologia , Metaloproteínas/biossíntese , Camundongos , Camundongos Transgênicos , Proteínas Proto-OncogênicasRESUMO
The LIM-only protein Lmo2, originally identified as an oncogenic protein in human T cell leukemia, is essential for erythropoiesis. A possible role for Lmo2 in transcription during erythropoiesis has been investigated. Direct interaction of Lmo2 was observed in vitro and in vivo with the zinc finger transcription factor GATA-1, as well as with the basic helix-loop-helix (bHLH) transcription factor Tall. By using mammalian two-hybrid analysis, E47/Tall/Lmo2/GATA-1 protein complex could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis. This data suggest that variations in amounts of complexes involving Lmo2, Tall, and GATA-1 could be important for erythroid differentiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Eritropoese , Metaloproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células COS , Proteínas de Ligação a DNA/isolamento & purificação , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Sequências Hélice-Alça-Hélice , Humanos , Proteínas com Domínio LIM , Leucemia Eritroblástica Aguda , Leucemia de Células T/metabolismo , Metaloproteínas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATA1 or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Eritropoese , Proteínas de Homeodomínio/metabolismo , Metaloproteínas/metabolismo , Proteínas Proto-Oncogênicas , Fatores de Transcrição/metabolismo , Dedos de Zinco , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Sequências Hélice-Alça-Hélice , Proteínas com Domínio LIM , Leucemia Eritroblástica Aguda , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Células Tumorais CultivadasRESUMO
The octanucleotide recognition site for the endonuclease SwaI was introduced into the Escherichia coli bicistronic expression vector pTiSDT by mutating a single position in the coupling SD sequence between a truncated form of the cro-gene and the multicloning site. This mutation does not influence the expression rate. The introduction of this restriction site allows high level production of proteins, that are modified only by an N-terminal methionine incorporated as the start codon.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Vetores Genéticos , Oligonucleotídeos/metabolismo , Proteínas/genética , Sequência de Bases , Clonagem Molecular , Códon , Escherichia coli/genética , Dados de Sequência Molecular , Biossíntese de ProteínasRESUMO
BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-abl protein-tyrosine kinase gene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, p210BCR-ABL and p190BCR-ABL, are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. Transformed murine hematopoietic cells can be used as a model of BCR-ABL function since these cells can be made growth factor independent and tumorigenic by the action of the BCR-ABL oncogene. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. Furthermore, BCR-ABL-expressing cells revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These results help to explain the ability of BCR-ABL oncogenes to synergize with c-myc in cell transformation.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Fusão bcr-abl/genética , Oncogenes , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Apoptose/genética , Linhagem Celular , Humanos , Interleucina-3/farmacologia , Masculino , Camundongos , Camundongos Nus , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2 , TransfecçãoRESUMO
Peptides derived from the CDRs of the anti-TNF alpha monoclonal antibody Di62 were tested for inhibition of binding of Di62 to TNF alpha as well as of TNF alpha to its 55 and 75 kDa receptor. A peptide derived from the CDR1 of the light chain was shown to specifically inhibit Di62 binding to TNF alpha with markedly higher activity (Ki = 4 microM) than all other CDR-derived peptides. This peptide also significantly inhibited binding of TNF alpha to its 55 and 75 kDa receptor and protected L929 cells from the cytotoxic effect of TNF alpha (IC50 = 6 microM). The C-terminal region of this peptide, which is homologous to the 55 and 75 kDa TNF receptor, was found to be essential for activity.
Assuntos
Anticorpos Monoclonais/imunologia , Região Variável de Imunoglobulina/imunologia , Fator de Necrose Tumoral alfa/imunologia , Sequência de Aminoácidos , Animais , Ligação Competitiva/imunologia , Testes Imunológicos de Citotoxicidade , Ensaio de Imunoadsorção Enzimática , Células L , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Radioimunoensaio , Receptores do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Among a panel of nearly 3,000 IgM-producing hybridomas obtained from 22 independent fusions of human fetal lymphocytes (liver/spleen; 15th-36th gestational week) a high number (5-10%) produced autoantibodies, independently of the gestational age. A significant portion of these autoantibodies was found to be polyreactive, i.e. capable of binding to more than two antigens, when tested against a set of five antigens of the internal (ssDNA, thrombocytes, keratin) and external (lipid A, tetanus toxoid) environment. Analyzing the IgVH genes utilized in eight polyreactive and two putatively nonpolyreactive hybridomas, members of the VHI, III, IV and VI families were found once, seven times, once and once, respectively, mostly with germline identity. All but one of the utilized gene elements could be related to the biased VH gene repertoire said to be expressed during the early ontogeny of the human immune system. We also noted a bias for the utilization of DN1 (3/10), DHQ52 (3/10), JH2 (4/10) and JH6 (4/10) elements, whereas all heavy-chain CDR3 regions manifest a diversity by addition of N nucleotides and/or exonuclease activity on coding segments. In addition, VL segments which belong to different subgroups of both isotypes were found to be used. The molecular basis of polyreactive immunoglobulin specificities in human fetuses is discussed.
Assuntos
Linfócitos B/imunologia , Desenvolvimento Embrionário e Fetal/imunologia , Região Variável de Imunoglobulina/genética , Anticorpos Biespecíficos/biossíntese , Sequência de Bases , Southern Blotting , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/biossíntese , Dados de Sequência MolecularRESUMO
An ELISA is described for the detection of HIV-1 protease activity using an immobilized gag-related polyprotein as substrate. Proteolytic activity was demonstrated with either bacterial lysates expressing HIV-1 protease or purified protease. No cleavage was observed with a protein preparation from control bacteria not expressing HIV-1 protease. Under these conditions the aspartyl-type protease inhibitor, pepstatin A, was found to inhibit HIV-1 protease cleavage by > 90% at a concentration of 0.1 mM. This assay may be a useful tool for the study of both synthetic and natural inhibitors of HIV-1 protease.