RESUMO
D-bifunctional protein deficiency (D-BPD) is a rare, autosomal recessive peroxisomal disorder that affects the breakdown of long-chain fatty acids. Patients with D-BPD typically present during the neonatal period with hypotonia, seizures, and facial dysmorphism, followed by severe developmental delay and early mortality. While some patients have survived past two years of age, the detectable enzyme activity in these rare cases was likely a contributing factor. We report a D-BPD case and comment on challenges faced in diagnosis based on a narrative literature review. An overview of Romania's first patient diagnosed with D-BPD is provided, including clinical presentation, imaging, biochemical, molecular data, and clinical course. Establishing a diagnosis can be challenging, as the clinical picture is often incomplete or similar to many other conditions. Our patient was diagnosed with type I D-BPD based on whole-exome sequencing (WES) results revealing a pathogenic frameshift variant of the HSD17B4 gene, c788del, p(Pro263GInfs*2), previously identified in another D-BPD patient. WES also identified a variant of the SUOX gene with unclear significance. We advocate for using molecular diagnosis in critically ill newborns and infants to improve care, reduce healthcare costs, and allow for familial counseling.
Assuntos
Proteína Mitocondrial Trifuncional/deficiência , Proteína Multifuncional do Peroxissomo-2 , Humanos , Proteína Multifuncional do Peroxissomo-2/deficiência , Proteína Multifuncional do Peroxissomo-2/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Recém-Nascido , Lactente , Masculino , Feminino , Sequenciamento do Exoma , Mutação da Fase de Leitura , 17-Hidroxiesteroide Desidrogenases/deficiência , 17-Hidroxiesteroide Desidrogenases/genética , Região de Recursos Limitados , Miopatias Mitocondriais , Cardiomiopatias , Doenças do Sistema Nervoso , RabdomióliseRESUMO
Background and Objectives: Colorectal cancer (CRC) continues to be an essential public health problem. Our study aimed to evaluate the prognostic significance of classic prognostic factors and some less-studied histopathological parameters in CRC. Materials and Methods: We performed a retrospective study on 71 colorectal carcinoma patients who underwent surgery at the "Pius Brînzeu" County Clinical Emergency Hospital in TimiÈoara, Romania. We analyzed the classic parameters but also tumor budding (TB), poorly differentiated clusters (PDCs) of cells, tumor-infiltrating lymphocytes (TILs), and the configuration of the tumor border on hematoxylin-eosin slides. Results: A high degree of malignancy (p = 0.006), deep invasion of the intestinal wall (p = 0.003), an advanced stage of the disease (p < 0.0001), lymphovascular invasion (p < 0.0001), perineural invasion (p < 0.0001), high-grade TB (p < 0.0001), high-grade PDCs (p < 0.0001), infiltrative tumor border configuration (p < 0.0001) showed a positive correlation with lymph node metastases. Conclusions: The analyzed parameters positively correlate with unfavorable prognostic factors in CRC. We highlight the value of classic prognostic factors along with a series of less-known parameters that are more accessible and easier to evaluate using standard staining techniques and that could predict the risk of relapse or aggressive evolution in patients with CRC.
Assuntos
Neoplasias Colorretais , Humanos , Metástase Linfática , Neoplasias Colorretais/patologia , Estadiamento de Neoplasias , Estudos Retrospectivos , Recidiva Local de Neoplasia/patologia , PrognósticoRESUMO
Background and Objectives: This study aimed to assess the clinical-pathological profile of patients with invasive cutaneous melanomas and to identify the parameters with a prognostic role in the lymph nodal spread of this malignant tumor. Materials and Methods: We performed a retrospective study on patients with invasive cutaneous melanomas who underwent surgery in the "Pius Brînzeu" County Clinical Emergency Hospital from TimiÈoara, Romania, and were evaluated for the status of loco-regional lymph nodes. We selected and analyzed some parameters searching for their relationship with lymph node metastases. Results: We identified 79 patients with invasive cutaneous melanomas (29 men and 50 women, mean age 59.36 years). A percentage of 58.3% of melanomas had Breslow tumor thickness >2 mm; 69.6% of melanomas showed a Clark level IV-V. Tumor ulceration was present in 59.5% of melanomas. A mitotic rate of ≥5 mitoses/mm2 was observed in 48.1% of melanomas. Tumor-infiltrating lymphocytes (TILs), non-brisk, were present in 59.5% of cases and 22.8% of patients had satellite/in-transit metastasis (SINTM). Tumor regression was identified in 44.3% of cases. Lymph nodes metastases were found in 43.1% of patients. Statistical analysis showed that lymph node metastases were more frequent in melanomas with Breslow thickness >2 mm (p = 0.0002), high Clark level (p = 0.0026), mitotic rate >5 mitoses/mm2 (p = 0.0044), ulceration (p = 0.0107), lymphovascular invasion (p = 0.0182), SINTM (p = 0.0302), and non-brisk TILs (p = 0.0302). Conclusions: The Breslow thickness >2 mm, high Clark level, high mitotic rate and ulceration are the most important prognostic factors for lymph nodal spread in cutaneous melanomas. However, some melanomas without these clinical-pathological features can have an unexpected, aggressive evolution, which entails the necessity of close and prolonged clinical follow-up of patients, including those with lesions considered without risk.
Assuntos
Melanoma , Neoplasias Cutâneas , Masculino , Humanos , Feminino , Pessoa de Meia-Idade , Melanoma/patologia , Neoplasias Cutâneas/patologia , Metástase Linfática/patologia , Prognóstico , Estudos Retrospectivos , Biópsia de Linfonodo Sentinela , Linfonodos/patologia , Melanoma Maligno CutâneoAssuntos
Falso Aneurisma/etiologia , Mordeduras e Picadas/complicações , Rajidae , Animais , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The aim of our study was to assess the prognostic value of the two new grading systems based on the quantification of tumor budding - TB (GBd) and poorly differentiated clusters - PDCs (PDCs-G) in colorectal carcinomas (CRC). We performed a retrospective study on 71 CRC patients who underwent surgery at the Emergency County Hospital, TimiÈoara. CRC cases were classified based on haematoxylin-eosin slides, using the conventional grading system, GBd and PDCs-G, respectively. We used two-tier and three-tier grading schemes for each system. Subsequently, we evaluated associations with other prognostic factors in CRC. Based on the three-tier GBd (GBd-3t) most cases (34/69, 49.27%) were classified as G3Bd-3t, while based on the conventional grading system, the majority of the cases (55/69, 79.71%) were considered G2. On the other hand, based on the three-tier PDCs-G system (PDCs-G-3t), most cases (31/69, 44.93%) were PDCs-G2-3t. We also noted a more significant association of GBd-3t with other prognostic parameters analyzed, as compared to the conventional grading system. Nodal status, tumor stage, and lymphovascular invasion were strongly correlated with GBd-3t (p=0.0001). Furthermore, we noted that PDCs-G-3t correlated more significantly than the conventional grading system with nodal status (p<0.0001), tumor stage (p=0.0003), lymphovascular invasion (p<0.0001), perineural invasion (p=0.005) and the tumor border configuration (p<0.0001). High GBd and PDCs-G grades correlate directly with other negative prognostic factors in CRC.Thus, these new parameters/classification methods could be used as additional tools for risk stratification in patients with CRC.
Assuntos
Neoplasias Colorretais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Humanos , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Estudos RetrospectivosRESUMO
Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.
Assuntos
Ciclo Celular , Ciclinas/biossíntese , Transcrição Gênica , Células 3T3 , Animais , Composição de Bases , Sequência de Bases , Divisão Celular , Sequência Conservada , Ciclinas/genética , Pegada de DNA , Primers do DNA , Sondas de DNA , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Biblioteca Genômica , Humanos , Luciferases/biossíntese , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência do Ácido Nucleico , Pele/citologiaRESUMO
E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120E4F expression induces cell cycle arrest in G1. Although p21WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120E4F can induce a G1 block in p21(-/-) cells, suggesting that other proteins are essential for the p120E4F-dependent block in G1. We show here that cyclin A promoter activity can be repressed by p120E4F and that this repression correlates with p120E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120E4F-arrested cells from the G1 block. These data identify the cyclin A gene as a cellular target for p120E4F and suggest a mechanism for p120E4F-dependent cell cycle regulation.
Assuntos
Ciclina A/metabolismo , Fase G1/fisiologia , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , Ciclina A/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Fator de Transcrição de Proteínas de Ligação GA , Expressão Gênica , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The instability of oncogenic mRNA such as c-fos mRNA is controlled in cis by sequences present in both the coding and the 3' untranslated regions (3'UTR). The latter contains AU-rich elements (ARE) which, depending on the cellular context, mediate either their rapid degradation or inhibit their translation. These observations, along with the known increase of the life spans of many unstable mRNA promoted by inhibitors of protein synthesis, raise the possibility that both processes are linked. To investigate further the putative involvement of translation in both coding region and ARE-mediated rapid decay of c-fos mRNA, we designed an expression vector based on the use of the ferritin mRNA iron regulatory element (IRE). The latter structure links translation to intracellular iron concentration when inserted at the proper location within the 5'UTR. Rapid degradation of a beta-globin/c-fos 3'UTR construct was prevented by Desferrioxamine, an iron chelator, and facilitated by ferric ammonium citrate or hemin, while stability of other mRNAs not containing the IRE or the ARE were unchanged. The same conclusion was reached when the stability of a c-fos mRNA devoid of ARE was assessed in function of iron availability.
Assuntos
Éxons , Íntrons , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Adenina/metabolismo , Animais , Sequência de Bases , Citoplasma/metabolismo , Estabilidade de Medicamentos , Ferritinas/biossíntese , Ferritinas/genética , Genes fos , Meia-Vida , Humanos , Ferro/metabolismo , Proteínas Reguladoras de Ferro , Camundongos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Ratos , Uracila/metabolismoRESUMO
Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5' element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3' element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.
Assuntos
Ciclo Celular/genética , Ciclina A/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Linfócitos T/metabolismo , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Hidroxiureia/farmacologia , Substâncias Macromoleculares , Camundongos , Microinjeções , Dados de Sequência Molecular , Peso Molecular , Fase de Repouso do Ciclo Celular , Fase S/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies have demonstrated that the promoter of its gene is under transcriptional repression in quiescent cells. Whereas the DNA sequences mediating this effect have been clearly delineated, the nature of the proteins acting in trans is still debated. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). However, the precise role of these proteins has been difficult to assess, since most experiments designed to analyse their function have been carried out in transformed cell lines. Nevertheless, a current model has emerged whereby the role of the p130 protein would be restricted to resting and early G1 cells and p107, absent in quiescent cells, would be involved later in the control of the G1/S transition, whilst pRb would be effective throughout the cell cycle. We show here that cyclin A transcriptional inhibition is relieved in primary fibroblasts from pRb(-/-) embryos and not in fibroblasts from p13O(-/-), p107(-/-) or even p130(-/-)/p107(-/-) double mutant embryos. This suggests a unique role for pRb in controlling the extinction of specific genes in G0, providing thus the first example of non-overlapping functions achieved by the different pocket proteins.
Assuntos
Ciclina A/genética , Regulação para Baixo , Proteínas , Proteína do Retinoblastoma/metabolismo , Animais , Sequência de Bases , Células Cultivadas , DNA , Camundongos , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-Like , Transcrição GênicaRESUMO
Many cells, when cultured in suspension, fail to express cyclin A, a regulatory component of cell cycle kinases cdc2 and cdk2 and as a consequence, do not enter S phase. However, many cell type-specific differences are disclosed between not only normal and transformed cells, but also between cell lines whose proliferation is strictly anchorage-dependent. These apparent discrepancies are seen in established cell lines most probably because of adaptative events that have occurred during cell culture. We have therefore used primary cells to understand how cyclin A transcription is controlled by cell anchorage properties. To this aim, we have used embryonic fibroblasts from either wild type, Rb(-/-) or p107(-/-)/p130(-/-) mice and tested the effect of an ectopic expression of Rb mutants. In the experiments reported here, we show that anchorage-dependent expression of cyclin A (i) is reflected by the in vivo occupancy of a negative DNA regulatory element previously shown to be instrumental in the down regulation of cyclin A transcription in quiescent cells (Cell Cycle Responsive Element: CCRE) (ii) requires a functional Rb but neither p107 nor p130 (iii) mutation of the CCRE abolishes both adhesion-dependent regulation and response to Rb.
Assuntos
Ciclina A/genética , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Proteínas , Proteína do Retinoblastoma/fisiologia , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-LikeRESUMO
Transforming growth factor beta (TGF-beta) is a potent inhibitor of the proliferation of many cell lines. The expression of Cyclin A is down-regulated by TGF-beta 1 in Chinese hamster lung fibroblasts and most of this effect is mediated at the transcriptional level through a cAMP-responsive element (CRE), but does not require a functional cAMP-dependent protein kinase. However, activation of the cAMP pathway in these cells gives rise to a strong inhibition of proliferation, paralleled by a down-regulation of Cyclin A promoter activity. This effect requires the integrity of the CRE, suggesting a role for CRE-binding proteins in late G1/S controls.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , AMP Cíclico/fisiologia , Ciclinas/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Fator de Crescimento Transformador beta/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , Cricetulus , Ciclinas/metabolismo , DNA , Humanos , Microinjeções , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , Ligação ProteicaRESUMO
Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Interferon gama/fisiologia , Receptores de Interferon/fisiologia , Animais , Anticorpos Antifúngicos/sangue , Candida albicans/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Candidíase/sangue , Candidíase/microbiologia , Contagem de Colônia Microbiana , Citocinas/metabolismo , Feminino , Deleção de Genes , Humanos , Imunidade nas Mucosas , Imunoglobulina A Secretora/imunologia , Ativação Linfocitária , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Nasal/microbiologia , Receptores de Interferon/genética , Baço/citologia , Baço/imunologia , Receptor de Interferon gamaRESUMO
Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J. (1994) J. Biol. Chem. 269, 26559-26567). Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower Km value for the co-substrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMP-dependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser32, a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects.
Assuntos
AMP Cíclico/metabolismo , Dopamina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Drosophila/enzimologia , Compostos Ferrosos/farmacologia , Heparina/farmacologia , Ferro/farmacologia , Fosforilação , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
The abnormally high rate of proliferation described in cultured psoriatic fibroblasts could result from inappropriate expression of cellular oncogenes (c-onc) associated to the control of cell division. Four c-onc (c-Myc, c-Myb, c-Erb-B, c-H-Ras) were studied in cultured fibroblasts from lesional and nonlesional psoriatic skin (n = 6) and compared with normal subjects (n = 3). RNA was analyzed by hybridization with nick-translated cloned human DNA probes after extraction by the guanidinium thiocyanate/LiCl procedure, electrophoresis and transfer on nitrocellulose. No difference in the level of c-Myc and c-Myb mRNA could be detected in psoriatic skin compared with controls. N-Ras did not give a detectable signal and c-Erb-B exhibited individual variations which were not linked to the disease. These results do not rule out subtle qualitative changes of these genes; moreover, an abnormal mRNA expression of other c-onc remains possible in psoriasis.
Assuntos
Oncogenes , Psoríase/genética , RNA Mensageiro/genética , Pele , Divisão Celular , Células Cultivadas , Feminino , Fibroblastos/patologia , Humanos , Masculino , Psoríase/patologia , Pele/patologiaRESUMO
Serum stimulation of resting cells is mediated at least in part at the transcriptional level by the activation of numerous genes among which c-fos constitutes a model. Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory protein p62TCF/Elk-1. Both proteins are the targets of multiple phosphorylation events and their role is still unknown in the amino terminus of SRF. While the transcriptional activation domain has been mapped between amino acids 339 and 508, the DNA-binding and the dimerization domains have been mapped to between amino acids 133-235 and 168-235, respectively, no role has been proposed for the amino-terminal portion of the molecule. We demonstrate in the present work that amino acids 95 to 100 contain a stretch of basic amino acids that are sufficient to target a reporter protein to the nucleus. Moreover, this sequence appears to be the only nuclear localization signal operating in SRF. Finally, whereas the global structure around this putative nuclear location signal is reminiscent of what is found in the SV40 T antigen, the casein kinase II phosphorylation site does not determine the rate of cyto-nuclear protein transport of this protein.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Transporte Biológico Ativo , Caseína Quinase II , Linhagem Celular , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , DNA/genética , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Resposta Sérica , Transfecção , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The expression of cyclin A, one of the key regulators of cell cycle progression in association with cdc2/cdk2 protein kinases and which undergoes cyclic accumulation during the cell cycle, has been investigated in CCL39 Chinese hamster lung fibroblasts and in two transformed variants, A71 and 39Py. Whereas A71 (selected after tumor induction in nude mice) is subject to growth arrest (less than 5% of labeled nuclei after 24 h of serum starvation), 39Py (obtained after transformation by polyoma virus) is not (more than 50% of labeled nuclei). In both cells, cyclin A expression was correlated with establishment of S phase, with a progressive deregulation of its G1 controls. This deregulation was not detected with the two early response genes c-fos and c-myc. The kinetics of accumulation of cyclin A lagged behind that of [3H]thymidine incorporation, thereby questioning a direct role for cyclin A in S phase triggering. Moreover, transforming growth factor beta 1, which is known to inhibit alpha-thrombin or fibroblast growth factor-induced mitogenicity in G0-arrested CCL39 cells, is shown here to down-regulate cyclin A expression in both CCL39 and A71 cells but has no effect on 39Py cells. These data establish cyclin A as a sensitive marker for the loss of growth factor requirement.