RESUMEN
Proteomic patterns of myocardial tissue in different etiologies of heart failure were investigated using a direct analytical approach with High Performance Liquid Chromatography (HPLC)/Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Right atrial appendages from 20 patients, 10 with hemodynamically significant isolated aortic valve disease and 10 with symptomatic coronary artery disease were collected during elective cardiac surgery. After preparation of tissue samples and tryptic digestion of proteins, the peptide mixture was HPLC-separated and on-line analyzed by electrospray FT-ICR MS. Data obtained from HPLC / FT-ICR MS runs were compared for classification. To extract the classification features, the selection of best individual features was applied and the "nearest mean classifier" was used for the classification of test samples and the sample projection onto classification patterns. The pattern distribution characteristics of aortic and coronary diseases were clearly different. No interference between samples of both disease categories was registered, even if the distribution of unsupervised classified test samples were closer. Samples representing aortic valve disease showed a closer accumulation pattern of spots compared to the samples representing coronary disease, which indicated a more specific protein classification. Through selective identification of specific peptides and protein patterns with FTMS, valvular and coronary heart disease is for the first time clearly distinguished at molecular level. The described methodology could also be feasible in search for specific biomarkers in plasma or serum for diagnostic purposes.
Asunto(s)
Biomarcadores/metabolismo , Cromatografía Liquida , Enfermedades de las Válvulas Cardíacas/diagnóstico , Isquemia Miocárdica/diagnóstico , Proteómica , Espectrometría de Masa por Ionización de Electrospray , Adulto , Anciano , Anciano de 80 o más Años , Válvula Aórtica/cirugía , Cromatografía Líquida de Alta Presión , Ciclotrones , Femenino , Análisis de Fourier , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/cirugía , Humanos , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/cirugía , Proteínas/análisisRESUMEN
BACKGROUND: The presence of lymphocytic infiltration in prostate carcinomas has been shown to have prognostic relevance. However, it is not yet clear if this infiltrate represents a tumor-specific activated cell population or not. Therefore, the aim of the present study was to characterize the activation status of freshly isolated tumor infiltrating lymphocytes (TIL) from prostate carcinomas (PCa) and benign hyperplasia (BPH) with respect to the mRNA expression of cytokines and apoptotic factors. METHODS: TIL were isolated from mechanically disaggregated tumor material by gradient centrifugation. The cells of the interphase were depleted from epithelial cells with anti-human epithelial antigen magnetic beads and then CD3(+)- lymphocytes were selected with magnetic beads against this determinant. In these pure lymphocyte preparations the mRNA expression of IL-1, IL-10, IFN-gamma, TNF-alpha, Fas and Fas ligand was determined by using a semiquantitative RT-PCR. Contamination with tumor cells was excluded by a PCR for PSA and PSMA. RESULTS: The CD3(+)-TIL from 21 patients with PCa and 20 patients with BPH expressed significantly higher levels of IL-10- and Fas ligand-mRNA compared to the autologous CD3(+)- PBL, whereas the expression of IL-1-, TNF-alpha- and Fas-mRNA was not different in either cell population. In contrast, the mRNA levels of IFN-gamma were significantly higher only in the CD3(+)-TIL from the carcinomas but not from the BPH compared to autologous CD3(+)-PBL. CONCLUSIONS: Since high levels of IFN-gamma have been reported to be produced by specifically lytic lymphocytes, our results suggest the presence of specifically activated TIL in the prostate carcinomas but not in the BPH, whereas inflammatory activated TIL are present both in the carcinomas and the BPH.
Asunto(s)
Adenocarcinoma/inmunología , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos/inmunología , Hiperplasia Prostática/inmunología , Neoplasias de la Próstata/inmunología , Adenocarcinoma/sangre , Adenocarcinoma/metabolismo , Anciano , Anciano de 80 o más Años , Complejo CD3/inmunología , Citocinas/biosíntesis , Citocinas/genética , Proteína Ligando Fas , Expresión Génica , Humanos , Linfocitos/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Hiperplasia Prostática/sangre , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/sangre , ARN Mensajero/genética , Receptor fas/biosíntesis , Receptor fas/sangre , Receptor fas/genéticaRESUMEN
The mRNA expression of the cytokines IFN-gamma, IL-10 and TNF-alpha and the proapoptotic factor Fas ligand (FasL) was compared in freshly isolated CD4(+)and CD8(+)tumour-infiltrating lymphocytes (TIL) and simultaneously obtained autologous CD4(+)and CD8(+)peripheral blood lymphocytes (PBL) from 20 patients with renal cell carcinomas (RCC). TIL were isolated from mechanically disaggregated tumour material and PBL from peripheral blood by gradient centrifugation. The cells of the interphase were depleted from tumour cells with anti-human epithelial antigen magnetic beads and then positive selection was performed with anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of cytokine and FasL mRNAs was determined by using a PCR-assisted mRNA amplification assay. In the CD4(+)TIL from the 20 patients with RCC, levels of mRNAs encoding for IFN-gamma (P
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Células Renales/metabolismo , Interferón gamma/biosíntesis , Neoplasias Renales/metabolismo , Subgrupos Linfocitarios/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , ARN Mensajero/biosíntesis , Adenocarcinoma de Células Claras/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteína Ligando Fas , Femenino , Humanos , Interferón gamma/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Activación de Linfocitos , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3+ tumor-infiltrating lymphocytes (CD3+ TIL) and in autologous CD3+ peripheral blood lymphocytes (CD3+ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4+ TIL and CD8+ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon gamma (IFNgamma), and tumor necrosis factor alpha (TNFalpha) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3+ TIL, levels of mRNA for IFNgamma, IL-10, IL-1 and TNFalpha were significantly higher than in the autologous CD3+ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4+ TIL and simultaneously obtained CD8+ TIL revealed a significantly higher expression of IFNgamma in the CD8+ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8+ TIL.
Asunto(s)
Carcinoma de Células Renales/inmunología , Citocinas/genética , Neoplasias Renales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Complejo CD3/análisis , Antígenos CD4/análisis , Antígenos CD8/análisis , Citocinas/biosíntesis , Humanos , ARN Mensajero/análisisRESUMEN
As a decreased production of Th1 cytokines by stimulated peripheral blood leukocytes has recently been shown in patients with various carcinomas, the present study was performed to determine whether these patients also exhibit a Th1/Th2 imbalance compared to healthy controls. We measured the production of the Th1 cytokines IL-2 and IFN-gamma as well as the Th2 cytokines IL-4, IL-6 and IL-10 in mitogen-stimulated peripheral blood mononuclear cell (PBMC) cultures of patients with urinary bladder carcinomas (n = 47), prostate carcinomas (n = 111) and renal cell carcinomas (n = 67) as compared to 40 age-matched healthy controls. In the PBMC cultures of the tumor patients, the levels of the Th1 cytokines IL-2 and IFN-gamma were lower as compared to the controls. For IFN-gamma, the differences were highly significant and in the patients with renal cell carcinomas it could be shown that the values decreased with increasing tumor mass. In contrast, the levels of the Th2 cytokines IL-4, IL-6 and IL-10 were comparable in the PBMC cultures of tumor patients and controls. From these results, it is concluded that there is only a malfunction in Th1 cells but no switch from a Th1 type to a Th2 type cytokine profile in the PBMCs of cancer patients.
Asunto(s)
Carcinoma de Células Renales/sangre , Interferón gamma/metabolismo , Interleucinas/metabolismo , Neoplasias Renales/sangre , Leucocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/sangre , Neoplasias de la Vejiga Urinaria/sangre , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
IFN-gamma production in whole blood cell cultures (WBCC), and TNF-receptor p75 (TNF-R-75) plasma levels were measured as two independent immunological parameters in a group of 67 untreated renal cell carcinoma (RCC) patients at different clinical stages, and 40 age matched healthy controls. In the blood cell cultures of the tumor patients the levels of IFN-gamma were significantly lower compared to the controls and the values decreased with increasing tumor mass. In contrast, TNF-R-75 plasma levels were significantly higher in the tumor patients and increased with tumor stage. Additionally serial assessments of these parameters were studied in another group of 15 patients with advanced RCC during treatment with IL-2, IFN-alpha and retinoic acid according to three different protocols in order to search for any correlation between the biological marker values and the clinical response to treatment. During each 5 day cycle of high dose IL-2/IFN-alpha combination therapy (protocol 1) IFN-gamma-levels in the WBCC were markedly decreased, whereas the plasma levels of TNF-R-75 were increased. During low dose, long-term continuous IFN-alpha/IL-2 administration (protocol 2) in two patients a clear increase of the ex vivo leukocyte IFN-gamma production was seen for the first 5 and 6 months of treatment, respectively, which could be correlated to stable disease for this time. When progression was diagnosed, IFN-gamma levels in the WBCC decreased. In the WBCC of the other four patients with progressive IFN-gamma levels were rather low throughout (< 10 ng/ml) and no clear changes were measured. During low does IFN-alpha and 13-cis-retinoic acid therapy in repetitive weekly cycles (protocol 3) two patients had stable disease for 6 and 14 months respectively. In the WBCC cultures of these patients IFN-gamma production was higher during stable than during progressive disease. The other two patients with tumor progression had a very low leukocyte IFN-gamma production and high plasma levels of TNF-R-75. It is concluded that IFN-gamma levels in WBCC and TNF-R-75 plasma levels may be useful parameters for the immunological monitoring of therapies with biological response modifiers. Low IFN-gamma values and high TNF-R-75 levels may be predictive of tumor progression and bad prognosis.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/inmunología , Interferón-alfa/administración & dosificación , Interleucina-2/administración & dosificación , Neoplasias Renales/inmunología , Neoplasias Renales/terapia , Tretinoina/administración & dosificación , Adulto , Anciano , Antígenos CD/sangre , Biomarcadores/sangre , Carcinoma de Células Renales/terapia , Estudios de Evaluación como Asunto , Femenino , Humanos , Interferón gamma/sangre , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Receptores del Factor de Necrosis Tumoral/sangre , Receptores Tipo II del Factor de Necrosis Tumoral , Factores de TiempoRESUMEN
The aim of the present study was to compare the biological effects of 12 different clinically applied mistletoe preparations (I, II, III and IV) from the host trees "pinus" (P), "malus" (M), "abies" (A) and "quercus" (Q) on human leukocytes. When the preparations I-P, II-P, III-P and IV-A were added to the whole blood cell cultures of 37 cancer patients (breast cancer, n = 22, colorectal cancer, n = 15) and 34 healthy controls, a significant induction of the cytokines IL-1-beta, IL-2, IL-6, IL-10 and TNF-alpha was found with preparation I-P. A significant induction of IL-1-beta and TNF-alpha was obtained with the preparations II-P and III-P as compared to the nonstimulated control cultures. Induction of IFN-gamma was not found with any preparation. Cytokine induction was comparable in the blood cell cultures of the tumor patients and the healthy controls. When the clinical preparations I-P, I-M, I-Q, II-P, II-M, II-A, III-P, III-M, III-A and IV-P, IV-M, IV-A were tested in cultures of peripheral blood mononuclear cells from 5 healthy donors, differences in the induction of cytokine production and apoptosis were seen after addition of the mistletoe preparations from different host trees. Increased levels of IL-1-beta were found after addition of the preparations I-P and I-M, increased levels of TNF-alpha were measured after addition of preparations I-P and III-A. Induction of apoptosis was most evident with the preparations I-M, I-Q, III-M and IV-A. Neither cytokine induction nor apoptosis could be correlated to the amount of lectins found in the preparations. Stimulation of separated CD4(+)-, CD8(+)- and CD14(+)-cells from 5 healthy donors with the above noted preparations revealed an induction of IL-1-beta and TNF-alpha production by the preparations I-P, I-M and I-Q mainly in monocytes and to a minimal extent in lymphocytes. Also apoptosis was seen mainly in CD14(+)-monocytes. From these results it is concluded that both, apoptosis and cytokine production are induced differentially in leukocyte cultures by clinically applied mistletoe preparations. However, there is no correlation between the biological effects and the lectin content of the various preparations and none of them were comparable with respect to the extent of these effects. Therefore, it may be expected that clinical studies with different preparations are not comparable either.
Asunto(s)
Leucocitos/efectos de los fármacos , Muérdago/química , Extractos Vegetales/farmacología , Plantas Medicinales , Adulto , Anciano , Apoptosis , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Técnicas In Vitro , Receptores de Lipopolisacáridos/metabolismo , Subgrupos Linfocitarios/efectos de los fármacos , Masculino , Persona de Mediana Edad , Neoplasias/sangreRESUMEN
The molecular events governing the differentiation pathway of natural killer (NK) cells are not well understood. The phenotype of mature NK cells is specified by the expression of the low affinity Fc receptor for IgG (human FcgammaRIII, CD16) encoded by the FcgammaRIII-A gene. Here we report that the Pprox promoter (-198/-10) of FcgammaRIII-A stimulated by its own intron enhancer (+10/+712) was only one of the cis-elements that target the expression of a reporter gene in the immature NK cell line, YT. The transcription start sites of the FcgammaRIII-A a2/3 and a5/6 splice alternatives in NK cells were mapped to the medial -1817/-850 FcgammaRIII-A control region. Two promoters, Pmed1 (-942/-850) and Pmed2 (-1376/-1123) resided in this region and controlled for the initiation of these transcript classes encoding the known FcgammaRIII-A receptor protein. Deletion mapping studies demonstrated that the 93 base pairs -942/-850 Pmed1 sequence was sufficient to confer cell type-specific expression in YT cells. The 5' end of Pmed1 (-942 to -921) was required for full promoter function indicating the presence of an important sequence motif recognized by a YT-specific factor. Our data suggest that this motif might be a useful tool for subsequent identification of putative transcription factors uniquely active in YT and NK cells.
Asunto(s)
Células Asesinas Naturales/fisiología , Regiones Promotoras Genéticas , Receptores de IgG/genética , Empalme Alternativo , Secuencia de Bases , Genes , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
The expression of the human Fc receptor with low affinity for IgG (Fc gamma RIII, CD16) encoded by the Fc gamma RIII-A or Fc gamma RIII-B genes is restricted to natural killer (NK), a subset of T cells and macrophages or neutrophils (PMN). The genetic heterogeneity of Fc gamma RIII generates alternative membrane-anchored proteins with distinct signaling capacities when cross-linked by immune complexes. Of great importance is the characterization of the regulatory gene elements directing the expression of a particular Fc gamma RIII isoform to a given specific cell type. Molecular characterization of the Fc gamma RIII-A and Fc gamma RIII-B genes has revealed that the promoter regions display distinct tissue-specific transcriptional activities. In addition, the differential Fc gamma RIII-A/B gene activation can be regulated by enhancer elements located in the more upstream and intron regions. Transcription initiation in NK cells occurs also outside the normal promoter region by a second independent Fc gamma RIII-A promoter. Analysis of the additional Fc gamma RIIIa2-4 transcripts suggests the expression of novel, as yet unknown Fc gamma RIII receptor isoforms on NK cells.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Receptores de IgG/biosíntesis , Receptores de IgG/genética , Secuencia de Bases , Desoxirribonucleasa I , Elementos de Facilitación Genéticos/inmunología , Humanos , Intrones/inmunología , Células Asesinas Naturales/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/inmunología , Receptores de IgG/química , Transcripción Genética/inmunología , Activación TranscripcionalRESUMEN
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect of hematopoietic stem cells in which affected cells are characterized by the lack of glycosylphosphatidylinositol (GPI)-anchored proteins. The lesion in PNH lies in the defective synthesis of N-acetyl-D-glucosaminyl-phosphatidylinositol (GlcNAc-Pl), the first intermediate in GPI biosynthesis. Reintroduction of the PIG-A gene into GPI(-) patient cells reportedly complements this defect. We have analyzed here PIG-A transcripts of six PNH patients. GPI+ and GPI- cell lines from each individual were used, ie, Epstein-Barr virus-transformed B-lymphoblastoid cell lines, T-cell lines, and natural killer cell clones. Reverse transcriptase polymerase chain reaction and sequencing showed three different PIG-A splicing variants in GPI+ cell lines, in which the largest transcript contained the wild-type PIG-A coding region sequence. GPI-deficient cell lines showed abnormal splicing variants. Sequencing of PIG-A complementary DNA and genomic DNA showed heterogeneous mutations ranging from different point mutations to small deletions. Two lymphocyte cell lines (T- and B-cell lines) of one patient presented with the same mutation. For another patient, two different mutations were detected in one natural killer cell line. Therefore, different cell lineages have somatic mutations in PIG-A that lead to PNH.
Asunto(s)
Glicosilfosfatidilinositoles/genética , Hemoglobinuria Paroxística/genética , Proteínas de la Membrana/genética , Mutación , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Bases , Línea Celular , Humanos , Proteínas de la Membrana/deficiencia , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The human Fc receptor with low affinity for IgG (Fc gamma RIII, CD16) is encoded by two nearly identical genes, Fc gamma RIII-A and Fc gamma RIII-B, resulting in tissue-specific expression of alternative membrane-anchored isoforms. The transmembrane CD16 receptor forms a heteromeric structure with the Fc epsilon RI (gamma) and/or CD3 (zeta) subunits on the surface of activated monocytes/macrophages, NK cells, and a subset of T cells. The expression of the glycosylphosphatidylinositol-anchored CD16 isoform encoded by the Fc gamma RIII-B gene is restricted to polymorphonuclear leukocytes and can be induced by Me2SO differentiation of HL60 cells. We have isolated and sequenced genomic clones of the human Fc gamma RIII-A and Fc gamma RIII-B genes, located their transcription initiation sites, identified a different organization of their 5' regions, and demonstrated four distinct classes of Fc gamma RIII-A transcripts (a1-a4) compared with a single class of Fc gamma RIII-Bb1 transcripts. Both CD16 promoters (positions -198 to -10) lack the classical "TATA" positioning consensus sequence but confer transcriptional activity when coupled to the human lysozyme enhancer. Both promoters also display different tissue-specific transcriptional activities reflecting the expected gene expression of Fc gamma RIII-A and Fc gamma RIII-B in NK cells versus polymorphonuclear leukocytes. Within the -198/-10 fragments, the sequences of the two CD16 genes have been identified to differ in 10 positions. It is suggested that these nucleotide differences might contribute to cell type-specific transcription of Fc gamma RIII genes.
Asunto(s)
Inmunoglobulina G/metabolismo , Regiones Promotoras Genéticas , Receptores de IgG/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Receptores de IgG/metabolismo , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
PAb1614, an SV40-specific monoclonal antibody of the panel PAb1601-1636 reacts with large and small tumor antigens of SV40, BK and JC virus, and with polyoma virus large and middle tumor antigens, but not with the large tumor antigen of the lymphotropic papova virus. Using immunofluorescence and immunoblot competition assays and ELISA with synthetic peptides, it is shown that the epitope is represented by the SV40 tumor antigen undecapeptide, K39-E49. This peptide comprises the tumor antigen consensus sequence, H42-G47, of the polyoma viruses. However, the epitope of PAb1614 probably does not exactly coincide with this hexapeptide. This explains why some cross-reactions are less strong, or absent, as in the case of the lymphotropic papova virus. Further antibodies of the PAb1601-1636 panel that cross-react with the JC virus large tumor antigen are PAb1602, 1604, 1606, 1618, 1621, 1622, 1623, 1624, 1626, 1629, and 1633.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Transformadores de Poliomavirus/inmunología , Epítopos/inmunología , Poliomavirus/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Virus BK/inmunología , Línea Celular Transformada , Reacciones Cruzadas , Virus JC/inmunología , Datos de Secuencia Molecular , Oligopéptidos/inmunología , Poliomavirus/química , Alineación de Secuencia , Virus 40 de los Simios/inmunologíaRESUMEN
In the chromatin domain of the chicken lysozyme gene of myeloid and oviduct cells, which both have the potential to activate the gene, a developmentally stable DNase I-hypersensitive site is formed around 6.1 kb upstream of the gene. This implies that this DNA region, which has previously been demonstrated to function as a transcriptional enhancer element in myeloid cells, is intimately involved in the cell-type-specific activation of the lysozyme gene locus. Deletion analysis identifies a 157-bp minimal fragment that confers the same promacrophage-specific enhancer activity as the originally described 562-bp -6.1-kb enhancer fragment. By introducing specific point mutations, we demonstrate in transient gene transfer experiments that the minimal fragment consists of at least six adjacent elements, each substantially contributing to enhancer function. The compact multifactorial enhancer complex includes a nuclear factor I (NF-I)/TGGCA binding site, homologies to AP1, and octanucleotide or enhancer core consensus motifs. Point mutation of the NF-I binding site results in the loss of NF-I binding in vitro and enhancer activity in vivo after gene transfer. Surprisingly, four overlapping oligonucleotides, each consisting of at least two elements of the -6.1-kb enhancer, confer myeloid-cell-specific enhancer activity. We found several myeloid-cell-specific DNA-binding proteins interacting with the -6.1-kb enhancer, a result consistent with that described above. Therefore, we suggest that more than a single trans-acting factor mediates the cell type specificity of the -6.1-kb enhancer.
Asunto(s)
Elementos de Facilitación Genéticos , Muramidasa/genética , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Embrión de Pollo , Pollos , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Cromatina/fisiología , Deleción Cromosómica , Desoxirribonucleasa I , Femenino , Regulación Enzimológica de la Expresión Génica , Cinética , Luciferasas/genética , Luciferasas/metabolismo , Macrófagos/enzimología , Datos de Secuencia Molecular , Muramidasa/metabolismo , Músculos/enzimología , Mutagénesis Sitio-Dirigida , Oviductos/enzimología , Plásmidos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , TransfecciónRESUMEN
Affinity-purified medium T antigen of wild-type polyomavirus and dl8, a transforming mutant with a deletion in the medium T gene, is associated with three cellular proteins with apparent molecular weights of 88,000 (88K protein), 61,000 (61K protein), and 37,000 (37K protein). Medium T antigen encoded by the nontransforming hrt mutants fails to associate with these proteins, whereas medium T antigen of the nontransforming mutant dl1015 is able to do so. Medium T antigen of the nontransforming mutant dl23 binds to the 61K and 37K proteins; however, binding to the 88K protein is uncertain. The pattern of complex formation between these proteins and medium T antigen resembles that of pp60c-src and medium T antigen. The binding of medium T antigen to the 88K, 61K, and 37K proteins, as well as to pp60c-src, might represent a necessary but insufficient step in transformation. By mixing extracts from infected and uninfected cells, complex formation between medium T antigen and the 88K, 61K, and 37K proteins can be demonstrated in vitro. Pulse-chase experiments indicated that in vivo the association between medium T antigen and the 61K and 37K proteins is a slow process. The latter two proteins are probably bound to each other in uninfected cells. On two-dimensional gels of whole-cell extract, the 61K protein comigrated with a minor protein with an isoelectric point of 5.2. The 61K protein was neither phosphorylated nor glycosylated. Polyomavirus tumor serum precipitated the 61K and 37K proteins independently of medium T antigen. Therefore, the 61K protein or the 37K protein or both have the properties of a cellular tumor antigen.
Asunto(s)
Antígenos Transformadores de Poliomavirus/inmunología , Poliomavirus/inmunología , Proteínas/metabolismo , Animales , Línea Celular , Inmunoensayo , Peso Molecular , Mutación , Poliomavirus/genética , Proteínas/inmunologíaRESUMEN
Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-Arg-Ser-Arg-His-Phe (KF), and an internal region, Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp60c-src and a new protein of approximately equal to 61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of approximately equal to 72,000 Da.
Asunto(s)
Antígenos Virales de Tumores , Poliomavirus/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Virales/metabolismo , Animales , Técnicas de Inmunoadsorción , Sustancias Macromoleculares , Ratones , Peso Molecular , Poliomavirus/inmunología , Unión Proteica , Proteínas Proto-Oncogénicas pp60(c-src)RESUMEN
Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit.