RESUMEN
SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.
Asunto(s)
Ciclohexanos/farmacología , Inhibidores Enzimáticos/farmacología , Inmunosupresores/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Esteroide Isomerasas/antagonistas & inhibidores , Secuencia de Aminoácidos , División Celular/efectos de los fármacos , Farmacorresistencia Microbiana/genética , Ergosterol/biosíntesis , Proteínas Fúngicas/genética , Eliminación de Gen , Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Esteroide Isomerasas/genética , Transformación GenéticaRESUMEN
P73, a p53-homologue gene, has been studied for its possible role in head and neck squamous epithelium (HNSE) differentiation and carcinogenesis. P73 RNA and protein were analysed in 50 biopsies, including well- and moderately-differentiated carcinomas, and 21 matched normal adjacent tissues. P73 immunohistochemical analyses revealed intense p73 nuclear staining in basal and parabasal cells of normal squamous epithelium, in contrast with complete absence of staining in the more superficial cell layers. Moderately-differentiated carcinomas demonstrated homogeneous and diffuse staining in all tumour cells, while only basal cells were stained in well-differentiated carcinomas as in normal tissue. No correlation was observed between p73 and p53 protein expression. Immunostaining for p63, another p53-related protein previously described as being involved in HNSE morphogenesis and overexpressed in head and neck squamous cell carcinomas (HNSCC), was found to be similar to p73 labelling in carcinomas, but spread to the more differentiated layers in normal epithelium. Biallelic expression of p73 was found in tumours as well as in matched normal tissues. Comparison of p73 transcript levels between tumours and normal tissues showed decreased mRNA expression in 5/17 (30%) tumours independently of the differentiation status. Mutation and loss of heterozygosity analyses of the p73 gene revealed wild type status and no deletion. Our results strongly suggest that: (i) p73 is associated with homeostasis and control of differentiation of head and neck squamous epithelium probably in concert with p53 and p63; (ii) down-regulation of p73 expression could participate in HNSE carcinogenesis.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Proteínas de Unión al ADN/biosíntesis , Células Epiteliales/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas de la Membrana , Proteínas Nucleares/biosíntesis , Fosfoproteínas/biosíntesis , Transactivadores/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Alelos , Diferenciación Celular , Regulación hacia Abajo , Genes Supresores de Tumor , Heterocigoto , Humanos , Neoplasias Hipofaríngeas/metabolismo , Inmunohistoquímica , Pérdida de Heterocigocidad , Modelos Genéticos , Mutación , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
The p73 gene is a p53 homologue located at 1p36-33, a region submitted to deletions in breast cancer (BC) and putatively imprinted. To study whether p73 was associated with breast carcinogenesis, loss of heterozygosity (LOH), allele expression and transcript levels were assessed in 59 BC, including 39 BC presenting no inflammatory symptoms (NBC) and 20 inflammatory BC (IBC). IBC is a rare but aggressive form of cancer with a very poor prognosis. Normal breast epithelium (BE) and lymphocytes from patients were used as controls. StyI polymorphism generating GC and/or AT alleles was used to select 22 heterozygous patients. p73 LOH was significantly higher in IBC than in NBC [five of eight cases (62%) versus two of 14 cases (14%); Fisher's exact test, P=0.05]. p73 was biallelically expressed in all BE. In contrast, 12 of 16 (75%) BC were monoallelically expressed, showing that allele silencing was significantly associated with breast carcinogenesis (P=0.012), AT being the preferential silent allele (10 out of 12 tumours). p73 mRNA levels in NBC and IBC were two- and threefold lower than in BE, respectively, suggesting that decreased expression could be related to tumour aggressiveness. In conclusion, LOH, allele silencing and decreased expression of the p73 gene may play a role in breast carcinogenesis.
Asunto(s)
Alelos , Empalme Alternativo , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Pérdida de Heterocigocidad/genética , Proteínas Nucleares/genética , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/inmunología , Carcinoma Ductal de Mama/epidemiología , Carcinoma Ductal de Mama/inmunología , Femenino , Francia/epidemiología , Genes Supresores de Tumor , Humanos , Prevalencia , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
C6.9 rat glioma cells undergo a cell death program when exposed to 1, 25-dihydroxyvitamin D3 (1,25-D3). As a global analytical approach, we have investigated gene expression in C6.9 engaged in this cell death program using differential screening of a rat brain cDNA library with probes derived from control and 1,25-D3-treated cells. Using this methodology we report the isolation of 61 differentially expressed cDNAs. Forty-seven cDNAs correspond to genes already characterized in rat cells or tissues. Seven cDNAs are homologous to yeast, mouse or human genes and seven are not related to known genes. Some of the characterized genes have been reported to be differentially expressed following induction of programmed cell death. These include PMP22/gas3, MGP and beta-tubulin. For the first time, we also show a cell death program induced up-regulation of the c-myc associated primary response gene CRP, and of the proteasome RN3 subunit and TCTP/mortalin genes. Another interesting feature of this 1,25-D3 induced-cell death program is the down-regulated expression of transcripts for the microtubule motor dynein heavy chain/MAP 1C and of the calcium-binding S100beta protein. Finally 15 upregulated cDNAs encode ribosomal proteins suggesting a possible involvement of the translational apparatus in this cell program. Alternatively, these ribosomal protein genes could be up-regulated in response to altered rates of cellular metabolism, as has been demonstrated for most of the other isolated genes which encode proteins involved in metabolic pathways. Thus, this study presents to our knowledge the first characterization of genes which are differentially expressed during a cell death program induced by 1, 25-D3. Therefore, this data provides new information on the fundamental mechanisms which participate in the antineoplastic effects of 1,25-D3 and on the machinery of a cell death program in a glioma cell line.
Asunto(s)
Apoptosis/efectos de los fármacos , Biomarcadores de Tumor , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Proteínas de la Matriz Extracelular , Glioma , Vitamina D/farmacología , Animales , Apoptosis/fisiología , Huesos/fisiología , Proteínas de Unión al Calcio/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Cisteína , Cisteína Endopeptidasas/genética , ADN/análisis , ADN Complementario , Dineínas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Proteínas HSP70 de Choque Térmico/genética , Complejos Multienzimáticos/genética , Proteínas de la Mielina/genética , Proteínas de Neoplasias/genética , Osteonectina/genética , Complejo de la Endopetidasa Proteasomal , Biosíntesis de Proteínas/fisiología , ARN Mensajero/análisis , Ratas , Proteínas Ribosómicas/genética , Tubulina (Proteína)/genética , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/fisiología , Proteína Tumoral Controlada Traslacionalmente 1 , Proteína Gla de la MatrizRESUMEN
Thioredoxin (TR) is a small ubiquitous dithiol-reductase enzyme first identified in bacteria and plants. In recent years, this protein has been recognized as playing an important role in the growth control of eukaryotic cells, especially in lymphocytes. It was first cloned from a human Epstein-Barr virus-transformed lymphoblastoid B-cell line by our group in 1988 [Wollman et al., J. Biol. Chem. 263 (1988) 15506-15512] and localized on chromosome 3 p11-p12 by in situ hybridization [Lafage-Pochitaloff-Huvalé et al., FEBS Lett. 255 (1989) 89-91]. The present work was performed to study the genomic organization of the human thioredoxin (hTR)-encoding gene (hTR). The screening of a human genomic library in lambda EMBL4 phage led to the identification of two genomic clones which encompassed the entire gene, including the promoter region. The coding region of hTR spans over 13 kb and is organized into five exons separated by four introns which were 60% sequenced. We determined the transcription start point (tsp) by primer extension. This tsp located, in lymphocytes, 22-bp downstream from a TATA box (TATAA) defines a 5' untranslated region of 74 bp. We analyzed 2149-bp upstream from the promoter for sequence motifs which could bind regulatory proteins. This promoter contains many possible regulatory elements compatible with both a basal constitutive expression and a regulated inducible transcription, especially by cytokines such as interleukin-6 and interferons. Finally, Southern hybridization of genomic DNAs from several donors detected only one active gene encoding hTR.
Asunto(s)
Regiones Promotoras Genéticas , Tiorredoxinas/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , TATA BoxRESUMEN
To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator. The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption. It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA. Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active. Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA. Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals.
Asunto(s)
Escherichia coli/genética , Genes Sintéticos , Inmunotoxinas/genética , Ricina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular/efectos de los fármacos , Glicosilación , Humanos , Inmunotoxinas/farmacocinética , Operón Lac , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ricina/farmacocinéticaRESUMEN
A hybrid gene consisting of the sequences coding for the signal peptide of human growth hormone and the mature form of interleukin-1 beta (IL-1 beta) was chemically synthesized. This sequence was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of recombinant IL-1 beta, which was secreted into the culture medium mainly as a 22-kDa form. Expression in the presence of tunicamycin, an inhibitor of N-glycosylation, led to the complete disappearance of the 22-kDa form and the appearance of a new form of 17.5 kDa, indicating that the hybrid protein had been both processed and N-glycosylated. However, transformed cells producing mature IL-1 beta without a signal peptide produced the predicted 17.5-kDa nonglycosylated form. These results suggest that fusion to a heterologous leader sequence allowed IL-1 beta to be translocated across the membrane of the endoplasmic reticulum and to be transported and secreted by the exocytotic pathway.
Asunto(s)
Hormona del Crecimiento/genética , Interleucina-1/genética , Señales de Clasificación de Proteína/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , Expresión Génica , Glicosilación , Humanos , Interleucina-1/metabolismo , Datos de Secuencia Molecular , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tunicamicina/farmacologíaRESUMEN
A cDNA library was generated from rat brain tissues and organized into 1536-well plates, using a fluorescence activated cell sorter (FACS), acting as a single cell deposition system. The organized library containing 10,000 clones, with 60% full-length cDNA inserts, allowed the generation of multiple identical membrane replicas. Each replica was hybridized with a complex probe obtained from a particular brain tissue or a given cultured cell. The signal intensity for each of the clones present on the membrane, quantified with a standard image-analysis software, is proportional both to the abundance of the corresponding mRNA in the probe and to the amount of plasmid template on the membrane. The latter value was thus used to normalize the signals produced with complex probes, to optimize the comparison of mRNA expression levels for the different systems under study. The construction of high-quality cDNA libraries, the generation of identical membrane replicas and comparable probes, and the utilization of an image-analysis software package, coupled with the normalization of the spot intensity by assaying plasmid quantity, significantly improves the differential screening approach. Altogether, these technical improvements open the possibility to compare a great number of different probes and, in consequence, to accumulate biological information for each clone present in an organized cDNA library. The functional information obtained should complement data from DNA sequencing projects.
Asunto(s)
Secuencia de Bases , Encéfalo/metabolismo , ADN Complementario , Biblioteca de Genes , Transcripción Genética , Animales , Células Cultivadas , Clonación Molecular , Citometría de Flujo , Masculino , Especificidad de Órganos , Sondas ARN , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Programas InformáticosRESUMEN
We report that the rat beta 3-adrenergic receptor (beta 3-AR) gene has an intron. The intron starts with an in-frame stop codon with the result that unspliced transcripts will encode a C-terminal truncated protein. The reported protein sequences of mouse and human beta 3-AR were both deduced from genomic DNA sequences. Given the heterogeneity at the C-termini of the otherwise highly similar rat, mouse and human sequences, we discuss the intriguing possibility that the beta 3-AR gene of the latter two species also contain an intron near the extremity of the open reading frame. A beta-adrenergic receptor (beta-AR) cDNA we have cloned from rat colonic tissue which has a sequence essentially identical to that previously reported for the rat adipose beta 3-AR cDNA [(1991) J. Chem. 266, 24053], encodes the spliced version of the beta 3-AR.
Asunto(s)
Intrones , Receptores Adrenérgicos beta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Codón , ADN/química , ADN/genética , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , RatasRESUMEN
ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [33P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [33P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.
Asunto(s)
Activación Plaquetaria/fisiología , Receptores Purinérgicos P2/fisiología , Adenosina Difosfato/fisiología , Animales , Calcio/metabolismo , Clopidogrel , Humanos , Técnicas In Vitro , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Reacción en Cadena de la Polimerasa , Conejos , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y1 , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
Corticotrophin-releasing factor (CRF) is the principal hypothalamic factor governing the pituitary-adrenal axis, but the wide extra-pituitary distribution of CRF and its receptors suggest a major role for this neuropeptide in the integration of the overall physiological and behavioral responses of an organism to stress. We have cloned a CRF receptor complementary DNA (cDNA) by expression in COS-7 cells of a cDNA library from the AtT20 mouse pituitary tumour cell line. The cloned mouse cDNA was then as a probe to isolate a human CRF receptor cDNA from a human brain cDNA library. The mouse and human cDNAs both encode 415 amino acid proteins that are 97% identical, containing seven putative transmembrane domains characteristic of G protein-coupled receptors. The CRF receptor shows homology with the receptors for growth hormone-releasing factor, vasoactive intestinal peptide, secretin, parathyroid hormone, and calcitonin. COS-7 cells transfected with the mouse CRF receptor cDNA bind radiolabelled ovine CRF with high affinity and respond specifically to CRF by accumulation of intracellular cAMP. A 2.7 kb mRNA coding for the CRF receptor could be detected in AtT20 cells and human cortex tissue. PCR analysis also detected the receptor transcript in human pituitary, brainstem, and testis.
Asunto(s)
Encéfalo/metabolismo , Hipófisis/metabolismo , Receptores de Hormona Liberadora de Corticotropina/química , Receptores de Hormona Liberadora de Corticotropina/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Hormona Liberadora de Corticotropina/metabolismo , Hormona Liberadora de Corticotropina/farmacología , AMP Cíclico/metabolismo , ADN Complementario/genética , Expresión Génica , Humanos , Riñón , Ratones , Datos de Secuencia Molecular , Hipófisis/química , ARN Mensajero , TransfecciónRESUMEN
We report the molecular cloning of a beta 3-adrenergic receptor [beta 3-AR] cDNA from human brown adipose tissue. The cDNA-encoded protein is identical to the previously cloned beta 3-AR but with 6 additional amino acids at the C-terminus. The C-terminus is shared by the beta 3 receptors expressed in human neuroblastoma cells [SK-N-MC] [Mol. Pharmacol. 42 (1992) 964-970]. Furthermore, using a polymerase chain reaction strategy we have cloned and sequenced the beta 3-AR introns. Sequence analysis demonstrates that the human beta 3-AR gene comprises at least 3 exons and 2 introns and that the most abundant beta 3-AR transcripts encode a protein with an exon 3-derived C-terminus. Interestingly, although a similar organization has been found in rodent genes, the rat beta 3-AR transcripts encode a receptor with an exon 2-derived C-terminus.
Asunto(s)
Receptores Adrenérgicos beta/genética , Simpatomiméticos/metabolismo , Tejido Adiposo Pardo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Exones/genética , Humanos , Intrones/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Receptores Adrenérgicos beta/biosíntesis , Proteínas Recombinantes/biosíntesisRESUMEN
A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR [Neuron, 4 (1990) 847-854]. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.
Asunto(s)
Neurotensina/metabolismo , Receptores de Neurotransmisores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , Línea Celular , Clonación Molecular , ADN , Humanos , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores de Neurotensina , Receptores de Neurotransmisores/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
A search for sequences homologous to the neurotensin receptor cDNA in a rat hypothalamic library has identified a novel neurotensin receptor (NTR-2). The 1539 bp cDNA encodes a 416 amino acid protein and shows highest homology to the previously cloned neurotensin receptor (NTR-1) (64% homology and 43% identity). Binding and pharmacological studies demonstrate that NTR-2 expressed in COS cells recognizes neurotensin (NT) with high affinity as well as several other agonists and antagonists. However, a fundamental difference was found; unlike NTR-1, NTR-2 recognizes, with high affinity, levocabastine, a histamine H1 receptor antagonist previously shown to compete with NT for low-affinity binding sites in brain.
Asunto(s)
Piperidinas/farmacología , Receptores de Neurotensina/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Hipotálamo/metabolismo , Datos de Secuencia Molecular , ARN Mensajero , Ratas , Receptores de Neurotensina/efectos de los fármacos , Receptores de Neurotensina/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
A 7.275-kb DNA fragment which encodes resistance by abortive infection (Abi+) to bacteriophage was cloned from Lactococcus lactis subsp. cremoris S114. The genetic determinant for abortive infection was subcloned from this fragment. This gene was found to confer a reduction in efficiency of plating and plaque size for prolate-headed bacteriophage phi 53 (group I homology) and for small isometric-headed bacteriophage phi 59 (group III homology). This new gene, termed abiN, is predicted to encode a polypeptide of 178 amino acid residues with a deduced molecular mass of 20,461 Da and an isoelectric point of 4.63. No homology with any previously described genes was found. A probe was used to determine the presence of this gene only in S114 from 31 strains tested.
Asunto(s)
Cromosomas Bacterianos , Genes Bacterianos , Lactococcus/genética , Bacteriófagos , Secuencia de Bases , Lactococcus/virología , Datos de Secuencia Molecular , Sistemas de Lectura AbiertaRESUMEN
The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.
Asunto(s)
Neurotensina/farmacología , Receptores de Neurotensina/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Cricetinae , ADN Complementario/análisis , Humanos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores de Neurotensina/agonistas , Receptores de Neurotensina/biosíntesis , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Distribución TisularRESUMEN
We have isolated a cDNA (NC28) transcribed from a mRNA which is transiently induced in U937 promonocytic cells by PMA and super-induced by cycloheximide. NC28 cDNA encodes a new member of the chemokine family, MCP-3, recently purified from MG-63 osteosarcoma cells by Van Damme et al. [1]. The MCP-3 protein sequence shows 74% identity with human monocyte chemoattractant protein 1 (MCP-1) and, like MCP-1, recombinant MCP-3 protein shows chemotactic activity for monocytes but not for neutrophils. However the secreted MCP-3 protein differs from MCP-1 in being N-glycosylated. The 3' noncoding regions of MCP-3 and MCP-1 mRNAs are more diverged (44%), allowing specific cDNA probes to be made, and indicating that the two genes are evolutionarily distant. Sequence comparisons of the 3' noncoding regions suggest that MCP-3 may be the human homologue of the mouse MARC gene [2], and that MCP-1 and MCP-3 genes arose by a gene duplication event before the mammalian radiation. Both MCP-1 and MCP-3 mRNAs are expressed by PBMC, principally by monocytes, with MCP-1 mRNA being expressed at levels 2-4 times that of MCP-3 mRNA. However, while MCP-1 mRNA is also expressed at high levels in fibroblast or astrocytoma cell lines after IL-1 and TNF stimulation, MCP-3 mRNA is expressed only at very low levels in these cells. The cellular origin of MCP-3 is thus more restricted than that of MCP-1. In our experiments on PBMC, LPS is not a consistent inducer of MCP-1 and MCP-3 mRNAs. In some experiments, it actually decreases levels of these two mRNAs, while concomitantly increasing IL-6 and TNF-alpha mRNA levels. Levels of MCP-1 and MCP-3 mRNAs in PBMC are both increased by IFN-gamma, although IL-6 mRNA is not induced. They are also increased by PHA-P and are decreased, in most cases, by IL-13 [3]. MCP-1 and MCP-3 mRNAs are thus co-ordinately regulated in monocytes in response to a number of inducing or inhibitory agents, in a manner differing in several respects from that of other monokines such as IL-6.
Asunto(s)
Factores Quimiotácticos/genética , Citocinas , Proteínas Quimioatrayentes de Monocitos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Células Cultivadas , Quimiocina CCL7 , Clonación Molecular , ADN/genética , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Monocitos/inmunología , ARN Mensajero/genética , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
E.Tn sequences form a family of long moderately repeated sequences which are abundantly transcribed in the pluripotent cell lineage between day 3.5 and 7.5 of early mouse embryogenesis. The structure of the long terminal repeat (LTR) bordering the E.Tn sequences has been investigated by nucleotide sequencing, primer extension and S1 mapping experiments. This has allowed the identification of U3, R and U5 domains, and of several other structural features all of which are characteristics of retroviral LTRs.
Asunto(s)
Elementos Transponibles de ADN , Retroviridae/genética , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Enzimas de Restricción del ADN , ADN de Cadena Simple/genética , Endonucleasas , Ratones , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Endonucleasas Específicas del ADN y ARN con un Solo FilamentoRESUMEN
Two-hybrid screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitinas/química , Ubiquitinas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genes Reporteros , Genes Supresores de Tumor , Humanos , Leupeptinas/farmacología , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Transporte de Proteínas , Proteína SUMO-1 , Transfección , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos Híbridos , Ubiquitinas/genéticaRESUMEN
Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human cancers. Recent identification of two human homologues of p53 has raised the prospect of functional interactions between family members via a conserved oligomerization domain. Here we report in vitro and in vivo analysis of homo- and hetero-oligomerization of p53 and its homologues, p63 and p73. The oligomerization domains of p63 and p73 can independently fold into stable homotetramers, as previously observed for p53. However, the oligomerization domain of p53 does not associate with that of either p73 or p63, even when p53 is in 15-fold excess. On the other hand, the oligomerization domains of p63 and p73 are able to weakly associate with one another in vitro. In vivo co-transfection assays of the ability of p53 and its homologues to activate reporter genes showed that a DNA-binding mutant of p53 was not able to act in a dominant negative manner over wild-type p73 or p63 but that a p73 mutant could inhibit the activity of wild-type p63. These data suggest that mutant p53 in cancer cells will not interact with endogenous or exogenous p63 or p73 via their respective oligomerization domains. It also establishes that the multiple isoforms of p63 as well as those of p73 are capable of interacting via their common oligomerization domain.