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1.
Stat Med ; 32(1): 124-30, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22825881

RESUMO

The analysis of gap times in recurrent events requires an adjustment to standard marginal models. One can perform this adjustment with a modified within-cluster resampling technique; however, this method is computationally intensive. In this paper, we describe a simple adjustment to the standard Cox proportional hazards model analysis that mimics the intent of within-cluster resampling and results in similar parameter estimates. This method essentially weights the partial likelihood contributions by the inverse of the number of gap times observed within the individual while assuming a working independence correlation matrix. We provide an example involving recurrent mammary tumours in female rats to illustrate the methods considered in this paper.


Assuntos
Análise por Conglomerados , Funções Verossimilhança , Modelos de Riscos Proporcionais , Animais , Simulação por Computador , Feminino , Neoplasias Mamárias Animais/patologia , Recidiva Local de Neoplasia/prevenção & controle , Ratos , Fatores de Tempo
2.
Stat Med ; 32(14): 2374-89, 2013 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-23172770

RESUMO

Methods for dealing with tied event times in the Cox proportional hazards model are well developed. Also, the partial likelihood provides a natural way to handle covariates that change over time. However, ties between event times and the times that discrete time-varying covariates change have not been systematically studied in the literature. In this article, we discuss the default behavior of current software and propose some simple methods for dealing with such ties. A simulation study shows that the default behavior of current software can lead to biased estimates of the coefficient of a binary time-varying covariate and that two proposed methods (Random Jitter and Equally Weighted) reduce estimation bias. The proposed methods can be easily implemented with existing software. The methods are illustrated on the well-known Stanford heart transplant data and data from a study on intimate partner violence and smoking.


Assuntos
Modelos de Riscos Proporcionais , Viés , Bioestatística , Simulação por Computador , Violência Doméstica/estatística & dados numéricos , Feminino , Transplante de Coração/mortalidade , Transplante de Coração/estatística & dados numéricos , Humanos , Masculino , Fumar , Software , Processos Estocásticos , Análise de Sobrevida , Fatores de Tempo
3.
Curr Opin Cell Biol ; 11(6): 678-82, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10600708

RESUMO

Recent advances in identifying molecular signals that dictate liver development and differentiation have come from analysis of several experimental systems including the developing embryo, cell and tissue culture, knockout mice and transplantation of hepatic precursor cells. Fibroblast growth factors and several families of transcription factors including hepatocyte nuclear factors 1, 3 and 4 and CCAAT/enhancer-binding protein have been shown to be important components of the differentiation process that culminates in the fully functional liver.


Assuntos
Fígado/embriologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Proteínas de Ligação a DNA/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Fator 3-beta Nuclear de Hepatócito , Fator 4 Nuclear de Hepatócito , Fígado/metabolismo , Camundongos , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Isoformas de Proteínas , Transdução de Sinais , Fatores de Transcrição/fisiologia
4.
J Exp Med ; 188(6): 1173-84, 1998 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-9743535

RESUMO

Cytokines stimulate granulopoiesis through signaling via receptors whose expression is controlled by lineage-specific transcription factors. Previously, we demonstrated that granulocyte colony-stimulating factor (G-CSF) receptor mRNA was undetectable and granulocyte maturation blocked in CCAAT enhancer binding protein alpha (C/EBPalpha)-deficient mice. This phenotype is distinct from that of G-CSF receptor-/- mice, suggesting that other genes are likely to be adversely affected by loss of C/EBPalpha. Here we demonstrate loss of interleukin 6 (IL-6) receptor and IL-6-responsive colony-forming units (CFU-IL6) in C/EBPalpha-/- mice. The observed failure of granulopoiesis could be rescued by the addition of soluble IL-6 receptor and IL-6 or by retroviral transduction of G-CSF receptors, demonstrating that loss of both of these receptors contributes to the absolute block in granulocyte maturation observed in C/EBPalpha-deficient hematopoietic cells. The results of these and other studies suggest that additional C/EBPalpha target genes, possibly other cytokine receptors, are also important for the block in granulocyte differentiation observed in vivo in C/EBPalpha-deficient mice.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Granulócitos/fisiologia , Hematopoese , Proteínas Nucleares/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/biossíntese , Receptores de Interleucina-6/biossíntese , Fatores de Transcrição/fisiologia , Regulação para Cima/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/genética , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Elementos Facilitadores Genéticos , Feto , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-6/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores de Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Receptores de Fator Estimulador de Colônias de Granulócitos/deficiência , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/deficiência , Receptores de Interleucina-6/genética , Solubilidade , Fatores de Transcrição/genética , Regulação para Cima/genética
5.
Spinal Cord ; 48(1): 60-4, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19581916

RESUMO

STUDY DESIGN: Longitudinal, non-experimental. OBJECTIVES: To determine the following: (1) prevalence of supplement use in a representative sample of the chronic spinal cord injury (SCI) population; (2) most frequently consumed supplements; and (3) characteristics of consistent supplement users. SETTING: Ontario, Canada. METHODS: A structured questionnaire was used to collect demographic information from 77 community-dwelling adults with chronic SCI (50.6% paraplegia, 81.8% male, 42.4 + or - 11.9 years, body mass index (BMI) 25.4 + or - 5.1 kg m(-2)). A standardized form was used to record dietary intake, including supplements, in the previous 24 h, at three time points (baseline, 6 months and 18 months). Logistic regression and multivariate logistic regression were used to determine which characteristic(s) was (were) associated with consistent supplement use. RESULTS: Seventy-one percent of the sample reported using supplements at least once, with 50.6% being classified as consistent supplement users (at least twice across the three time points). The top three supplements consumed were multivitamins (25%), calcium (20%) and vitamin D (16%). Supplement use status was not associated with gender, level of injury, age, education, physical activity, BMI, smoking or alcohol intake. CONCLUSIONS: Dietary supplement use was common in our sample of individuals with long-standing SCI, but no common characteristics distinguished users from non-users. We suggest that health practitioners be aware of the high dietary supplement use in this population so that they can probe for type, dose and frequency, as supplements may have an important influence on dietary assessment results.


Assuntos
Cálcio/administração & dosagem , Suplementos Nutricionais/estatística & dados numéricos , Traumatismos da Medula Espinal , Vitaminas/administração & dosagem , Adulto , Idoso , Antropometria/métodos , Índice de Massa Corporal , Doença Crônica , Feminino , Inquéritos Epidemiológicos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Adulto Jovem
6.
J Cell Biol ; 103(3): 787-93, 1986 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3017995

RESUMO

Human hepatoma cells mimic the acute phase response after treatment with monocyte-conditioned medium. Levels of secreted fibrinogen, alpha-1 acid glycoprotein, C-reactive protein, haptoglobin, and the third component of complement were elevated compared with control levels after 48 h of incubation with conditioned supernatant medium from an enriched fraction of normal peripheral monocytes. Albumin levels declined and alpha-1 antitrypsin remained unchanged. Levels of specific mRNA were measured by hybridization to slot blots and Northern blots and changed in correspondence with protein alterations. Interleukin-1 and tumor necrosis factor stimulated the third component of complement, but did not elevate any other member of the acute phase group and were therefore only partially active in this system. The identification of an in vitro model of the human acute phase response will permit analysis of the molecular basis for coordinate regulation of this group of facultative genes.


Assuntos
Proteína C-Reativa/biossíntese , Glicoproteínas/farmacologia , Interleucina-1/farmacologia , Fígado/efeitos dos fármacos , Linfocinas/farmacologia , Carcinoma Hepatocelular , Linhagem Celular , DNA , Humanos , Inflamação , Neoplasias Hepáticas , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa
7.
Science ; 185(4154): 859-62, 1974 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-4367319

RESUMO

Murine hepatoma cells that secreted mouse serum albumin were fused with human leukocytes that did not produce albumin. The resulting hybrids secreted both mouse and human serum albumin, as demonstrated by immuno-electrophoretic techniques. The activation of the human genome suggests that mapping genes governing specialized functions in somatic cell hybrids may be accomplished by using adifferentiated human cells as a parental line.


Assuntos
Genes , Células Híbridas/metabolismo , Fenótipo , Albumina Sérica/biossíntese , Animais , Carcinoma Hepatocelular/enzimologia , Linhagem Celular , Mapeamento Cromossômico , Repressão Enzimática , Ligação Genética , Genótipo , Guanina , Humanos , Células Híbridas/enzimologia , Hipoxantinas , Imunoeletroforese , Leucócitos/enzimologia , Neoplasias Hepáticas , Camundongos , Pentosiltransferases/isolamento & purificação , Albumina Sérica/metabolismo
8.
Science ; 176(4042): 1429-31, 1972 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-4113665

RESUMO

The segregation of the human peptidase-C phenotype in five different series of human-mouse hybrid clones was examined. The chromosome constitution of these hybrids was determined by quinacrine mustard fluorescence, Giemsa banding, and constitutive heterochromatin staining. That the clones could be classified without exception either as human peptidase C positive/ A-1 positive (14 clones), or as peptidase C negative/ A-1 negative (12 clones) indicates that peptidase C can be assigned to the human A-i chromosome. Data from an extensive series of human-mouse clones used provide support for the syntenic association between peptidase C and phosphoglucomutase-1 and by inference a linkage of both to Rh factor group.


Assuntos
Mapeamento Cromossômico , Cromossomos Humanos 1-3 , Genes , Peptídeo Hidrolases/biossíntese , Sistema do Grupo Sanguíneo Rh-Hr/biossíntese , Animais , Células Clonais , Ligação Genética , Humanos , Células Híbridas/enzimologia , Cariotipagem , Camundongos , Quinacrina , Coloração e Rotulagem
9.
Science ; 219(4590): 1335-7, 1983 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-6828861

RESUMO

Flow cytometry revealed that, in the presence of tritiated thymidine, a greater percentage of phytohemagglutinin-stimulated lymphocytes from old human donors were arrested in the G2 or M phase than were cells from young donors. Furthermore, lymphocytes from old donors showed significantly more chromosomal damage than did lymphocytes from young donors. Lymphocyte cultures from old or young donors not exposed to tritiated thymidine had the same percentage of cycling lymphocytes in G2 or M, although the number of lymphocytes stimulated by phytohemagglutinin to enter the cell cycle was significantly lower in cultures from old donors. Thus, the impaired incorporation of tritiated thymidine by phytohemagglutinin-exposed lymphocytes from old humans reflects both an impaired proliferative response to phytohemagglutinin and an increased sensitivity to the radiobiological effects of tritiated thymidine.


Assuntos
Envelhecimento , Ciclo Celular/efeitos da radiação , Cromossomos/efeitos da radiação , Adulto , Idoso , Cromossomos/ultraestrutura , Reparo do DNA/efeitos da radiação , Humanos , Pessoa de Meia-Idade , Timidina/efeitos adversos , Trítio
10.
Science ; 269(5227): 1108-12, 1995 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-7652557

RESUMO

Mice homozygous for the targeted deletion of the c/ebp alpha gene, which expresses the CCAAT/enhancer-binding protein alpha (C/EBP alpha), did not store hepatic glycogen and died from hypoglycemia within 8 hours after birth. In these mutant mice, the amounts of glycogen synthase messenger RNA were 50 to 70 percent of normal and the transcriptional induction of the genes for two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was delayed. The hepatocytes and adipocytes of the mutant mice failed to accumulate lipid and the expression of the gene for uncoupling protein, the defining marker of brown adipose tissue, was reduced. This study demonstrates that C/EBP alpha is critical for the establishment and maintenance of energy homeostasis in neonates.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Metabolismo Energético , Proteínas Nucleares/fisiologia , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Animais Recém-Nascidos , Glicemia/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica , Glucose-6-Fosfatase/genética , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Homeostase , Humanos , Canais Iônicos , Metabolismo dos Lipídeos , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais , Proteínas Nucleares/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , RNA Mensageiro/metabolismo , Albumina Sérica/genética , Proteína Desacopladora 1
11.
Curr Opin Genet Dev ; 5(5): 565-70, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8664543

RESUMO

The role of C/EBPalpha in the developmental expression of a subset of genes governing essential metabolic processes has recently been elucidated using a mutant mouse model that lacks this transcription factor. The mutation results in a failure of the liver and white and brown adipose tissue to develop normal metabolic functions in the immediate perinatal period, including hepatic glycogen synthesis and gluconeogenesis and the synthesis and deposition of triglyceride in adipose tissue. The metabolic alterations are very similar to those of human infants born prior to the third trimester and suggest that many of the medical complications of prematurity are a result of the lack of activation of C/EBPalpha in development.


Assuntos
Tecido Adiposo Marrom/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout
13.
Mol Cell Biol ; 18(12): 7269-77, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9819413

RESUMO

Members of the C/EBP (CCAAT/enhancer binding protein) family of transcription factors play important roles in mediating the acute-phase response (APR), an inflammatory process resulting from infection and/or tissue damage. Among the C/EBP family of proteins, C/EBPbeta and -delta were thought to be the primary mediators of the APR. The function of C/EBPalpha in the APR has not been fully characterized to date. Here, we investigate the role of C/EBPalpha in the APR by using neonatal mice that lack C/EBPalpha expression. Northern blot analysis of acute-phase protein gene expression in neonatal mice treated with purified bacterial lipopolysaccharide or recombinant interleukin 1beta as an inflammation stimulus showed a strong APR in wild-type mice, but a response in C/EBPalpha null animals was completely lacking. The C/EBPalpha knockout and wild-type mice demonstrated elevations in C/EBPbeta and -delta mRNA expression and DNA binding as well as increased DNA binding of NF-kappaB, all of which are known to be important in the APR. Null mice, however, failed to activate STAT3 binding in response to lipopolysaccharide. Our results provide the first evidence that C/EBPalpha is absolutely required for the APR in neonatal mice, is involved in STAT3 regulation, and cannot be compensated for by other C/EBP family members.


Assuntos
Reação de Fase Aguda/genética , Proteínas de Ligação a DNA/genética , Inflamação/genética , Proteínas Nucleares/genética , Proteínas de Fase Aguda/genética , Reação de Fase Aguda/induzido quimicamente , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/metabolismo , Genótipo , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/genética , RNA Mensageiro/genética , Fator de Transcrição STAT3 , Transativadores/metabolismo , Regulação para Cima/genética
14.
Mol Cell Biol ; 19(3): 1695-704, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10022857

RESUMO

C/EBPalpha and C/EBPbeta are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPbeta has been reported to produce four isoforms: full-length 38-kDa C/EBPbeta, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. In this report, we investigated the mechanisms by which C/EBPbeta isoforms are generated in the liver and in cultured cells. Using an in vitro translation system, we found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism-specific proteolytic cleavage of full-length C/EBPbeta. Studies of mice in which the C/EBPalpha gene had been deleted (C/EBPalpha-/-) showed that the regulation of C/EBPbeta proteolysis is dependent on C/EBPalpha. The induction of C/EBPalpha in cultured cells leads to induced cleavage of C/EBPbeta to generate the LIP isoform. We characterized the cleavage activity in mouse liver extracts and found that the proteolytic cleavage activity is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBPalpha-/- animals. The lack of cleavage activity in the livers of C/EBPalpha-/- mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration showed that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. We propose that there are two mechanisms by which C/EBPbeta isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPalpha-dependent, specific proteolytic cleavage of full-length C/EBPbeta. The latter mechanism implicates C/EBPalpha in the regulation of posttranslational generation of the dominant negative C/EBPbeta isoform, LIP.


Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/biossíntese , Animais , Animais Recém-Nascidos , Proteína beta Intensificadora de Ligação a CCAAT , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Sistema Livre de Células , Inibidores de Cisteína Proteinase/farmacologia , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dimerização , Ativação Enzimática , Fígado/metabolismo , Fígado/fisiologia , Regeneração Hepática , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Oligopeptídeos/farmacologia , Biossíntese de Proteínas , Isoformas de Proteínas , Inibidores de Serina Proteinase/farmacologia , Extratos de Tecidos
15.
Mol Cell Biol ; 19(4): 2936-45, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10082561

RESUMO

We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBPalpha knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level was reduced in the knockout animals compared to that in wild-type littermates. Here we show additional cell cycle-associated proteins that are affected by C/EBPalpha. We have observed that C/EBPalpha controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBPalpha mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPalpha knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fígado/embriologia , Proteínas Nucleares/biossíntese , Proteínas Nucleares/metabolismo , Fase S/fisiologia , Fatores de Transcrição/biossíntese , Animais , Animais Recém-Nascidos , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ciclo Celular/análise , Núcleo Celular/metabolismo , Ciclina A/análise , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/análise , Ciclinas/biossíntese , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/análise , Proteína do Retinoblastoma/análise , Proteína 1 de Ligação ao Retinoblastoma , Proteína p107 Retinoblastoma-Like , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Fator de Transcrição DP1 , Fatores de Transcrição/análise , Fatores de Transcrição/química
16.
Mol Cell Biol ; 8(9): 3857-63, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2464743

RESUMO

We characterized alpha-amylase expression in the hepatoma cell line Hepa 1-6 and in normal mouse liver. Both Amy-1 and Amy-2 were expressed in Hepa 1-6 and were regulated by glucocorticoids. Transcription in the hepatoma cells was initiated at the same start sites as in mouse tissues. Glucocorticoid treatment increased the abundance of Amy-1 and Amy-2 transcripts by 10 to 20-fold. This increase was detected within 4 h and was maximal by 24 h. The pattern of amylase expression in this hepatoma cell line accurately reflects amylase expression in the liver in vivo. During liver development, we observed a large increase in the abundance of Amy-1 transcripts just before birth, at a time when circulating glucocorticoids are also elevated. Adult mouse liver expressed Amy-1 and Amy-2 at levels comparable to those of fully induced hepatoma cells. Liver is thus a likely source of both amylase isozymes in mouse serum. These studies demonstrate that Amy-2 expression is not limited to the pancreas but also occurs at a low level in liver cells.


Assuntos
Amilases/genética , Dexametasona/farmacologia , Neoplasias Hepáticas Experimentais/genética , Fígado/enzimologia , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Amilases/biossíntese , Animais , Células Cultivadas , Regulação da Expressão Gênica , Genes , Isoenzimas/biossíntese , Isoenzimas/genética , Neoplasias Hepáticas Experimentais/enzimologia , Camundongos , Especificidade de Órgãos , Pâncreas/enzimologia , RNA Mensageiro/efeitos dos fármacos , Valores de Referência
17.
Mol Cell Biol ; 13(7): 4233-41, 1993 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7686619

RESUMO

Alu repeats are short interspersed elements (SINEs) of dimeric structure whose transposition sometimes leads to heritable disorders in humans. Human cells contain a poly(A)- small cytoplasmic transcript of -120 nucleotides (nt) homologous to the left Alu monomer. Although its monomeric size indicates that small cytoplasmic Alu (scAlu) RNA is not an intermediary of human Alu transpositions, a less abundant poly(A)-containing Alu transcript of dimeric size and specificity expected of a transposition intermediary is also detectable in HeLa cells (A. G. Matera, U. Hellmann, M. F. Hintz, and C. W. Schmid, Mol. Cell. Biol. 10:5424-5432, 1990). Although its function is unknown, the accumulation of Alu RNA and its ability to interact with a conserved protein suggest a role in cell biology (D.-Y. Chang and R. J. Maraia, J. Biol. Chem. 268:6423-28, 1993). The relationship between the -120- and -300-nt Alu transcripts had not been determined. However, a B1 SINE produces scB1 RNA by posttranscriptional processing, suggesting a similar pathway for scAlu. An Alu SINE which recently transposed into the neurofibromatosis 1 locus was expressed in microinjected frog oocytes. This neurofibromatosis 1 Alu produced a primary transcript followed by the appearance of the scAlu species. 3' processing of a synthetic -300-nt Alu RNA by HeLa nuclear extract in vitro also produced scAlu RNA. Primer extension of scAlu RNA indicates synthesis by RNA polymerase III. HeLa-derived scAlu cDNAs were cloned so as to preserve their 5'-terminal sequences and were found to correspond to polymerase III transcripts of the left monomeric components of three previously identified Alu SINE subfamilies. Rodent x human somatic cell hybrids express Alu RNAs whose size, heterogeneous length, and chromosomal distribution indicate their derivation from SINEs. The coexpression of dimeric and monomeric Alu RNA in several hybrids suggests a precursor-product relationship.


Assuntos
Citoplasma/metabolismo , RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , DNA , Células HeLa , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , RNA Polimerase III/metabolismo , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Xenopus laevis
18.
Mol Cell Biol ; 11(9): 4423-30, 1991 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1715019

RESUMO

Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT was expressed when transferred to pancreatic cell lines but not when transferred to GPT-fibroblast (L) cells or hepatoma cells. When the transformed L cells and hepatoma cells were fused with pancreatic cell lines, GPT was activated in the hybrid cells. Endogenous pancreas-specific genes from the L-cell and hepatoma parents were also activated in the hybrids. In addition, a pancreas-specific nuclear protein, PTF1, was produced in pancreatic and hybrid cells, correlating with GPT expression. The transformed L cells and hepatoma cells thus contained a nonexpressed construct which could be activated in trans by factors present in pancreatic cells. The hepatoma hybrid also continued to produce albumin, demonstrating the coexpression of liver and pancreas-specific genes in the hybrid-cell population. Cell lines carrying the amylase/elastase/GPT construct may be useful as a selection system for cloning of pancreatic transcription activators.


Assuntos
Amilases/genética , Carboxipeptidases , Pâncreas/enzimologia , Elastase Pancreática/genética , Ativação Transcricional , Albuminas/metabolismo , Animais , Southern Blotting , Carboxipeptidase B , Clonagem Molecular , Elementos Facilitadores Genéticos , Humanos , Células Híbridas , Neoplasias Hepáticas Experimentais , Camundongos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Proteínas/metabolismo , Transcrição Gênica , Transfecção , Transformação Genética , Células Tumorais Cultivadas
19.
Mol Cell Biol ; 15(3): 1192-202, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7862113

RESUMO

The human C/EBP alpha gene promoter shares significant sequence homology with that of the mouse but has a different mechanism of autoregulation. Activation of the murine promoter by direct binding of C/EBP alpha to a site within 200 bp of the transcriptional start was shown to elevate activity by approximately threefold (R. J. Christy, K. H. Kaestner, D. E. Geiman, and M. D. Lane, Proc. Natl. Acad. Sci. USA 88:2593-2597, 1991; K. Legraverend, P. Antonson, P. Flodby, and K. G. Xanthapoulos, Nucleic Acids Res. 21:1735-1742, 1993). Unlike its murine counterpart, the human C/EBP alpha gene promoter does not contain a cis element that binds the C/EBP alpha protein. Neither C/EBP alpha nor C/EBP beta (NF-Il-6) binds the human C/EBP alpha promoter within 437 bp. However, cotransfection studies show that C/EBP alpha stimulates transcription of a reporter gene driven by 437 bp of the C/EBP alpha promoter. Our studies show that the human C/EBP alpha protein stimulates USF to bind to a USF consensus element within C/EBP alpha promoter and activates it by two- to threefold. We propose that the human gene employs the ubiquitously expressed DNA-binding protein factor USF to carry out autoregulation. Autoregulation of the human C/EBP alpha promoter was abolished by deletion of the USF binding site, CACGTG. Expression of human C/EBP beta following transfection did not stimulate USF binding. These studies suggest a mechanism whereby tissue-specific autoregulation can be achieved via a trans-acting factor that is expressed in all cell types. Thus, direct binding of the C/EBP alpha protein to the promoter of the C/EBP alpha gene is not required for autoregulation.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Carcinoma Hepatocelular , Linhagem Celular , Primers do DNA , Homeostase , Humanos , Immunoblotting , Neoplasias Hepáticas , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição , Células Tumorais Cultivadas
20.
Mol Cell Biol ; 6(4): 969-75, 1986 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2431276

RESUMO

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.


Assuntos
Amilases/genética , Genes , Isoenzimas/genética , Neoplasias Hepáticas Experimentais/enzimologia , Pâncreas/enzimologia , Saliva/enzimologia , Transcrição Gênica , Amilases/biossíntese , Animais , Sequência de Bases , Linhagem Celular , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Glândula Parótida/enzimologia , Regiões Promotoras Genéticas/efeitos dos fármacos
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