RESUMEN
Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase.
Asunto(s)
ADN Viral/efectos de los fármacos , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Integración Viral/efectos de los fármacos , Vitamina B 12/farmacología , Secuencia de Bases , Células Cultivadas , Cobamidas/farmacología , Cartilla de ADN/genética , ADN Viral/genética , ADN Viral/fisiología , VIH-1/genética , VIH-1/fisiología , Humanos , Hidroxocobalamina/farmacología , Técnicas In Vitro , Linfocitos/efectos de los fármacos , Linfocitos/virología , Monocitos/efectos de los fármacos , Monocitos/virología , Reacción en Cadena de la Polimerasa , Vitamina B 12/análogos & derivadosRESUMEN
Upon cytokine induction, inducible nitric oxide synthase (iNOS) mRNA and enzyme activity in DLD-1 cells reached maximal levels at 6 and 8 h, respectively. A 3.7 kb 5'-flanking region of the human iNOS gene was used to prepare luciferase reporter constructs. Upon transfection of these constructs into DLD-1 cells and primary monkey hepatocytes, significant promoter activity was detected in the absence of cytokines, and this activity decreased with successive truncations of the human iNOS promoter. No increase in luciferase activity was observed after cytokine treatment, in spite of the fact that nuclear run-on analysis indicated that iNOS induction in DLD-1 cells was due, in part, to an increase in transcription rate. These results suggest that 3.7 kb of 5'-flanking DNA do not contain all of the elements required for transcriptional induction of the human iNOS gene. This differs from the mouse iNOS gene for which 1.7 kb of 5'-flanking DNA contain most or all of the elements that control iNOS expression in mouse macrophages. Thus, important cell- and species-specific mechanisms may exist for the control of iNOS expression.
Asunto(s)
Adenocarcinoma/enzimología , Neoplasias Colorrectales/enzimología , Hígado/enzimología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Células Cultivadas , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Dactinomicina/farmacología , Semivida , Haplorrinos , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Interleucina-6/farmacología , Lipopolisacáridos/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Ratones , Óxido Nítrico Sintasa/efectos de los fármacos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/efectos de los fármacos , ARN/biosíntesis , ARN/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Transcripción Genética , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Nitric oxide produced in endothelial cells affects vascular tone. To investigate the role of endothelial nitric oxide synthase (eNOS) in blood pressure regulation, we have generated mice heterozygous (+/-) or homozygous (-/-) for disruption of the eNOS gene. Immunohistochemical staining with anti-eNOS antibodies showed reduced amounts of eNOS protein in +/- mice and absence of eNOS protein in -/- mutant mice. Male or female mice of all three eNOS genotypes were indistinguishable in general appearance and histology, except that -/- mice had lower body weights than +/+ or +/- mice. Blood pressures tended to be increased (by approximately 4 mmHg) in +/- mice compared with +/+, while -/- mice had a significant increase in pressure compared with +/+ mice (approximately 18 mmHg) or +/- mice (approximately 14 mmHg). Plasma renin concentration in the -/- mice was nearly twice that of +/+ mice, although kidney renin mRNA was modestly decreased in the -/- mice. Heart rates in the -/- mice were significantly lower than in +/- or +/+ mice. Appropriate genetic controls show that these phenotypes in F2 mice are due to the eNOS mutation and are not due to sequences that might differ between the two parental strains (129 and C57BL/6J) and are linked either to the eNOS locus or to an unlinked chromosomal region containing the renin locus. Thus eNOS is essential for maintenance of normal blood pressures and heart rates. Comparisons between the current eNOS mutant mice and previously generated inducible nitric oxide synthase mutants showed that homozygous mutants for the latter differ in having unaltered blood pressures and heart rates; both are susceptible to lipopolysaccharide-induced death.
Asunto(s)
Presión Sanguínea , Endotelio Vascular/enzimología , Hipertensión/genética , Hipertensión/fisiopatología , Óxido Nítrico Sintasa/deficiencia , Análisis de Varianza , Animales , Bovinos , Quimera , Cartilla de ADN , Femenino , Genotipo , Heterocigoto , Isoenzimas/deficiencia , Isoenzimas/genética , Riñón/enzimología , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Renina/biosíntesis , Renina/sangre , Células Madre , Transcripción GenéticaRESUMEN
The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN-gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN-gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa/genética , Animales , Secuencia de Bases , Humanos , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/fisiología , Ratones , Datos de Secuencia Molecular , FN-kappa B/fisiología , Óxido Nítrico Sintasa/biosíntesis , Fagocitos/metabolismo , Regiones Promotoras Genéticas/fisiología , Proteínas Recombinantes , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaAsunto(s)
Encéfalo/enzimología , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/farmacología , Neuronas/enzimología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/aislamiento & purificación , Placenta/enzimología , Adenocarcinoma , Arginina/metabolismo , Radioisótopos de Carbono , Línea Celular , Cromatografía de Afinidad/métodos , Cromatografía en Gel/métodos , Neoplasias Colorrectales , Citocinas/farmacología , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática/efectos de los fármacos , Femenino , Humanos , Indicadores y Reactivos , Isoenzimas/análisis , Isoenzimas/antagonistas & inhibidores , Isoenzimas/aislamiento & purificación , Cinética , Óxido Nítrico Sintasa/análisis , Embarazo , Técnica de Dilución de RadioisótoposRESUMEN
Chinese hamster ovary cells were transfected with the human inducible nitric oxide synthase (iNOS) cDNA under the control of the hCMV promoter. The NOS inhibitor, S-ethyl isothiourea, was utilized to minimize toxicity from NO production. Selected colonies were grown in the absence of S-ethyl isothiourea, after which nitrite levels in the medium were measured. NOS activities in cell lysates were determined, and 9 colonies had iNOS activities of over 1 nmol of citrulline formed/min/mg protein. iNOS expression was further increased by gene amplification and the use of sodium butyrate, resulting in two cell lines with stable activity of greater than 3 nmol/min/mg. The iNOS purified from these cells had a specific activity of over 500 nmol/min/mg, and its properties were similar to native iNOS purified from cytokine-induced DLD-1 cells. This is the most efficient expression system reported to date for iNOS.
Asunto(s)
Óxido Nítrico Sintasa/genética , Animales , Butiratos/farmacología , Ácido Butírico , Células CHO , Cricetinae , Inducción Enzimática , Humanos , Cinética , Peso Molecular , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Proteínas RecombinantesRESUMEN
Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock. To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon gamma, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shock.
Asunto(s)
Lipopolisacáridos/toxicidad , Óxido Nítrico Sintasa/deficiencia , Choque Séptico/enzimología , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Resistencia a Medicamentos , Immunoblotting , Interferón gamma/farmacología , Macrófagos Peritoneales/enzimología , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Nitratos/sangre , Óxido Nítrico Sintasa/genética , Nitritos/sangre , Óxidos de Nitrógeno/sangre , ARN Mensajero/análisis , Recombinación Genética , Choque Séptico/etiología , Choque Séptico/genética , Células Madre , Análisis de SupervivenciaRESUMEN
S-Ethylisothiourea was a potent competitive inhibitor of human nitric oxide synthase (NOS), with Ki values of 17, 36, and 29 nM for the inducible (i), endothelial (e), and neuronal (n) isozymes, respectively. Unlike some potent inhibitors of NOS, no time dependence was observed. S-Ethylisothiourea was not a detectable substrate for eNOS. S-Ethylisothiourea was also a potent inhibitor of mouse iNOS (Ki value of 5.2 nM), and its binding perturbed the spectrum of iNOS consistent with its altering the environment of the bound heme. The optimum binding of S-ethyl- and S-isopropylisothiourea relative to 70 other analogs suggested that these alkyl substitutions fit into a small hydrophobic pocket. Most isothioureas were 2-6-fold selective for the human iNOS (Ki for iNOS versus Ki for eNOS), with one being 19-fold selective. The cyclized mimics of S-ethylisothiourea, 2-NH2-thiazoline, and 2-NH2-thiazole, were also competitive inhibitors of human NOS. A third structural class of inhibitors, bisisothioureas, were, in general, the most selective in their inhibition of human iNOS. S,S'-(1,3-Phenylenebis(1,2-ethanediyl))bisisothiourea was 190-fold selective (Ki value of 0.047 microM against iNOS versus 9.0 microM against eNOS). These results demonstrate that potent and selective inhibition of human NOS isozymes is achievable.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Arginina/análogos & derivados , Isoenzimas/antagonistas & inhibidores , Neuronas/enzimología , Tiourea/análogos & derivados , Animales , Endotelio Vascular/enzimología , Inducción Enzimática , Humanos , Isotiuronio/análogos & derivados , Isotiuronio/metabolismo , Isotiuronio/farmacología , Cinética , Ratones , Modelos Químicos , Óxido Nítrico Sintasa , Especificidad de la Especie , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
A combination of cytokines induced the expression of nitric oxide synthase (NOS) in a human colorectal adenocarcinoma cell line, DLD-1. We have purified the enzyme and examined some of its biochemical properties. An antiserum to an inducible NOS from murine macrophages cross-reacted with the DLD-1 NOS. The purified human and murine enzymes displayed a similar lack of dependence on exogenous calcium and calmodulin for activity, which contrasts with the requirement for calcium and calmodulin of purified brain and endothelial isoforms of NOS. We have also isolated a cDNA for a cytokine-induced NOS from DLD-1 cells. Sequence analysis of this cDNA and NOS cDNAs from human liver, smooth muscle, and macrophages suggests that, at the genetic level, there is a single isoform of human-inducible NOS.
Asunto(s)
Aminoácido Oxidorreductasas/biosíntesis , Adenocarcinoma/enzimología , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Neoplasias Colorrectales/enzimología , Citocinas/farmacología , ADN Complementario/química , Inducción Enzimática , Humanos , Macrófagos/enzimología , Ratones , Datos de Secuencia Molecular , Óxido Nítrico Sintasa , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
Polymerase chain reaction amplification of reverse transcribed RNA (RNA-PCR) was used to isolate overlapping cDNA clones encoding an inducible form of nitric oxide synthase (NOS) from purified rat hepatocytes. NOS gene expression in hepatocytes is dependent upon bacterial lipopolysaccharide (LPS) and cytokine treatment. The predicted amino acid sequence of rat hepatocyte inducible NOS is 94% identical with that from the mouse macrophage cell line, RAW264.7. Differences between the two sequences occur as sporadic amino acid substitutions with no major gaps or insertions. These differences are most likely the result of species variability which suggests that hepatocytes and macrophages express the same NOS gene upon induction.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Hígado/enzimología , Macrófagos/enzimología , Secuencia de Aminoácidos , Animales , Citocinas/farmacología , Inducción Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Óxido Nítrico Sintasa , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Ratas , Alineación de SecuenciaRESUMEN
Using purified integration protein (IN) from human immunodeficiency virus (HIV) type 1 and oligonucleotide mimics of viral and target DNA, we have investigated the DNA sequence specificity of the cleaving and joining reactions that take place during retroviral integration. The first reaction in this process is selective endonucleolytic cleaving of the viral DNA terminus that generates a recessed 3' OH group. This 3' OH group is then joined to a 5' phosphoryl group located at a break in the target DNA. We found that the conserved CA located close to the 3' end of the plus strand of the U5 viral terminus (also present on the minus strand of the U3 terminus) was required for both cleaving and joining reactions. Six bases of HIV U5 or U3 DNA at the ends of model substrates were sufficient for nearly maximal levels of selective endonucleolytic cleaving and joining. However, viral sequence elements upstream of the terminal 6 bases could also affect the efficiencies of the cleaving and joining reactions. The penultimate base (C) on the minus strand of HIV U5 was required for optimal joining activity. A synthetic oligonucleotide mimic of the putative in vivo viral "DNA" substrate for HIV IN, a molecule that contained a terminal adenosine 5'-phosphate (rA) on the minus strand, was indistinguishable in the cleaving and joining reactions from the DNA substrate containing deoxyadenosine instead of adenosine 5'-phosphate at the terminal position. Single-stranded DNA served as an in vitro integration target for HIV IN. The DNA sequence specificity of the joining reaction catalyzed in the reverse direction was also investigated.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , ADN Nucleotidiltransferasas/metabolismo , VIH-1/metabolismo , Oligonucleótidos/metabolismo , Proteínas de los Retroviridae/metabolismo , Secuencia de Bases , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Escherichia coli/genética , Transcriptasa Inversa del VIH , Humanos , Integrasas , Datos de Secuencia Molecular , ADN Polimerasa Dirigida por ARN/metabolismo , Especificidad por Sustrato , Integración ViralRESUMEN
The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Elementos Transponibles de ADN , Escherichia coli/genética , VIH/enzimología , Secuencia de Bases , Clonación Molecular/métodos , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/aislamiento & purificación , Escherichia coli/enzimología , Expresión Génica , VIH/genética , Integrasas , Cinética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The human class II gene, HLA-DR alpha, contains an octanucleotide sequence ATTTGCAT located approximately 40 base pairs upstream of the transcription initiation site. We have investigated the transcriptional function of the DR alpha octamer in human B-lymphoblastoid cells and non-B cells. Deletion and substitution mutagenesis of the octamer sequence greatly reduced the activity of the DR alpha promoter in both in vivo and in vitro cell-free transcription systems of B-cell origin. Conversely, these mutations did not affect promoter activity in several non-B-cell lines that express the DR alpha gene. Removal of octamer-binding proteins by in vivo titration with an octamer-containing competitor plasmid reduced DR alpha promoter activity in B-lymphoblastoid cells. These results suggest that a protein-octamer interaction, most likely involving the B-cell-specific octamer binding protein (OTF-2), is required for DR alpha promoter function in B-lymphoblastoid cells but not in non-B cells.
Asunto(s)
Linfocitos B/inmunología , Genes MHC Clase II , Genes Reguladores , Antígenos HLA-DR/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Línea Celular , Deleción Cromosómica , ADN de Neoplasias/genética , Humanos , Datos de Secuencia Molecular , Mutación , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide. Each of several DNA polymerases exhibited unique reaction characteristics with the oligonucleotide template primers, which was attributed to the differing exonuclease activities associated with these various enzymes. Assay optimization therefore included matching the polymerase with the template primer to obtain the lowest background reaction and highest sensitivity. This modified assay is particularly well suited for keeping cell sample size to a minimum in experimental protocols which generate large numbers of data points or require careful timing of sampling. With this technique, we measured the levels of all four deoxyribonucleoside triphosphates in extracts from as few as 2 x 10(4) cultured cells.
Asunto(s)
Desoxirribonucleótidos/análisis , Oligonucleótidos/genética , Secuencia de Bases , Células Cultivadas , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiadenina/análisis , Nucleótidos de Desoxicitosina/análisis , Nucleótidos de Desoxiguanina/análisis , Humanos , Micrococcus/genética , Percloratos/farmacología , Poli dA-dT/metabolismo , Estándares de Referencia , Moldes Genéticos , Nucleótidos de Timina/análisis , TritioRESUMEN
The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DR alpha gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DR alpha promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. This Y box-binding protein was present in nuclear extracts of all cell types examined, including human B and T cells and HeLa cells. The molecular mass of the protein, as determined by photoactivated protein-DNA cross-linking, was approximately 40 to 50 kilodaltons. Mutagenesis of the Y box that decreased protein binding also decreased promoter activity, implying that protein binding to this DNA sequence is important for DR alpha promoter function.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes MHC Clase II , Antígenos HLA-DR/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Sondas de ADN/análisis , Proteínas de Unión al ADN/análisis , Endonucleasas , Regulación de la Expresión Génica , Humanos , Metilación , Conformación de Ácido Nucleico , Virus 40 de los Simios/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Transfección , Células Tumorales CultivadasRESUMEN
In this report, we determined that induction of the DR alpha-chain by recombinant human interferon-gamma (IFN-gamma) in a human glioblastoma multiform cell line is transcriptionally regulated and showed that protein synthesis is not necessary for this to occur. The regions of the DR alpha-chain gene that are responsible for basal and recombinant IFN-gamma-induced gene transcription have been determined by gene transfer of a series of 5' deletion mutants in which the upstream region of the DR alpha chain was linked to a reporter gene, chloramphenicol acetyltransferase. Chloramphenicol acetyltransferase transcript and protein levels were determined by S1 nuclease protection and chloramphenicol acetyltransferase enzyme assays, respectively. By using these deletion mutants, we were able to draw the following conclusions. (i) One hundred and nine base pairs of upstream sequence contains the basic DR alpha-chain gene promoter and represents the minimal amount of sequence necessary for basal gene expression. (ii) An additional 9 base pairs of upstream sequence can mediate recombinant IFN-gamma induction. (iii) Maximal recombinant IFN-gamma induction requires at most an additional 23 base pairs of upstream sequence. (iv) The sequence between positions -267 and -141 does not appear to contain any additional positive or negative regulatory elements. These results suggest that the region between positions -141 and -109 contains a critical IFN-gamma-responsive element. Substitution mutagenesis was performed to confirm this suggestion.
Asunto(s)
Regulación de la Expresión Génica , Genes MHC Clase II , Glioblastoma/inmunología , Antígenos HLA-DR/genética , Interferón gamma/inmunología , Proteínas Recombinantes/inmunología , Secuencia de Bases , Línea Celular , Deleción Cromosómica , Glioblastoma/genética , Humanos , Datos de Secuencia Molecular , Mutación , TransfecciónRESUMEN
We have used in vitro deletion mutagenesis in combination with DNA transfection to search for cis-acting regulatory elements involved in the tissue-specific expression of a human class II major histocompatibility complex gene. A 140-base-pair 5' flanking fragment that contains the class II box consensus sequences and an octamer sequence (ATTTGCAT) confers tissue specificity on the promoter of the HLA-DR alpha gene. Recombinant DNA plasmids containing this DR alpha gene segment fused to the coding sequence of the bacterial chloramphenicol acetyltransferase gene are expressed at higher levels in human B-cell lines than in human T-cell lines. We have demonstrated that the most 5' of the class II boxes is essential for tissue-specific DR alpha promoter function. In addition, using an electrophoretic mobility shift assay to identify DNA binding proteins, we have detected binding of nuclear proteins to DNA probes containing the class II boxes and the octamer sequence. A protein that binds to the octamer is present at higher levels in nuclear extracts of B-cell lines than in other cell lines examined. This protein may be important for the tissue-specific expression of the HLA-DR alpha gene.
Asunto(s)
Genes Reguladores , Genes , Antígenos HLA-D/genética , Antígenos HLA-DR/genética , Transcripción Genética , Linfocitos B/inmunología , Secuencia de Bases , Línea Celular , Humanos , Mutación , Plásmidos , Regiones Promotoras Genéticas , Linfocitos T/inmunologíaRESUMEN
The expression of class II major histocompatibility (MHC) antigens is central to the mounting of a successful immune response. Understanding the molecular mechanisms involved in the induction of class II MHC expression may therefore provide the knowledge necessary to manipulate the immune system appropriately. We are particularly interested in the induction of class II MHC antigens on cells in the brain, because of the potential involvement of such brain cells in the initiation and perpetuation of autoimmune-like diseases of the central nervous system. We examined the mechanisms involved in interferon-gamma (IFN-gamma) induction of class II MHC antigens on a human glioblastoma multiforme cell line. This paper describes the identification of a 297-bp (base pair) fragment of the class II MHC DR alpha chain gene which is involved in IFN-gamma induction. We were able to identify this IFN-gamma responsive sequence by preparing recombinant plasmids containing 5' flanking pieces of the human DR alpha chain gene placed upstream of the indicator gene, chloramphenicol acetyltransferase (CAT). These recombinant plasmids were transfected into human glioma cells which were then cultured in the presence or absence of IFN-gamma. After 48 hr, transient expression of CAT was assayed by thin layer chromatography. CAT enzyme activity was significantly increased only in IFN-gamma-treated cells. This increase was also reflected at the RNA level in that IFN-gamma treatment resulted in higher CAT transcripts. A computer homology search revealed a possible consensus sequence shared among different IFN-gamma-inducible genes.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Antígenos HLA-D/genética , Antígenos HLA-DR/genética , Interferón gamma/farmacología , Secuencia de Bases , Línea Celular , ADN Recombinante , Genes , Antígenos HLA-DR/biosíntesis , Humanos , Caperuzas de ARN/genética , Proteínas Recombinantes de Fusión/biosíntesis , TransfecciónRESUMEN
Several seemingly unrelated procedures used to elicit the latent ATPase activity of soluble spinach coupling factor 1 can be correlated to the release of tightly-bound ADP from the uncoupled enzyme. This ADP release is further enhanced by the presence of medium nucleotides, especially substrate ATP, and may or may not involve release from the catalytic site itself. Similarly, the light/dithiothreitol activation of membrane-bound CF1 ATPase is reported to be accompanied by energy-dependent ADP dissociation. Further indication that ADP release is involved in the ATPase activation mechanism is the observation that a pyruvate kinase phosphoenolpyruvate trap for ADP released during light/dithiothreitol treatment greatly retards the decay of membrane-bound ATPase activity that occurs in the dark, presumably by preventing reassociation of ADP. The time course of CF1 reactivation by light, after light/dithiothreitol activation followed by dark decay, allows a distinction to be made between the apparently rate-limiting dithiol modification and the more rapid dissociation of tightly bound ADP.
Asunto(s)
Adenosina Difosfato/metabolismo , Plantas/enzimología , ATPasas de Translocación de Protón/metabolismo , Adenosina Trifosfato/farmacología , Catálisis , Ditiotreitol/farmacología , Activación Enzimática , Luz , Unión Proteica , SolubilidadRESUMEN
This is a retrospective review of 335 elective sterilizations performed in a private community practice. One hundred sixteen minilaparotomy sterilizations and 219 laparoscopic Falope-Ring sterilizations were reviewed. The study indicates a significantly greater mean hospital stay (1.78 versus 1.05 days), requirement for postoperative analgesics (3.34 doses versus 1.76), operative time (23.35 versus 18.82 minutes), and number of days (5.2 versus 2.84) required to resume normal functional activity on a subjective basis in the minilaparotomy group. There were two subsequent pregnancies among the laparoscopic group and none in the minilaparotomy cases. Technical failures occurred in 2.7% of the laparoscopic group. The authors offer suggestions, based on their results, for counseling patients interested in sterilization procedures.