RESUMO
p58cdc2L1, a protein kinase implicated in apoptotic signaling, is one of eight separate kinases encoded by three tandemly duplicated and linked genes, which we have termed PITSLRE A, B and C. One allele of this complex on chromosome 1 was either deleted or translocated in each of 18 neuroblastoma cell lines with cytogenetically apparent 1p alterations. A protein encoded by this locus, PITSLRE gamma 1, was absent in three of the lines and a smaller, apparently truncated, PITSLRE polypeptide was found in another line. These findings identify a novel gene complex on chromosome 1 that encodes a protein kinase subfamily. We suggest that the PITSLRE locus may harbour one or more tumour suppressor genes affected by chromosome 1p36 modifications in neuroblastoma.
Assuntos
Cromossomos Humanos Par 1 , Genes Supressores de Tumor , Família Multigênica , Neuroblastoma/genética , Proteínas Quinases/genética , Sequências Repetitivas de Ácido Nucleico , Alelos , Criança , Pré-Escolar , Quinases Ciclina-Dependentes , Genes , Humanos , Hibridização in Situ Fluorescente , Lactente , Monossomia , Neuroblastoma/enzimologia , Proteínas Quinases/deficiência , Proteínas Serina-Treonina Quinases , Deleção de Sequência , Translocação Genética , Células Tumorais CultivadasRESUMO
Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.
Assuntos
Caspases/genética , Amplificação de Genes , Inativação Gênica , Genes myc , Neuroblastoma/genética , Antineoplásicos/farmacologia , Apoptose , Caspase 8 , Caspase 9 , Caspases/biossíntese , Criança , Metilação de DNA , Doxorrubicina/farmacologia , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Recombinantes/biossíntese , Retroviridae/genética , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Bcl-2 can both promote and attenuate tumorigenesis. Although the former function is relatively well characterized, the mechanism of the latter remains elusive. We report here that enforced Bcl-2 expression in MCF7 cells stabilizes p53, induces phosphorylation of p53 serine 15 (p53pSer15) and inhibits MCF7 cell growth. Consistent with p53 Ser15 being a target of ataxia telangiectasia mutated protein(ATM)/ATR (ATM- and rad3-related) in the DNA damage response, Bcl-2 activates ATM by inducing ATM Ser1981 phosphorylation, which is accompanied with the phosphorylaton of two additional ATM substrates, Chk2 Thr68 and H2AX Ser139. Downregulation of ATM using a specific small interference RNA fragment (ATMRNAi) abolished Bcl-2-induced p53pSer15 and Bcl-2-mediated growth inhibition of MCF7 cells. Ectopic expression of a dominant-negative p53 mutant, p53175H, partially rescued this growth inhibition. Taken together, these observations demonstrate the contribution of ATM-p53 function to Bcl-2-mediated inhibition of MCF7 cell growth, indicating an ATM-mediated surveillance system for regulating Bcl-2 overexpression. Consistent with this concept, we found that MCF7 cells express Bcl-2 heterogeneously with 34.5% of cells being Bcl-2 negative. In general, Bcl-2-positive MCF7 cells proliferate slower than those of Bcl-2 negative. Thus, we provide evidence suggesting that activation of ATM suppresses Bcl-2-induced tumorigenesis, and that attenuation of ATM function may be an important event in breast cancer progression.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Supressoras de Tumor/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular/genética , Linhagem Celular Tumoral , Primers do DNA , Imunofluorescência , Humanos , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/fisiologiaRESUMO
BACKGROUND: Antimicrobial stewardship programmes (ASPs) have been effective in optimizing antibiotic use for inpatients. However, an emergency department's fast-paced clinical setting can be challenging for a successful ASP. AIM: In April 2015, an ASP was implemented in our emergency department and we aimed to determine its impact on antimicrobial use for outpatients. METHODS: This was a single-centre study comparing the quality of antibiotic prescriptions between a one-year period before ASP implementation (November 2012 to October 2013) and a one-year period after its implementation (June 2015 to May 2016). For each period, antimicrobial prescriptions for all adult outpatients (hospitalized for <24h) were evaluated by an infectious disease specialist and an emergency department physician to assess compliance with local prescribing guidelines. Inappropriate prescriptions were then classified. FINDINGS: Before and after ASP, 34,671 and 35,925 consultations were registered at our emergency department, of which 25,470 and 26,208 were outpatients. Antimicrobials were prescribed in 769 (3.0%) and 580 (2.2%) consultations, respectively (P < 0.0001). There were 484 (62.9%) and 271 (46.7%) (P < 0.0001) instances of non-compliance with guidelines before and after ASP implementation. Non-compliance included unnecessary antimicrobial prescriptions, 197 (25.6%) vs 101 (17.4%) (P<0.0005); inappropriate spectrum, 108 (14.0%) vs 54 (9.3%) (P=0.008); excessive treatment duration, 87 (11.3%) vs 53 (9.1%) (P>0.05); and inappropriate choices, 11 (1.4%) vs 15 (2.6%) (P>0.05). CONCLUSION: The implementation of an ASP markedly decreased the number of unnecessary antimicrobial prescriptions, but had little impact on most other aspects of inappropriate prescribing.
Assuntos
Anti-Infecciosos/uso terapêutico , Gestão de Antimicrobianos , Infecções Comunitárias Adquiridas/tratamento farmacológico , Uso de Medicamentos/normas , Serviço Hospitalar de Emergência , Pacientes Ambulatoriais , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Pesquisa sobre Serviços de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: Clostridium difficile infection (CDI) has become an emerging infectious disease, especially in community settings. Little data is available on its frequency and characteristics in France. We aimed to describe CDI case patients consulting at the emergency department and to compare community-acquired and nosocomial CDIs. PATIENTS AND METHODS: We conducted a multicenter retrospective study over a three-year period of community-acquired and nosocomial CDI case patients seen at the emergency department and compared their characteristics. RESULTS: A total of 2055 patients consulted for diarrhea during the study period and had a stool culture performed: 66 (3.2%) presented with a CDI, of which 28 were community-acquired and 26 were nosocomial. Community-acquired CDI patients had a mean age of 57.7years (18-91), with a sex-ratio of 0.65. At least one risk factor was observed in 24 patients (85.7%), of whom 22 (78.6%) had been prescribed a previous antimicrobial treatment. Diabetes mellitus and renal failure were more frequently observed in patients presenting with nosocomial CDI. They required fluid replacement and needed be to re-hospitalized more often than community-acquired patients. CONCLUSION: Community-acquired CDIs in the emergency department account for approximately 1.4% of patients presenting with diarrhea. One risk factor is present in 85.7% of cases. In our study, their presentation and outcome seemed less severe than nosocomial CDIs.
Assuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/microbiologia , Infecções Comunitárias Adquiridas/microbiologia , Serviço Hospitalar de Emergência/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Infecções por Clostridium/epidemiologia , Infecções Comunitárias Adquiridas/epidemiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Complicações do Diabetes/epidemiologia , Feminino , França/epidemiologia , Humanos , Falência Renal Crônica/epidemiologia , Masculino , Pessoa de Meia-Idade , Readmissão do Paciente , Inibidores da Bomba de Prótons/efeitos adversos , Estudos Retrospectivos , Fatores de Risco , Adulto JovemRESUMO
OBJECTIVES: The proper use of antibiotics is a public health priority to preserve their effectiveness. Little data is available on outpatient antibiotic prescriptions, especially in the emergency department. We aimed to assess the quality of outpatient antibiotic prescriptions in our hospital. PATIENTS AND METHODS: Retrospective monocentric study of antibiotic prescriptions written to adult patients managed at the emergency department without hospitalization (November 15th, 2012-November 15th, 2013). Prescriptions were evaluated by an infectious disease specialist and an emergency physician on the basis of local recommendations compiled from national and international guidelines. RESULTS: A total of 760 prescriptions were reviewed. The most frequent indications were urinary tract infections (n=263; 34.6%), cutaneous infections (n=198; 26.05%), respiratory tract infections (n=101; 13.28%), and ENT infections (n=62; 8.15%). The most frequently prescribed antibiotics were fluoroquinolones (n=314; 40.83%) and amoxicillin-clavulanic acid (n=245; 31.85%). Overall, 455 prescriptions (59.86%) did not comply with guidelines. The main reasons for inadequacy were the absence of an indication for antibiotic therapy (n=197; 40.7%), an inadequate spectrum of activity, i.e. too broad, (n=95; 19.62%), and excessive treatment duration (n=87; 17.97%). Rates of inadequate prescriptions were 82.26% for ENT infections, 71.2% for cutaneous infections, 46.53% for respiratory tract infections, and 38.4% for urinary tract infections. CONCLUSION: Antibiotic prescriptions written to outpatients in the emergency department are often inadequate. Enhancing prescribers' training and handing out guidelines is therefore necessary. The quality of these prescriptions should then be re-assessed.
Assuntos
Antibacterianos/uso terapêutico , Prescrições de Medicamentos/estatística & dados numéricos , Serviço Hospitalar de Emergência , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Bacterianas/tratamento farmacológico , Infecções Comunitárias Adquiridas/tratamento farmacológico , Grupos Diagnósticos Relacionados , Uso de Medicamentos , Feminino , França , Fidelidade a Diretrizes , Hospitais de Ensino/estatística & dados numéricos , Humanos , Prescrição Inadequada , Masculino , Erros de Medicação , Pessoa de Meia-Idade , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Adulto JovemRESUMO
Herpes simplex encephalitis is rarely caused by herpes simplex virus type 2 (HSV-2) after the neonatal period. The pathogenesis of HSV-2 encephalitis is not known and its treatment has not been discussed. We report a case of mild meningoencephalitis secondary to HSV-2 primary infection after sexual risk behaviour in a healthy young man. The diagnosis was established upon clinical, biological and electroencephalographic criteria. Aciclovir treatment led to rapid clinical improvement. This case highlights HSV-2 as a rare cause of meningoencephalitis, and questions the management of this rare manifestation of HSV-2 infection.
Assuntos
Encefalite por Herpes Simples/tratamento farmacológico , Encefalite por Herpes Simples/virologia , Herpes Genital/complicações , Herpesvirus Humano 2 , Meningoencefalite/tratamento farmacológico , Meningoencefalite/virologia , Aciclovir/uso terapêutico , Adulto , Anticorpos Antivirais/sangue , Antivirais/uso terapêutico , Encéfalo/diagnóstico por imagem , Encefalite por Herpes Simples/diagnóstico , Herpes Genital/virologia , Herpes Simples/diagnóstico , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/imunologia , Humanos , Imunocompetência , Masculino , Meningoencefalite/diagnóstico , Meningoencefalite/etiologia , Reação em Cadeia da Polimerase , RadiografiaRESUMO
Avian cyclin D2 (Cyl D2)-encoding cDNA clones were isolated from a chicken UG9 T-cell lambda gt10 library. Sequence analysis revealed a high degree of sequence conservation with both the mouse and human Cyl D2, and somewhat lower similarity with the mouse and human Cyl D1 and D3. The homology is highest between species in the Cyl-box domain which is well conserved among human, mouse and chicken. A single 6.0-kb CYL2 mRNA is produced in both avian B- and T-cells, as expected.
Assuntos
Ciclinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Ciclina D2 , DNA Complementar/genética , Humanos , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The human CASP8 gene, whose product is also known as caspase 8 and FLICE, encodes an interleukin-1beta converting enzyme (ICE)-related cysteine protease that is activated by the engagement of several different death receptors. Caspase 8 is immediately recruited to the Fas receptor once it oligomerizes, and its protease activity is crucial for the apoptotic response generated by the resulting death-inducing signaling complex (DISC). We report here that the CASP8 gene contains at least 11 exons spanning approximately 30kb on human chromosome band 2q33-34. This region of human chromosome 2 was previously reported as the location of the CASP10 gene, whose product is closely related to caspase 8. Chromosome 2 band q33-34 is also involved in tumorigenesis, with loss of heterogeneity (LOH) being reported in a number of tumors. We also report EcoRI and HindIII polymorphisms that may prove to be useful in disease analysis. Both caspases 8 and 10 contain long pro-domains with duplicated death effector domains (DEDs), as well as their corresponding cysteine protease catalytic domains. Thus, it appears that CASP8 and CASP10 have evolved by tandem gene duplication, much like the CASP1, CASP4 and CASP5 gene cluster on human chromosome 11q22.2-22.3.
Assuntos
Caspases/genética , Cromossomos Humanos Par 2 , Éxons , Íntrons , Northern Blotting , Southern Blotting , Caspase 8 , Caspase 9 , Mapeamento Cromossômico , DNA Complementar , Duplicação Gênica , Humanos , Hibridização in Situ Fluorescente , Polimorfismo de Fragmento de RestriçãoRESUMO
The human PITSLRE protein kinases (PK), members of the p34cdc2 kinase family named according to the single amino acid (aa) code of an important (PSTAIRE) regulatory region [Meyerson et al., EMBO J. 11 (1992) 2909-2917], are candidate tumor suppressor gene(s) localized to human chromosome 1p36.2 and a syntenic region of mouse chromosome 4 [Lahti et al., Nature Genet. 7 (1994) 370-375; Mock et al., Mammal. Genome 5 (1994) 191-192]. At least ten isoforms of this PK family are expressed from three duplicated and tandemly linked genes in humans [Xiang et al., J. Biol. Chem. 269 (1994) 15786-15794]. We have now isolated two different species of PITSLRE PK cDNAs from chicken that encode identical polypeptides, but are clearly expressed from different genes, based on nucleotide (nt) differences. Isolation of one of the corresponding chicken PITSLRE PK genes confirms that only one of the two species of PITSLRE mRNA is expressed from this gene. Comparison of the predicted avian PITSLRE PK aa sequence to human and mouse sequences shows a high degree of sequence identity (> 91%). Like humans, the PITSLRE PK genes in chickens must be closely linked, based on fluorescent in situ hybridization (FISH) localization of these genes to a single chicken microchromosome. PITSLRE PK mRNAs are expressed in two avian B- and T-cell lines. These results suggest that the PITSLRE PK gene family has been well conserved evolutionarily, that the gene duplication observed in humans is not a recent event, and that expression of redundant PITSLRE mRNAs is observed in different vertebrate species.
Assuntos
Galinhas/genética , Regulação Enzimológica da Expressão Gênica/genética , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Linfócitos B/química , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Quinases Ciclina-Dependentes , Fibroblastos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Família Multigênica , Proteínas Quinases/química , Proteínas Serina-Treonina Quinases , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Linfócitos T/química , Linfócitos T/enzimologiaRESUMO
In order to prevent the serious pancreatic fistulae which complicate anastomoses between the pancreatic tail and a jejunal loop after cephalic duodenopancreatectomy, we propose a pancreatic fluid derivation on a temporary drain equivalent to a controlled fistula of Wirsung's duct. This already known technique consists of a punctiform anastomosis on an Escat's supporting drain between the main pancreatic duct and the jejunum. No complication occurs when the drain is removed 12 days later, as shown by a series of 24 cephalic duodeno-pancreatectomies where this technique was used.
Assuntos
Ductos Pancreáticos/cirurgia , Drenagem/métodos , Duodeno/cirurgia , Humanos , Jejuno/cirurgia , Pancreatectomia , Fístula Pancreática/prevenção & controle , Complicações Pós-Operatórias/prevenção & controleRESUMO
The different techniques used for temporary jejunostomy are described. They are governed by the presence or absence of utilizable stomach. The advantages and drawbacks of each technique are examined. The simplest and most reliable one is transgastric jejunostomy. Two variants of jejunostomy after total gastrectomy and oesophagectomy are described.
Assuntos
Jejuno/cirurgia , Nutrição Enteral/métodos , Esôfago/cirurgia , Gastrectomia , Humanos , Métodos , EstômagoRESUMO
An angioplasty balloon was used to dilate 23 benign ureteral stenoses in 21 patients. The anterograde approach through percutaneous puncture of the kidney was used in 19 cases. Following dilatation, an 8 F to 24 F catheter was left in the ureter. Dilatation was successful in 13 out of 23 cases (56%) followed up for 1 month to 2 years. It failed in 8 cases, and 2 patients are undergoing ureteral remodelling. Thus, dilatation of benign ureteral stenoses after percutaneous renal puncture was effective in about 60% of the cases. The follow-up period is still too short for us to determine the indications of this technique according to the cause and duration of the stenosis.
Assuntos
Obstrução Ureteral/terapia , Adolescente , Adulto , Idoso , Angioplastia com Balão/instrumentação , Constrição Patológica/terapia , Dilatação/instrumentação , Humanos , Pessoa de Meia-Idade , Complicações Pós-Operatórias/terapia , Fatores de Tempo , Obstrução Ureteral/etiologiaRESUMO
To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.
Assuntos
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Piridinas/farmacologia , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/patologia , Roscovitina , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Hérnia Diafragmática/complicações , Hérnia Hiatal/complicações , Volvo Gástrico/etiologia , Idoso , Feminino , Hérnia Hiatal/diagnóstico por imagem , Hérnia Hiatal/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia , Volvo Gástrico/diagnóstico por imagem , Volvo Gástrico/cirurgiaRESUMO
Caffeine (CAF) but not 3-isobutyl 1-methyl-xanthine (IBMX) displayed a strong DNA anti-repair effect in G2 prophase, as estimated by the frequency of abnormal chromosomes in anaphase-telophase found shortly after treatment. IBMX is more effective than CAF in inhibiting cytokinesis, while the binucleate cells formed by a short treatment with any of these two xanthines have similar cycle kinetics.
Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Cafeína/farmacologia , Reparo do DNA/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Allium/citologia , Allium/efeitos dos fármacos , Ciclo Celular , Células Cultivadas , Prófase/efeitos dos fármacosRESUMO
The terminal end of the short arm of human chromosome 1, 1p36.3, is frequently deleted in a number of tumors and is believed to be the location of multiple tumor suppressor genes. Thus far, a bona fide tumor suppressor gene from this region has not been identified. The isolation and characterization of new 1p36 genes is, therefore, of some interest. Two novel matrix metalloproteinase genes, MMP21 and MMP22, have been identified in the Cdc2L1-2 locus, which spans approximately 120 kb on 1p36.3. These genes encode novel metalloproteinases that contain prepro, catalytic, cysteine-rich, interleukin-1 receptor-related, and proline-rich domains. Their catalytic domains are most closely related to stromelysin-3 and contain the consensus HEXXH zinc-binding region required for enzyme activation, while their cysteine-rich domains appear to be related to a number of human, mouse, and Caenorhabditis elegans metalloproteinase sequences. Of some possible interest is the absence of a highly conserved cysteine residue in the proenzyme domain, the so-called "cysteine switch," which has been shown to be involved in the autocatalytic activation of many metalloproteinases. The MMP genes are located less than 1 kb from the 3' regions of Cdc2L1 and Cdc2L2, suggesting that the MMP and Cdc2L genes are part of a larger region that has been duplicated. Finally, the MMP21/22 genes express multiple mRNAs, some of which are derived by alternative splicing, in a tissue-specific manner.
Assuntos
Cromossomos Humanos Par 1/genética , Ligação Genética , Metaloendopeptidases/química , Metaloendopeptidases/genética , Sequência de Aminoácidos , Northern Blotting , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , RNA/análise , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Mammalian glycosyltransferases have been implicated in a wide variety of functions besides N-linked glycosylation, including developmental processes. For this reason, we studied the effects of cell cycle and entry into the cell cycle on beta 1-4-galactosyltransferase gene expression. In this study we report that beta 1-4-galactosyltransferase (GalTase) gene expression is, indeed, regulated during the normal cell cycle, peaking during late G1-, S, and early G2 phase of the cell cycle. In addition, GalTase gene expression is regulated in a manner that resembles other "early response" genes such as jun and fos upon reentry into the cell cycle from quiescence. Finally, we show that the GalTase gene is differentially expressed during murine embryogenesis and in terminally differentiated adult tissues. It is most abundant in testis, followed by skeletal muscle and spleen. The reasons for this pattern of differential expression in adult tissues are unknown. These studies should provide important new information regarding GalTase gene expression, its regulation, and its potential link to other developmental functions.
Assuntos
Ciclo Celular/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , N-Acetil-Lactosamina Sintase/genética , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , Cicloeximida/farmacologia , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Músculos/enzimologia , Mapeamento por Restrição , Baço/enzimologia , Testículo/enzimologiaRESUMO
We have developed a reliable and reproducible method to induce synchrony of the DNA synthetic cycle in the Kinetoplastida. The method involves treatment of cultures with 20 mM hydroxyurea (HU) and fetal bovine serum. Both stationary-phase and exponential-phase cultures can be synchronized. However, in the case of exponential-phase cultures the population doubling time and rate of DNA synthesis of the population influenced the time of exposure to HU. The treatment of kinetoplastids with 20 mM HU did not adversely affect the cells as judged by oxygen consumption, RNA, and protein content. We postulate that the requirement for high HU levels, which would be toxic to vertebrate cells, may be due to a lower affinity of kinetoplastid ribonucleotide reductase, the target enzyme for HU. Some of the kinetoplastids are pathogens of man and his food chain. Consequently, the development of a reliable technique for synchronization of the kinetoplastids should not only permit a detailed analysis of their cellular and molecular biology but provide a means to collect and characterize biochemical and immunochemical substances relevant to the infectious process.