RESUMO
Aim: To determine indicators of oxidative stress in long-lived individuals. Methods: 120 subjects were studied and two groups were formed: 50 individuals older than 85 years of nuclear families and 70 adults under 60 years old taken as a control group, all belonging to the municipality of Santa Clara. Indicators of antioxidant defense status included enzymatic activities superoxide dismutase (SOD) and catalase (CAT), as well as reduced glutathione (GSH) concentrations. The determinations were made with the use of spectrophotometric techniques, and the comparisons between the groups were made through the statistical program SPSS with a level of significance of 95 percent. Results: The activity of the antioxidant enzyme SOD and GSH levels showed significant differences when comparing both study groups. In the case of the SOD enzyme, the group of long-lived individuals showed a significant reduction in their activity compared to the controls, while GSH levels also decreased in this group. The CAT enzyme activity showed no significant differences between the two study groups. Conclusions: The decrease in enzymatic activity SOD accompanied by a decrease in GSH levels could be an indicator of a state of oxidative imbalance in individuals older than 85 years, which increases their susceptibility to the action of reactive oxygen species(AU)
Assuntos
Humanos , Pessoa de Meia-Idade , Idoso , Cloranfenicol O-Acetiltransferase , Estresse Oxidativo , Indicadores e Reagentes , Antioxidantes , Espécies Reativas de OxigênioRESUMO
The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA Helicases/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA Antissenso/genética , Salmonella typhi/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Transcrição Gênica/genética , Ativação Transcricional/genética , Regulação para Cima/genéticaRESUMO
We investigated the effects of different directions of insertion of matrix attachment region (MAR) sequences on transgenic expression in stably transformed Chinese hamster ovary (CHO) cells. The MAR sequences were inserted in forward or reverse directions into the expression vectors, and transfected into CHO cells. The expression of the chloramphenicol acetyltransferase (CAT) reporter gene and the relative copy numbers of the CAT gene were analyzed. The CAT gene expression levels in the vector with the MAR sequence inserted in the forward or reverse directions increased compared with expression without the MAR sequence. The relative copy numbers of the CAT gene with MAR sequenced vectors inserted in the reverse and forward directions were lower, than in the control group. The direction of insertion of MAR sequences had no significant effect on expression levels. The expression levels were not proportional to the copy numbers of the gene.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , DNA Intergênico/genética , Vetores Genéticos/química , Regiões de Interação com a Matriz , Plasmídeos/química , Animais , Células CHO , Linhagem Celular Transformada , Cloranfenicol O-Acetiltransferase/metabolismo , Cricetulus , DNA Intergênico/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/metabolismo , Plasmídeos/metabolismo , TransgenesRESUMO
Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5' coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3' UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5' coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , Vetores Genéticos/genética , Neospora/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Coccidiose/parasitologia , Genes Reporter , Vetores Genéticos/metabolismo , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Neospora/crescimento & desenvolvimento , Neospora/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.
Assuntos
Chlorella vulgaris/genética , Expressão Gênica , Transformação Genética , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Resistência ao Cloranfenicol/genética , Chlorella vulgaris/efeitos dos fármacos , Chlorella vulgaris/enzimologia , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Nitrato Redutase/genética , Nitratos/farmacologia , Organismos Geneticamente Modificados , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Seleção Genética , Transformação Genética/efeitos dos fármacosRESUMO
Gene regulation in trypanosomatids occurs mainly by post-transcriptional mechanisms modulating mRNA stability and translation. We have investigated heat shock protein (HSP) 70 gene regulation in Trypanosoma cruzi, the causal agent of Chagas' disease. The HSP70 mRNA's half-life increases after heat shock, and the stabilization is dependent on protein synthesis. In a cell-free RNA decay assay, a U-rich region in the 3' untranslated region (UTR) is a target for degradation, which is reduced when in the presence of protein extracts from heat shocked cells. In a transfected reporter gene assay, both the 5'- and 3'-UTRs confer temperature-dependent regulation. Both UTRs must be present to increase mRNA stability at 37 degrees C, indicating that the 5'- and 3'-UTRs act cooperatively to stabilize HSP70 mRNA during heat shock. We conclude that HSP70 5'- and 3'-UTRs regulate mRNA stability during heat shock in T. cruzi.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Regiões 5' não Traduzidas/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/genética , RNA Mensageiro/metabolismo , Trypanosoma cruzi/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Northern Blotting , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Meia-Vida , Temperatura Alta , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Trypanosoma cruzi/metabolismoRESUMO
Se realizó un estudio sobre los marcadores de daño oxidativo y de defensa antioxidante en órganos de ratas jóvenes, adultas y senescentes; mediante los cuales se evidenció un incremento con el tiempo de la peroxidación lipídica en hígado, corazón, riñón y páncreas; así como una disminución en la actividad de la enzima antioxidante catalasa en todos los órganos, excepto el páncreas. Los cambios que se producen en el envejecimiento se han asociado a un desequilibrio en los mecanismos oxidantes y antioxidantes en las células. Los resultados demostraron un estado de estrés oxidativo que pudiera favorecer la aparición de enfermedades asociadas al envejecimiento.
A study of oxidative damage and anti-oxidizing defense markers in organs from young, adult and aged rats was conducted. It was evident that lipid peroxidation in liver, heart, kidneys and pancreas increases with the time as well as anti-oxidizing enzyme catalase reduces its action in all the organs except for pancreas. Changes at the time of aging have been associated with an imbalance in oxidizing and anti-oxidizing mechanisms within the cells. The results showed a state of oxidative stress that favors the occurrence of aging-related diseases.
Assuntos
Animais , Ratos , Cloranfenicol O-Acetiltransferase , Peroxidação de Lipídeos , Estresse OxidativoRESUMO
DNA replication mechanisms are poorly understood in most of trypanosomatids, in particular the replication of the peculiar mitochondrial DNA, the kinetoplast DNA (kDNA). To contribute to the knowledge on the mechanism of kDNA replication in Trypanosoma cruzi, we have previously characterized the Universal Minicircle Sequence Binding Protein of this parasite (TcUMSBP), which was first called PDZ5 [E.R. Coelho, T.P. Urmenyi, J. Franco da Silveira, E. Rondinelli, R. Silva, Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi, Int. J. Parasitol. 33 (2003) 853-858]. In this work, we describe two highly polymorphic alleles of the TcUMSBP locus in the T. cruzi reference clone CL Brener and the differential expression pattern of these alleles. A 62 bp sequence in the TcUMSBP upstream intergenic region in one of its alleles affects the efficiency of polycistronic RNA processing and the polyadenylation sites, and therefore regulates the differential expression of TcUMSBP alleles of this locus.
Assuntos
Alelos , DNA de Cinetoplasto , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Polimorfismo Genético , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Southern Blotting , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , Replicação do DNA , DNA de Cinetoplasto/genética , Eletroporação , Regulação da Expressão Gênica , Genoma , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , TransfecçãoRESUMO
Brazilian purpuric fever is a severe vascular disease caused by an invasive clone of Haemophilus influenzae biogroup aegyptius, which normally causes self-limiting eye infections. A previous genome subtraction procedure resulted in the isolation of a DNA fragment, which encodes a putative IgA1 protease, specific to the F3031 Brazilian purpuric fever type strain. Cloning and sequencing of the entire F3031 iga1 gene showed that the subtracted DNA fragment encompasses the iga1 region encoding the active site and the cleavage specificity determinant of the protein, which are different from the cognate regions of the proteases produced by other H. influenzae strains. Western and IgA cleavage assays together with clustering analysis showed that the F3031 IgA1 protease is most similar to the type 2 proteases produced by H. influenzae type c and e strains. Analysis of the promoter region of the F3031 iga1 gene revealed the presence of Fur binding sites. However, real-time PCR analysis and transcriptional fusion assays showed that the expression of iga1 is not regulated by iron or hemin under the conditions tested.
Assuntos
Haemophilus influenzae/enzimologia , Serina Endopeptidases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/análise , Clonagem Molecular , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Haemophilus influenzae/genética , Hemina/fisiologia , Ferro/fisiologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Púrpura , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
The multidrug resistance EhPgp1 gene is constitutively expressed in drug resistant trophozoites from Entamoeba histolytica. It has been demonstrated that two CCAAT/enhancer binding sites located in the EhPgp1 gene promoter control its transcriptional activation. However, functional assays of the 5' end of its promoter showed that region from -234 to -196 bp (38 bp) is also important for the EhPgp1 gene transcription. Here, we demonstrated that in the 38 bp region putative cis-activator sequences are located. In silico analysis showed the presence of GATA1, Gal4, Nit-2, and C/EBP consensus sequences. Additionally, we identified three specific DNA-protein complexes, which were competed by a C/EBP, GATA1, and HOX oligonucleotides. Finally, we partially purified three proteins of 64.4, 56.7, and 27.4 kDa. Further investigations are currently in progress to determine the identity of these nuclear factors and how they are interacting with the EhPgp1 gene promoter.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Entamoeba histolytica/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas de Protozoários/genética , Animais , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatografia de Afinidade , DNA de Protozoário/metabolismo , Resistência a Múltiplos Medicamentos/genética , Entamoeba histolytica/química , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Transfecção , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The Entamoeba histolytica Ehcp112 and Ehadh112 genes that encode the EhCPADH complex are separated by a non-coding 188pb region. Their proximity suggests a coordinated expression regulation for both genes. Here, we studied the structure and function of 996 bp (p996CAT) upstream the ATG start codon of the Ehadh112 gene. The p996CAT plasmid drove CAT transcription with a 78% of the activity showed by actin promoter. Deletion of 330 bp at the 5' end of p966CAT to produce the p776CAT plasmid, decreased activity to 40% in relation to actin promoter and to 50% of p996CAT, suggesting the presence of a silencer in this region. Interestingly, deletion of other 297 bp to the p776CAT to generate the p469CAT plasmid, augmented activity in 2.5-fold compared with p776CAT construction, showing the presence of a proximal enhancer promoter. Transcription initiation sites (-69 and -150 bp), TATA like box, GAAC, and Inr elements, as well as putative DNA binding motifs, were mapped in the -1 to -469 bp core promoter region.
Assuntos
Entamoeba histolytica/genética , Lectinas/genética , Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas de Protozoários/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , DNA de Protozoário/química , Lectinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/fisiologia , TransfecçãoRESUMO
Ehcp112 encodes the Entamoeba histolytica EhCP112 cysteine protease that is part of the EhCPADH complex. By in silico analysis we identified putative transcription factor-binding sites along 837 bp upstream the Ehcp112 gene ATG codon. A TATA-like motif (TATATAAA) was located at -36 to -29 bp, a GAAC box (GAACC) was found at -10 to -14 bp and an Inr sequence (TTCAAC) at -8 to -2 bp. These tripartite promoter elements are in non-canonical positions, downstream the transcription initiation site (-280 bp). We cloned four Ehcp112 promoter fragments in pBSCAT-ACT plasmid to obtain pI (355 bp), pII (681 bp), pIII (833 bp), and pIV (554 bp) constructs. In transfected trophozoites, only pIII drove CAT activity with 44% efficiency in relation to actin promoter activity. Our results showed the presence of a distal and weak promoter in the Ehcp112 gene. The active DNA region is inside the open reading frame of the Ehrab B gene, suggesting that expression of both genes could be coordinately regulated.
Assuntos
Cisteína Endopeptidases/genética , Entamoeba histolytica/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , DNA de Protozoário/química , Entamoeba histolytica/enzimologia , Regulação da Expressão Gênica , Dados de Sequência Molecular , Plasmídeos/química , Regiões Promotoras Genéticas/fisiologia , Ativação Transcricional/fisiologia , TransfecçãoRESUMO
Many types of evidence support a role of the sympathetic nervous system in the regulation of thyroid function, although there is no general consensus on the type of influence that catecholamines exert. Depending on the experimental approach, epinephrine and norepinephrine (NE) can stimulate, inhibit, or fail to act on thyroid function. The aim of this study was to determine the effect of NE on thyroglobulin (Tg) synthesis and gene expression in FRTL-5 cells. Tg content, measured by immunoprecipitation with a specific antibody, showed that NE caused a 45% inhibition of thyrotropin (TSH) effect. The content of Tg mRNA was analyzed by Northern blot, the relative inhibition in total Tg mRNA levels from NE-treated cells, compared to TSH alone, ran parallel with inhibition in Tg content, while total RNA did not change after incubation with NE. There was no alteration in Tg mRNA stability by NE. When plasmids harboring different sequences of Tg promoter fused to the CAT reporter gene were transfected into FRTL-5 cells, TSH treatment stimulated promoter activity while NE diminished this effect by 43%-55%. Northern blots were performed to analyze mRNA for thyroid transcription factors (TTF1, TTF2, Pax8), and no significant changes were observed with the different treatments. In conclusion these results suggest that NE inhibits Tg synthesis at the transcriptional level.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Norepinefrina/farmacologia , Tireoglobulina/biossíntese , Tireoglobulina/genética , Glândula Tireoide/metabolismo , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Processamento de Imagem Assistida por Computador , Metionina/metabolismo , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA Mensageiro/biossíntese , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/biossíntese , TransfecçãoRESUMO
BACKGROUND: Estrogens exert profound effects on target tissues. These effects are mediated by two estrogen receptors (ER(alpha) and ER(beta)) that bind to specific DNA sequences in estrogen-dependent genes. Other molecules such as growth factors, transcription factors and some oncoproteins might interact with the estrogen receptors and thus regulate the transcription of these genes. Currently there is no adequate cellular model to study these interactions. METHODS: We transfected the human wild-type ER(alpha) to an ER-negative rat epithelial endometrial cell line (Rentr01) using a tetracycline-regulated gene expression system. The exogenous receptor was correctly translated, had an appropriate hormone-binding affinity, and bound well to estrogen response elements containing DNA. RESULTS: We obtained a new stable cell line that is ER(beta) negative but ER(alpha) positive (R1-49E1). The expression of receptor alpha can be regulated in a dose-response manner by addition of tetracycline in the culture medium. Estradiol treatment of ER(alpha)-containing cells apparently diminished cellular proliferation, and the exogenous receptor can induce the transcription of the endogenous progesterone receptor isoform B (PgR-B) gene. CONCLUSIONS: This epithelial cellular model may be useful to study the interaction between estrogens and other cell signaling pathways in epithelial endometrial cell physiology.
Assuntos
Linhagem Celular , Endométrio/citologia , Receptor alfa de Estrogênio/biossíntese , Tetraciclina/farmacologia , Animais , Diferenciação Celular , Proliferação de Células , Cloranfenicol O-Acetiltransferase/metabolismo , Meios de Cultura/farmacologia , DNA/química , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Genes Reporter , Humanos , Imuno-Histoquímica , Camundongos , Células NIH 3T3 , Ligação Proteica , Ratos , Receptores de Progesterona/metabolismo , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
The multidrug resistance EhPgp5 gene promoter is active in drug resistant clone C2 trophozoites and its activity increases when trophozoites are cultured in the presence of emetine, suggesting that the EhPgp5 gene shows an inducible drug dependent mechanism. We analyzed different promoter fragments to detect those regions that activate transcription in the presence of emetine. Trophozoites were transfected with p375Pgp5, p259Pgp5, p187Pgp5, and p76Pgp5 plasmids and incubated with different emetine concentrations. p375Pgp5 and p259Pgp5 plasmids were able to drive CAT expression in A and C2 trophozoites only in the presence of emetine. CAT activity was turned off in the absence of drug. Interestingly, no CAT activity was detected in the presence or in the absence of emetine with p187Pgp5 plasmid in which 59 bp were deleted at the 5' end of the EhPgp5 minimal promoter (p259Pgp5). These results suggest that the overexpression of the EhPgp5 gene is a consequence of transcriptional activation of the gene promoter by putative drug responsive elements, located within the -111 to -170 bp of the transcription initiation site.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Amebicidas/farmacologia , Emetina/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Proteínas de Protozoários/genética , Ativação Transcricional/fisiologia , Animais , Cloranfenicol O-Acetiltransferase/metabolismo , Resistência a Múltiplos Medicamentos/genética , Eletroporação , Entamoeba histolytica/genética , Entamoeba histolytica/crescimento & desenvolvimento , Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , TransfecçãoRESUMO
The Human T-cell leukemia virus type I (HTLV-I) non-structural protein Tax plays a crucial role in cellular transformation. It activates the transcription factors of various cellular genes and interacts with cellular proteins. There is limited data available on the interaction between specific T-cell transcription factor GATA3 and Tax. Implications for the significance of GATA3 in T-cell development and function, T helper2 (Th2) differentiation, and a role of GATA3 during the immune response have been reported. To determine the effect of the Tax protein on GATA3 gene expression, we investigated the interaction between this protein and the GATA3 promoter and repressor regions. Results demonstrated an interaction between Tax and the GATA3 promoter via the transcription factor Sp1 and a role for Tax in the negative regulation of GATA3 expression, through its interaction with the repressor ZEB. This interaction may be involved in the pathophysiology of adult T-cell leukemia/lymphoma (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM).
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Regiões Promotoras Genéticas/genética , Transativadores/genética , Adulto , Cloranfenicol O-Acetiltransferase/metabolismo , Fator de Transcrição GATA3 , Humanos , Células Jurkat , Fator de Transcrição Sp1/fisiologia , Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Secreted as well as surface exposed proteins are assumed to play major roles in bacterial virulence. In this report we describe the construction of an N-terminal protein-capturing system and its use for the isolation of Brucella abortus S2308 genes coding for putative surface exposed or secreted proteins. For this purpose, a cloning vector that generates gene fusions to a ribosome binding site and start codon deficient Chloramphenicol Acetyl Transferase (CAT) reporter gene was constructed and the resulting library introduced into B. abortus S2308 and virB mutant strains. Secreted translational fusions were identified by determining CAT activity in culture supernatants. Secretion was confirmed by Western Blot using a polyclonal anti-CAT antibody. A total of 864 clones were screened and 10 genes encoding putative secreted/surface exposed proteins were identified. Seven are Brucella proteins with an assigned function, whereas three are hypothetical proteins. The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected. Secretion in a virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins seemed to require a full VirB system for their secretion.
Assuntos
Proteínas de Bactérias/genética , Brucella abortus/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Brucella abortus/metabolismo , Brucella abortus/patogenicidade , Clonagem Molecular , Vetores Genéticos , Mutagênese Insercional , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5'-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3beta or HNF3beta plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3beta binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Enzimológica da Expressão Gênica , Insulina/metabolismo , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Bases , Sítios de Ligação , Southern Blotting , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Genes Dominantes , Vetores Genéticos , Células HeLa , Fator 3-beta Nuclear de Hepatócito , Humanos , Dados de Sequência Molecular , Mutação , Fatores de Transcrição NFI , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA/química , RNA Mensageiro/metabolismo , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat with antiestrogens. This work was performed to determine if the re-expression of the human ER alpha could restore the hormone response of these cells. We have transfected the human wild-type ER alpha to an ER-negative breast cancer cell line (MDA-MB-231) using a tetracycline-regulated gene expression system. We obtained a new cell line, MDA-A4-5/2. Cell count and flow cytometry "S" phase cell fraction showed that 17-beta-estradiol induced an inhibition on the proliferation of these cells; on the contrary, the antiestrogens ICI 182 780, and tamoxifen blocked this effect. Finally, we demonstrated an induction of the endogenous progesterone receptor gene when ER alpha was present. These results suggest that the re-expression of ER alpha in ER-negative breast cancer cells recreate, at least partially, a hormone-responsive phenotype and may be useful as a therapeutic approach to control this pathology.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/fisiologia , Estrogênios/fisiologia , Tamoxifeno/análogos & derivados , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Cloranfenicol O-Acetiltransferase/genética , Células Clonais , Doxiciclina/farmacologia , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Citometria de Fluxo , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Luciferases , Receptores de Progesterona/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase S/efeitos dos fármacos , Tamoxifeno/farmacologia , TransfecçãoRESUMO
ZO-2 is a membrane-associated guanylate kinase (MAGUK) protein present at the tight junction (TJ) of epithelial cells. While confluent monolayers have ZO-2 at their cellular borders, sparse cultures conspicuously show ZO-2 at the nuclei. To study the role of nuclear ZO-2, we tested by pull-down assays and gel shift analysis the interaction between ZO-2 GST fusion proteins and different transcription factors. We identified the existence of a specific interaction of ZO-2 with Fos, Jun and C/EBP (CCAAT/enhancer binding protein). To analyze if this association is present "in vivo", we performed immunoprecipitation and immunolocalization experiments, which revealed an interaction of ZO-2 with Jun, Fos and C/EBP not only at the nucleus but also at the TJ region. To test if the association of ZO-2 with AP-1 (activator protein-1) modulates gene transcription, we performed reporter gene assays employing chloramphenicol acetyltransferase (CAT) constructs with promoters under the control of AP-1 sites. We observed that the co-transfected ZO-2 down-regulates CAT expression in a dose-dependent manner. Since ZO-2 is a multidomain protein, we proceeded to determine which region of the molecule is responsible for the modulation of gene expression, and observed that both the amino and the carboxyl domains are capable of inhibiting gene transcription.