RESUMO
Mitochondria are organelles known primarily for generating ATP via the oxidative phosphorylation process. Environmental signals are sensed by whole organisms or cells and markedly affect this process, leading to alterations in gene transcription and, consequently, changes in mitochondrial function and biogenesis. The expression of mitochondrial genes is finely regulated by nuclear transcription factors, including nuclear receptors and their coregulators. Among the best-known coregulators is the nuclear receptor corepressor 1 (NCoR1). Muscle-specific knockout of NCoR1 in mice induces an oxidative phenotype, improving glucose and fatty acid metabolism. However, the mechanism by which NCoR1 is regulated remains elusive. In this work, we identified the poly(A)-binding protein 4 (PABPC4) as a new NCoR1 interactor. Unexpectedly, we found that silencing of PABPC4 induced an oxidative phenotype in both C2C12 and MEF cells, as indicated by increased oxygen consumption, mitochondria content, and reduced lactate production. Mechanistically, we demonstrated that PABPC4 silencing increased the ubiquitination and consequent degradation of NCoR1, leading to the derepression of PPAR-regulated genes. As a consequence, cells with PABPC4 silencing had a greater capacity to metabolize lipids, reduced intracellular lipid droplets, and reduced cell death. Interestingly, in conditions known to induce mitochondrial function and biogenesis, both mRNA expression and PABPC4 protein content were markedly reduced. Our study, therefore, suggests that the lowering of PABPC4 expression may represent an adaptive event required to induce mitochondrial activity in response to metabolic stress in skeletal muscle cells. As such, the NCoR1-PABPC4 interface might be a new road to the treatment of metabolic diseases.
Assuntos
Receptores Citoplasmáticos e Nucleares , Fatores de Transcrição , Animais , Camundongos , Proteínas Correpressoras/metabolismo , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Fosforilação Oxidativa , Receptores Citoplasmáticos e Nucleares/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismoRESUMO
The human proteins FEZ1 (fasciculation and elongation protein zeta 1) and FEZ2 are orthologs of the protein UNC-76 from C. elegans, involved in the growth and fasciculation of the worms axon. Pull down assays showed that the protein FEZ1 interacts with other proteins (e.g., the protein SCOCO, short coiled-coil protein), mitochondria, and vesicles. These components may therefore represent cargoes to be transported along the microtubule, and the transport may be mediated through FEZ1 reported binding to kinesins (KIF3A). We previously showed that FEZ1 dimerizes in its N-terminal region and interacts with other proteins, including the candidate cargoe proteins, through its C-terminus. Here, we studied the fragment FEZ1(92-194) as well as full-length 6xHis-FEZ1 (1-392) in vitro and endogenous FEZ1 isolated from HEK 293 cells and were able to demonstrate the formation of an intermolecular disulfide bond through FEZ1 Cys-133, which appears to be essential for dimerization. This disulfide bond may be important for the FEZ1 role as a dimeric and bivalent transport adaptor molecule, since it establishes a strong link between the monomers, which could be a prerequisite for the simultaneous binding of two cargoes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Dissulfetos/química , Proteínas do Tecido Nervoso/química , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Sequência Conservada , Dissulfetos/metabolismo , Humanos , Espectrometria de Massas , Modelos Moleculares , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
In mouse pregnancy, pubic symphysis (PS) remodels into an elastic interpubic ligament (IpL) in a temporally regulated process to provide safe delivery. It restores at postpartum to assure reproductive tract homeostasis. Recently, macrophage localization in the IpL and dynamic changes in the expression of inflammatory mediators observed from the end of pregnancy (D18, D19) to early days postpartum (1dpp, 3dpp) highlighted the necessity of the identification of the key molecules involved in innate immune processes in PS remodeling. Therefore, this study uses morphological and high-sensitivity molecular techniques to identify both macrophage association with extracellular matrix (ECM) remodeling and the immunological processes involved in PS changes from D18 to 3dpp. Results showed macrophage association with active gelatinases and ECM components and 25 differentially expressed genes (DEGs) related to macrophage activities in interpubic tissues from D18 to 3dpp. Additionally, microarray and proteomic analysis showed a significant association of interpubic tissue DEGs with complement system activation and differentially expressed proteins (DEPs) with phagocytosis, highlighting the involvement of macrophage-related activities in mouse PS remodeling. Therefore, the findings suggest that PS ECM remodeling is associated with evidence of macrophage modulation that ensures both IpL relaxation and fast PS recovery postpartum for first labor.
Assuntos
Remodelação Óssea/imunologia , Macrófagos/citologia , Período Pós-Parto/fisiologia , Sínfise Pubiana/fisiologia , Animais , Matriz Extracelular/metabolismo , Feminino , Imunidade Inata , Camundongos , Período Pós-Parto/imunologia , Gravidez , Sínfise Pubiana/citologiaRESUMO
Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Centrossomo/metabolismo , Cílios/genética , Cílios/metabolismo , Sequência Conservada , Humanos , Modelos Moleculares , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/química , Miosina Tipo V/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas RecombinantesRESUMO
The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.
Assuntos
Antígenos da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Transativadores/metabolismo , Sítios de Ligação , Ligação Competitiva , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Enteropeptidase/metabolismo , Escherichia coli , Expressão Gênica , Vetores Genéticos , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/isolamento & purificação , Vírus da Hepatite B/genética , Hepatite B Crônica/epidemiologia , Ribonucleoproteína Nuclear Heterogênea D0 , Reação em Cadeia da Polimerase , Ligação Proteica , Sondas RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Transativadores/genética , Transativadores/isolamento & purificação , Proteínas Virais Reguladoras e AcessóriasRESUMO
In the last decade, imaging mass spectrometry has seen incredible technological advances in its applications to biological samples. One computational method of data mining in this field is the spatial segmentation of a sample, which produces a segmentation map highlighting chemically similar regions. An important issue for any imaging mass spectrometry technology is its relatively low spatial or lateral resolution (i.e. a large size of pixel) as compared with microscopy. Thus, the spatial resolution of a segmentation map is also relatively low, that complicates its visual examination and interpretation when compared with microscopy data, as well as reduces the accuracy of any automated comparison. We address this issue by proposing an approach to improve the spatial resolution of a segmentation map. Given a segmentation map, our method magnifies it up to some factor, producing a super-resolution segmentation map. The super-resolution map can be overlaid and compared with a high-res microscopy image. The proposed method is based on recent advances in image processing and smoothes the "pixilated" region boundaries while preserving fine details. Moreover, it neither eliminates nor splits any region. We evaluated the proposed super-resolution segmentation approach on three MALDI-imaging datasets of human tissue sections and demonstrated the superiority of the super-segmentation maps over standard segmentation maps.
Assuntos
Neoplasias do Colo/patologia , Mucosa Gástrica/patologia , Processamento de Imagem Assistida por Computador/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias do Colo/cirurgia , Neoplasias do Colo/ultraestrutura , Apresentação de Dados , Mucosa Gástrica/cirurgia , Mucosa Gástrica/ultraestrutura , Humanos , Microscopia/métodos , Reconhecimento Automatizado de Padrão/métodos , Sensibilidade e EspecificidadeRESUMO
In previous work, we generated four IgM, five IgG1, and one IgA1 mAbs to rabies virus using B cells from four subjects vaccinated with inactivated rabies virus, a thymus-dependent (TD) mosaic Ag, and sequenced the mAb V(H)DJ(H) genes. Here, we have cloned the V kappa J kappa and V lambda J lambda genes to complete the primary structure of the Ag-binding site of these mAbs. While the anti-rabies virus mAb selection of VA genes (2e.2.2 twice, DPL11, and DPL23) reflected the representation of the V lambda genes in the human haploid genome (stochastic utilization), that of V kappa genes (O2/O12 twice, O8/O18, A3/A19, A27, and L2) did not (p = 0.0018) (nonstochastic utilization). Furthermore, the selection of both V kappa and V lambda genes by the anti-rabies virus mAbs vastly overlapped with that of 557 assorted V kappa J kappa rearrangements, that of 253 V lambda J lambda rearrangements in lambda-type gammopathies, and that of other Abs to thymus-dependent Ags, including 23 anti-HIV mAbs and 51 rheumatoid factors, but differed from that of 43 Abs to Haemophilus influenzae type b polysaccharide, a prototypic thymus-independent (TI) Ag. The anti-rabies virus mAb V kappa J kappa and V lambda J lambda segments displayed variable numbers of somatic mutations, which, in mAb58 and the virus-neutralizing mAb57, entailed a significant concentration of amino acid replacements in the complementarity-determining regions (p = 0.0028 and p = 0.0023, respectively), suggesting a selection by Ag. This Ag-dependent somatic selection process was superimposed on a somatic diversification process that occurred at the stage of B cell receptor for Ag rearrangement, and that entailed V gene 3' truncation and N nucleotide additions to yield heterogeneous CDR3s.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Rearranjo Gênico de Cadeia Leve de Linfócito B , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Mutação , Vírus da Raiva/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/química , Diversidade de Anticorpos/genética , Sequência de Bases , Células Clonais , Clonagem Molecular , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Haemophilus influenzae tipo b/imunologia , Humanos , Imunoglobulina A/biossíntese , Imunoglobulina A/química , Imunoglobulina A/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Região de Junção de Imunoglobulinas/biossíntese , Região de Junção de Imunoglobulinas/química , Região de Junção de Imunoglobulinas/genética , Imunoglobulina M/biossíntese , Imunoglobulina M/química , Imunoglobulina M/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/química , Dados de Sequência Molecular , Paraproteinemias/genética , Paraproteinemias/imunologia , Fragmentos de Peptídeos/genética , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/imunologia , Fator Reumatoide/química , Fator Reumatoide/genéticaRESUMO
The activation marker CD30 is expressed on the cell surface of the malignant cells in Hodgkin's disease and a few non-Hodgkin lymphomas. We have analyzed the regulation of membrane-bound CD30 and found that the binding of a variety of anti-CD30 antibodies induced down-regulation of CD30 on cell lines. In addition, such down-modulation was also observed after treatment of the cell surface proteins with the sulfhydryl reagent iodoacetamide or after stimulation of the second messenger pathway with phorbol ester or calcium ionophore. This modulation was abolished at 4 degrees C and strongly inhibited by chelators like EDTA or 1,10-phenanthroline, whereas EGTA, a selective inhibitor of Ca(2+)-dependent proteinases and other inhibitors of serine, thiol and acid proteinases, showed no effect. The down-modulation was strengthened by Zn2+ or Cd2+, but not by other divalent cations such as Fe2+, Mn2+, Mg2+, Ca2+ or Co2+, thus indicating the involvement of a zinc metalloproteinase in CD30 modulation which can be activated by protein kinase C and by alkylation of sulfhydryl groups. Pulse-chase experiments, analysis of the CD30 glycosylation and specific measurement of the 90-kDa soluble form of CD30 (sCD30) with a sandwich radioimmunoassay revealed that CD30 down-modulation results from enhanced release of 90-kDa sCD30 by the site-specific cleavage of CD30 accomplished by a zinc metalloproteinase. This release occurs at the cell membrane without prior endocytosis.
Assuntos
Doença de Hodgkin/metabolismo , Antígeno Ki-1/metabolismo , Linfoma não Hodgkin/metabolismo , Metaloendopeptidases/metabolismo , Anticorpos Monoclonais/farmacologia , Calcimicina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Glicosilação , Doença de Hodgkin/enzimologia , Humanos , Iodoacetamida/farmacologia , Ionóforos/farmacologia , Cinética , Linfoma não Hodgkin/enzimologia , Solubilidade , Reagentes de Sulfidrila/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant delta 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca2+ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca2+ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Cálcio/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/química , Antígenos de Diferenciação de Linfócitos T/genética , Técnicas de Cultura de Células , Citoplasma/química , Humanos , Isomerismo , Células Jurkat , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Tirosina/genéticaRESUMO
The early phases of ontogeny are decisive for the development of the B-cell repertoire. Here, we demonstrate that maternal tertiary immunization of BALB/c mice with 2-phenyloxazolone (phOx) caused a drastic alteration of the primary antigen-specific repertoire of the offspring. Maternal tertiary immunization or quaternary antibodies, which exhibited an extremely weak cross-reactivity with the major Ox1 idiotype (IdOx1), induced a change in the proportion of IdOx1/non-IdOx1 antiphOx antibodies in the F1 and F2 primary repertoire. The observed variability in the level of IdOx1 expression (10-90%) exceeded even the seemingly genetically based differences between various mouse strains. In comparison with the non-IdOx1 of control mice, half of the non-IdOx1 antibodies showed a 5-100-fold enhanced affinity. Sixty per cent of these antibodies exhibited an affinity identical to that of IdOx1 antibodies, which are normally of the highest affinity, while the remaining 40% exceeded even that of IdOx1 by a factor of 10. The non-IdOx1 were encoded by VH/VL genes and/or combinations thereof which are either new, hitherto unobserved in the antiphOx response, or typical of memory responses in normal mice. The significance of these data is discussed with respect to the possibility that maternal antibodies, which are acquired through multiple immune maturation processes, may have an epigenetic (non-Mendelian) inheritable potential for the offspring.
Assuntos
Anticorpos Anti-Idiotípicos/biossíntese , Afinidade de Anticorpos/genética , Haptenos/imunologia , Troca Materno-Fetal/imunologia , Oxazolona/análogos & derivados , Sequência de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/sangue , Anticorpos Anti-Idiotípicos/genética , Sequência de Bases , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico de Cadeia Leve de Linfócito B , Haptenos/administração & dosagem , Haptenos/genética , Soros Imunes/análise , Imunização , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/sangue , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/sangue , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/sangue , Região Variável de Imunoglobulina/genética , Troca Materno-Fetal/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oxazolona/administração & dosagem , Oxazolona/imunologia , GravidezRESUMO
A novel antigen was identified by the cross-reactivity of the anti-CD30 antibody Ki-1. This 57-kD intracellular Ki-1 antigen (Ki-1/57) is induced upon activation of leukocytes and is transported to the nuclear compartment. We describe the partial cloning and sequencing of the Ki-1/57 cDNA from a lambda gt 11-cDNA library derived from the Hodgkin-analogous cell line L540. New monoclonal antibodies were produced against the recombinant Ki-1/57 protein fragment which were used to confirm that the Ki-1/57 antigen is associated with kinase activity and is expressed in a variety of tumor cell lines and in activated but not resting leukocytes. The Ki-1/57 gene was mapped to the bands 9q22.3-31 of human chromosome 9. This is an area which appears to be associated with secondary chromosomal aberrations in acute myeloid leukemias.
Assuntos
Antígeno Ki-1/genética , Sequência de Aminoácidos , Animais , Baculoviridae , Sequência de Bases , Linhagem Celular , Bandeamento Cromossômico , Cromossomos Humanos Par 9 , Clonagem Molecular , DNA Complementar/química , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Mapeamento por Restrição , SpodopteraRESUMO
Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and characterization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of mALCAM showed that the CD6 binding site is located in the N-terminal Ig-like domain and that mALCAM is capable of binding both human and mouse CD6. Mutagenesis studies on hALCAM suggested that residues critical for CD6 binding map to the predicted A'GFCC'C" beta-sheet of ALCAM's N-terminal binding domain. Residue differences in the N-terminal domains of mALCAM and hALCAM were analyzed with the aid of a molecular model of ALCAM. All residues critical for CD6 binding are conserved in both mALCAM and hALCAM, whereas residue differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale for the observed cross-species CD6/ALCAM interaction and the apparent inability to generate monoclonal antibodies (mAb) against the CD6 binding site. RNA blot analysis showed that mRNA transcripts encoding mALCAM are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two-color immunofluorescence studies was shown to bind to both activated CD4+ and CD8+ T cells.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/química , Glicoproteínas/química , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Molécula de Adesão de Leucócito Ativado , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Antígenos CD/imunologia , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Sequência Conservada , DNA Complementar/química , DNA Complementar/isolamento & purificação , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Células L , Ligantes , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/imunologia , Ligação Proteica/imunologia , Ratos , Especificidade da Espécie , Timo , Células Tumorais CultivadasRESUMO
The CD30-activation marker was detected as the Hodgkin-associated Ki-I antigen and is regarded as a target for the treatment of Hodgkin patients with immunotoxins. The CD30 is released from tumor cells and this soluble CD30 (sCD30) is an indicator of the disease activity. Since the shedding of sCD30 may be influenced by antibodies, we produced 6 new CD30-specific antibodies (Ki-2 to Ki-7) for the purpose of finding antibodies that might inhibit the formation of sCD30. Ki-2 to Ki-7 and the other anti-CD30 antibodies Ki-I, Ber-H2, HeFi-I, M44, M67, HRS-I, HRS-4 and C10 were employed for epitope mapping. The binding of a particular radio-labeled anti-CD30 antibody to Hodgkin's-disease-derived L540 cells was completed by addition of the various non-labeled anti-CD30 antibodies. Three non-overlapping regions, expressing different antigen-specific determinants, could be defined on the extracellular part of the CD30 molecule. Cluster A of determinants was recognized by Ki-2, Ki-4, Ki-6 and Ki-7, Ber-H2, HRS-I and HRS-4, while cluster B was detected by Ki-I, Ki-5 and M67. Cluster C, which probably contains the binding site for the CD30 ligand, was defined by Ki-3, M44, HeFi-I and C10. Co-culture experiments of L540 cells with the various antibodies followed by the isolation of sCD30 from culture supernatant fluids revealed that the release of sCD30 was most strongly increased by Ki-I and weakly enhanced by Ki-2, Ki-3, Ki-5 and HeFi-I, whereas it was almost completely inhibited by Ki-4 and to a slightly lesser extent by Ber-H2.
Assuntos
Anticorpos Monoclonais/biossíntese , Doença de Hodgkin/imunologia , Antígeno Ki-1/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/biossíntese , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Ligação Competitiva , Epitopos , Antígeno Ki-1/química , Antígeno Ki-1/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Solubilidade , Células Tumorais Cultivadas/imunologiaRESUMO
beta-Mannosidase from Trichoderma reesei, a 105 kDa glycoprotein, has been crystallized. The crystals belong to the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions a = b = 165.86, c = 122.46 A, and diffract beyond 2.75 A resolution. X-ray diffraction data were collected from a frozen crystal on a synchrotron X-ray source.