RESUMO
A major goal of hemoglobinopathy research is to develop treatments that correct the underlying molecular defects responsible for sickle cell disease and beta-thalassemia. One approach to achieving this goal is the pharmacologic induction of fetal hemoglobin (HbF). This strategy is capable of inhibiting the polymerization of sickle hemoglobin and correcting the globin chain imbalance of beta-thalassemia. Despite this promise, none of the currently available HbF-inducing agents exhibit the combination of efficacy, safety, and convenience of use that would make them applicable to most patients. The recent success of targeted drug therapies for malignant diseases suggests that this approach could be effective for developing optimal HbF-inducing agents. A first step in applying this approach is the identification of specific molecular targets. However, while >70 HbF-inducing agents have been described, neither molecular mechanisms nor target molecules have been definitively verified for any of these compounds. To help focus investigation in this area, we have reviewed known HbF-inducing agents and their proposed mechanisms of action. We find that in many cases, current models inadequately explain key experimental results. By integrating features of the erythropoietic stress model of HbF induction with data from recent intracellular signaling experiments, we have developed a new model that has the potential to explain several findings that are inconsistent with previous models and to unify most HbF-inducing agents under a common mechanism: cell stress signaling. If correct, this or related models could lead to new opportunities for development of targeted therapies for the beta-hemoglobinopathies.
Assuntos
Eritrócitos/metabolismo , Eritropoese/fisiologia , Hemoglobina Fetal/biossíntese , Regulação da Expressão Gênica/fisiologia , Globinas/biossíntese , Hemoglobinopatias/tratamento farmacológico , Modelos Genéticos , Estresse Fisiológico/genética , Adolescente , Animais , Butiratos/farmacologia , Butiratos/uso terapêutico , Ensaios Clínicos como Assunto/estatística & dados numéricos , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/uso terapêutico , Eritrócitos/patologia , Eritropoese/efeitos dos fármacos , Hemoglobina Fetal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Globinas/genética , Transplante de Células-Tronco Hematopoéticas , Hemoglobinopatias/sangue , Hemoglobinopatias/genética , Hemoglobinopatias/fisiopatologia , Inibidores de Histona Desacetilases , Humanos , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiopatologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in beta-thalassaemia and sickle cell anaemia.
Assuntos
Células Precursoras Eritroides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Globinas/biossíntese , Imunossupressores/farmacologia , Sirolimo/farmacologia , Talassemia beta/metabolismo , Adulto , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Células Precursoras Eritroides/patologia , Feminino , Hemoglobina Fetal/biossíntese , Hemoglobina Fetal/genética , Globinas/genética , Humanos , Imunossupressores/uso terapêutico , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Sirolimo/uso terapêutico , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , Talassemia beta/patologiaRESUMO
The first studies of the pharmacological induction of fetal hemoglobin were conducted in patients with sickle cell disease and thalassemia. Although hydroxyurea was approved by the FDA for the treatment of sickle cell disease in 1996, no similar pharmacological agent(s) has been approved for the treatment of patients with thalassemic disorders. The small-scale studies of the induction of fetal hemoglobin in thalassemia have been generally disappointing. The aim of this report is to provide a critical analysis of the factors that may be responsible for our failure to develop an effective fetal hemoglobin induction therapy for patients with thalassemia. We also describe several areas for future investigation that may be critically important for the development of an effective therapy for thalassemia.
Assuntos
Azacitidina/uso terapêutico , Hemoglobina Fetal/biossíntese , Expressão Gênica/efeitos dos fármacos , Globinas/biossíntese , Hidroxiureia/uso terapêutico , Talassemia/tratamento farmacológico , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Animais , Azacitidina/efeitos adversos , Azacitidina/farmacologia , Terapia Combinada , Aprovação de Drogas , Avaliação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Eritropoese/efeitos dos fármacos , Hemoglobina Fetal/genética , Regulação da Expressão Gênica , Globinas/genética , Humanos , Hidroxiureia/efeitos adversos , Hidroxiureia/farmacologia , Ferro/metabolismo , Células K562/efeitos dos fármacos , Células K562/metabolismo , Modelos Animais , Papio , Talassemia/sangue , Talassemia/genética , Talassemia/terapia , Reação Transfusional , Estados Unidos , United States Food and Drug AdministrationRESUMO
Reactivation of fetal hemoglobin (HbF) expression using pharmacological agents represents a potential strategy for the therapy of beta-thalassemia, sickle cell disease, HbE and other beta-hemoglobinopathies. However, the drugs currently available have low efficacy and specificity and are associated with high toxicity. We describe the development of stable cellular genomic reporter assays (GRAs) based on the green fluorescence protein (EGFP) gene under the Ggamma-globin promoter in the intact human beta-globin locus. We show that human erythroleukemic cell lines stably transfected with a Ggamma-EGFP beta-globin locus construct can maintain a uniform basal level of EGFP expression over long periods of continuous culture and that induction of EGFP expression parallels the induction of the endogenous globin genes. We compared the EGFP-induction potency of a number of chemotherapeutic agents, including histone deacetylase inhibitors and DNA-binding agents. We show that hydroxyurea and butyrate result in moderate levels of induction (70-80%) but with an additive inductive effect. Among the DNA-binding agents tested, cisplatin was the most potent inducer of HbF expression, (442+/-32%), a level which is comparable to hemin (764+/-145%). These results indicate that cellular GRAs containing Ggamma-EGFP-modified beta-globin locus constructs can be used to develop novel inducers of HbF synthesis for the therapy of beta-hemoglobinopathies.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Hemoglobina Fetal/efeitos dos fármacos , Butiratos/farmacologia , Linhagem Celular , Cisplatino/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genoma Humano , Globinas/biossíntese , Globinas/efeitos dos fármacos , Globinas/genética , Proteínas de Fluorescência Verde , Hemina/farmacologia , Inibidores de Histona Desacetilases , Humanos , Hidroxiureia/farmacologia , Células K562 , Proteínas Luminescentes/efeitos dos fármacos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismoRESUMO
Orally bioactive compounds that induce gamma globin gene expression at tolerable doses are needed for optimal treatment of the beta-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce gamma globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce gamma globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which gamma globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce gamma globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human beta globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, gamma globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate gamma globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the beta hemoglobinopathies and useful for the oral treatment of other anemias.
Assuntos
Eritropoese/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Globinas/biossíntese , Anemia/genética , Anemia/metabolismo , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Cromossomos Artificiais de Levedura , Avaliação Pré-Clínica de Medicamentos , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoetina/farmacologia , Ácidos Graxos Voláteis/farmacocinética , Genes Reporter , Globinas/genética , Meia-Vida , Humanos , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Transgênicos , Papio , Fenilpropionatos/farmacologia , Regiões Promotoras Genéticas , Proteínas Recombinantes , Contagem de Reticulócitos , TransfecçãoRESUMO
The biological activity of p53 in IW32 erythroleukemia cells was investigated. IW32 cells had no detectable levels of p53 mRNA and protein expression. By transfecting a temperature-sensitive mutant p53 cDNA, tsp53val135, into the cells, we have established several clones stably expressing the mutant p53 allele. At permissive temperature, these p53 transfectants were arrested in G1 phase and underwent apoptosis. Moreover, differentiation along the erythroid pathway was observed as evidenced by increased benzidine staining and mRNA expression of beta-globin and the erythroid-specific delta-aminolevulinic acid synthase (ALAS-E). Treatment of cells with protein tyrosine phosphatase inhibitor vanadate blocked the p53-induced differentiation, but not that of cell death or growth arrest. Increased protein tyrosine phosphatase activity as well as mRNA levels of PTPbeta2 and PTPepsilon could be observed by wildtype p53 overexpression. These results indicate that p53 induced multiple phenotypic consequences through separate signal pathways in IW32 erythroleukemia cells, and protein tyrosine phosphatase is required for the induced differentiation.
Assuntos
Genes p53 , Leucemia Eritroblástica Aguda/patologia , Proteínas de Neoplasias/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Fosfatases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , 5-Aminolevulinato Sintetase/biossíntese , 5-Aminolevulinato Sintetase/genética , Animais , Apoptose , Diferenciação Celular , DNA Complementar/genética , Indução Enzimática , Fase G1 , Regulação Leucêmica da Expressão Gênica , Globinas/biossíntese , Globinas/genética , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/genética , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fenótipo , Fosforilação , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
The development and evaluation of drugs to elevate fetal hemoglobin in the treatment of the genetic diseases of hemoglobin would be facilitated by the availability of reliable cell assays. We have used real-time, quantitative polymerase chain reaction (PCR) analyses of globin messenger RNA (mRNA) levels in a biphasic, erythropoietin-dependent primary culture system for human adult erythroid cells in order to assay compounds for their ability to modulate levels of adult (beta) and fetal (gamma) globin mRNA. Complementary DNA synthesized from total RNA extracted at timed intervals from aliquots of cells were assayed throughout the period that the culture was studied. gamma-globin mRNA levels were found to be much lower (less than 1%) than beta-globin mRNA levels. At concentrations of agents chosen for minimal effect on cell division, we find that the 3 drugs studied, 5-azacytidine (5 micromol/L), hydroxyurea (40 micromol/L), and butyric acid (0.5 mmol/L), significantly increase gamma-globin mRNA levels. Interestingly, hydroxyurea also had a small stimulatory effect on beta-globin mRNA levels, while butyric acid caused a twofold inhibition of beta-globin mRNA levels, and 5-azacytidine had little effect on beta-globin mRNA levels. The net result of all 3 drugs was to increase the gamma/(gamma + beta) mRNA ratios by threefold to fivefold. These data suggest that the mechanism is distinct for each drug. The profile of butyric-acid-induced changes on globin gene expression is also quite distinct from changes produced by trichostatin A, a known histone deacetylase inhibitor. Quantitative PCR analyses of human erythroid cells should prove useful for studying the mechanism(s) of action of known inducers of gamma-globin and identifying new drug candidates.
Assuntos
Células Precursoras Eritroides/efeitos dos fármacos , Hemoglobina Fetal/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Globinas/biossíntese , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Adulto , Azacitidina/farmacologia , Ácido Butírico/farmacologia , DNA Complementar/genética , Células Precursoras Eritroides/química , Eritropoetina/farmacologia , Hemoglobina Fetal/genética , Globinas/genética , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Hidroxiureia/farmacologiaRESUMO
Globin gene switching may be mediated by proteins expressed during different stages of development. Their identification may clarify the mechanisms of the conversion from fetal to adult globin production and lead to new approaches to reversing or retarding the gamma- to beta-globin gene switch. To explore this hypothesis, K562 erythroleukemia cells were induced to differentiate with 1.25, 2.5, and 5 mM sodium butyrate and gene expression was studied after 24, 48, and 72 h. Erythroid differentiation was verified by benzidine staining and by measuring the activity of a transduced A gamma-globin gene promoter linked to a luciferase reporter gene. Using differential display polymerase chain reaction (PCR), total mRNA extracted from induced cells at each time point of induction was reverse transcribed in the presence of A, G, and C anchored primers and 16 arbitrary primers, calculated to amplify approximately 50% of expressed genes. Amplified mRNAs from induced and uninduced cells were separated in polyacrylamide gels and compared. More than 110 cDNA fragments which appeared to represent either up- or downregulated mRNA species in induced K562 cells were identified. Sixty-four of these fragments had more than 95% homology to known GenBank sequences. Seventeen fragments with characteristics of transcription factors were cloned. These include differentiation-related gene-1 (drg-1), PAX 3/forkhead transcription factor, HZF2 which is a Kruppel-related zinc finger protein, three helix-loop-helix proteins (heir-1, Id3, and GOS8), alpha-NAC transcriptional coactivator, LIM domain protein, and trophoblast hypoxia regulating factor. Differential expression of all 17 fragments over 72 h was confirmed by reverse Northern dot blot analysis, semiquantitative PCR using nested primers, and Northern analysis. Erythroid maturation in induced K562 cells is associated with differential expression of numerous genes. Some encode transcription factors that could effect the initiation of HbF synthesis. Almost half of the differentially expressed clones contained cDNAs of unidentified open reading frames and these are the object of continued study.
Assuntos
Globinas/genética , Leucemia Eritroblástica Aguda/genética , Fatores de Transcrição/genética , Northern Blotting , DNA Complementar/análise , DNA Complementar/química , Hemoglobina Fetal/genética , Regulação da Expressão Gênica , Genes de Troca , Globinas/biossíntese , Humanos , Células K562 , Leucemia Eritroblástica Aguda/patologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
Stage-specific expression of the human beta-like globin genes is controlled by interactions between regulatory elements near the individual genes and additional elements located upstream in the Locus Control Region (LCR). Elucidation of the mechanisms that govern these interactions could suggest strategies to reactivate fetal (gamma) or embryonic (epsilon) genes in individuals with severe hemoglobinopathies. This study extends an earlier analysis of a transgenic construct, HS3epsilon gamma, testing: (A) the effect of substitution of HS2 for HS3 on stage-specific expression of the epsilon and gamma genes and, (B) the role of an evolutionarily conserved YY1 binding site in transcriptional regulation of the gamma gene. The data show that both HS3epsilon gamma and HS2epsilon gamma can individually support embryonic expression of epsilon and fetal expression of Agamma. Thus, the cis regulators of distinct expression patterns for epsilon and gamma are likely to reside near the genes, rather than in specific hypersensitive sites of the LCR. Alterations in Agamma expression patterns observed in transgenic lines carrying a construct with a mutation in a conserved YY1 binding site at -1086 indicate that this site might function to facilitate active transcription of the gamma gene in fetal life.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Globinas/genética , Região de Controle de Locus Gênico , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Sequência Conservada , Elementos Facilitadores Genéticos , Fatores de Ligação de DNA Eritroide Específicos , Globinas/biossíntese , Humanos , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Mapeamento por Restrição , Transcrição Gênica , Fator de Transcrição YY1 , Saco Vitelino/metabolismoRESUMO
Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glutationa Peroxidase/genética , RNA Mensageiro/biossíntese , Sequências Reguladoras de Ácido Nucleico , Selênio/farmacologia , Animais , Células CHO , Cricetinae , Regulação para Baixo , Globinas/biossíntese , Globinas/genética , Glutationa Peroxidase/biossíntese , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
Homozygous beta thalassemia affects thousands of people around the world. Current management of this condition includes regular transfusion of red cells, which leads to transfusional iron overload requiring chelation therapy: increasing hemoglobin levels while decreasing or eliminating iron overload is therefore a major therapeutic goal in the treatment of thalassemia. Bone marrow transplantation may achieve this goal, but it is not an option for most patients. This study reports on efforts to increase gamma-globin transcription and HbF production using sodium phenylbutyrate (SPB) and hydroxyurea (HU). It was found that 36% (4/11) of all patients or 50% (4/8) of non-transfused patients responded to SPB (increase in Hb levels of 1 g/dL). A positive correlation between baseline serum erythropoietin level and likelihood of response to SPB was observed. Since HU may also increase HbF production, evaluation of combination therapy with these drugs is underway and preliminary results are reported.
Assuntos
Antidrepanocíticos/uso terapêutico , Hemoglobina Fetal/biossíntese , Globinas/biossíntese , Hidroxiureia/uso terapêutico , Fenilbutiratos/uso terapêutico , Talassemia beta/tratamento farmacológico , Transfusão de Sangue , Eritropoetina/sangue , Humanos , Transcrição Gênica/efeitos dos fármacos , Talassemia beta/sangueRESUMO
Recent studies have indicated that nitric oxide may affect iron metabolism through disruption of the iron-sulfur complex of iron regulatory protein-1, a translational regulator. In the present study, we report that heterologous expression of murine macrophage nitric oxide synthase (NOS-2) in the human erythroleukemic K562 cell line results in constitutive production of nitric oxide associated with inhibition of hemoglobin expression. K562 cells were transfected with an episomally-maintained, hygromycin-selectable expression vector bearing the coding region of NOS-2. Constitutive NOS expression was detected by Western blotting of cell lysates and by the accumulation of nitrite in the culture media. Although NOS-transfected cells grew more slowly than control cells, they were able to maintain constitutive expression of NOS and production of nitric oxide for more than 1 month following transfection. The hemoglobin content of NOS-transfected K562 cells was less than one-fifth that of control cells, but increased markedly if NOS inhibitor was included in the culture media. The nitric oxide-mediated inhibition of hemoglobin expression was reversed by supplementing the culture media with 20 mumol/L hemin or 0.5 mmol/L 5-amino-levulinate, indicating that nitric oxide did not directly inhibit hemoglobin synthesis, but likely acted on a step in heme synthesis. mRNA levels for globin and erythroid aminolevulinic acid synthase (eALAS) were the same in both NOS-transfected and control cells. Our observations indicate that hemoglobin expression is inhibited by nitric oxide in NOS-transfected K562 cells by posttranscriptional repression of eALAS, the first enzyme of the heme biosynthetic pathway. The most likely mechanism is a nitric oxide-mediated translational repression of eALAS, as was recently demonstrated for ferritin synthesis. These observations further illustrate the potential for endogenously produced nitric oxide to regulate cellular posttranscriptional events. In particular, our observations may be relevant to the role of nitric oxide in anemia and lowered blood hemoglobin concentrations that are associated with chronic infections, such as tuberculosis or parasitic disease.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Hemoglobinas/biossíntese , Leucemia Eritroblástica Aguda/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/biossíntese , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/fisiologia , 5-Aminolevulinato Sintetase/genética , Ácido Aminolevulínico/farmacologia , Sequência de Bases , Butiratos/farmacologia , Ácido Butírico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Globinas/biossíntese , Globinas/genética , Heme/biossíntese , Hemina/farmacologia , Humanos , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Biossíntese de Proteínas , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The relative contribution of the transmembrane segments in the alpha-subunit of Shaker-type potassium channels was investigated in relation to potassium channel function. Starting from a wild-type Kv1.1 channel, four different deletion mutants were made, missing respectively transmembrane segments S1 and S2, S2 and S3, S1 to S3, and S1 to S4. To ensure the assembly of the different subunits, the hydrophylic N-terminal domain was always conserved. The lack of transmembrane segments S1 to S4 converts a depolarization-activated WT Kv1.1 channel with outward rectification into a hyperpolarization-activated channel with inward rectification. In contrast, mutant channels missing transmembrane segments S1 and S2, S2 and S3, or S1 to S3 did not reveal functional expression.
Assuntos
Oócitos/fisiologia , Peptídeos/fisiologia , Canais de Potássio/fisiologia , Deleção de Sequência , Animais , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Clonagem Molecular , DNA Complementar , Drosophila , Expressão Gênica , Globinas/biossíntese , Potenciais da Membrana , Modelos Estruturais , Mutagênese Sítio-Dirigida , Biossíntese Peptídica , Canais de Potássio/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Superfamília Shaker de Canais de Potássio , XenopusRESUMO
pSP64 derivatives were constructed to obtain cloning vectors suitable for in vitro transcription and subsequent in vitro synthesis of recombinant proteins equipped with N-terminal signal sequences. The amino acid sequence of the signal peptide was adapted and slightly modified from the one occurring in the neural cell adhesion molecule, NCAM. Its ability to direct recombinant proteins into secretory pathways was tested by in vitro translation in microsomal membrane-containing reticulocyte lysates and by injection of the pUMA-derived cRNAs into Xenopus laevis oocytes.
Assuntos
Globinas/biossíntese , Microssomos/metabolismo , Oócitos/metabolismo , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Vetores Genéticos , Globinas/isolamento & purificação , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , RNA Complementar/metabolismo , Proteínas Recombinantes/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Reticulócitos/metabolismo , Transcrição Gênica , Xenopus laevis , Dedos de ZincoRESUMO
Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.
Assuntos
Anticódon , Códon , Globinas/biossíntese , RNA de Transferência de Glutamina/metabolismo , Tetrahymena/genética , Zeína/biossíntese , Aminoacil-tRNA Sintetases/metabolismo , Animais , Composição de Bases , Sequência de Bases , DNA de Protozoário , Glutamina/metabolismo , Dados de Sequência Molecular , RNA de Protozoário/isolamento & purificação , RNA de Protozoário/metabolismo , RNA de Transferência de Glutamina/isolamento & purificação , Regiões Terminadoras GenéticasRESUMO
Inhibition of polypeptide chain elongation with the mRNA-complementary (antisense) oligonucleotide has been realized through a RNase H independent mechanism. Nuclease resistant complementary non-natural alpha-17-mer oligonucleotide did not inhibit cell-free protein biosynthesis of beta-globin in the wheat germ system because it did not elicit RNase H activity. Linkage of alkylating group [4-(N-2-chloroethyl-N-methyl)-aminobenzyl]-methylamine to the 5'-terminus of the alpha-oligomer led to the formation of its covalent adduct with mRNA which could not be translated in vitro. Linkage of hydrophobic residues to the terminal phosphates of natural oligonucleotides increased their stability against nucleases and uptake by human cancer cells. A porphyrin, substituted in the meso-position by aromatic groups, gave a rise to an approximately six-fold increase of uptake and cholesterol a 30-100-fold increase. Eighty percent of bound derivatives were found in cytoplasmic cellular fractions.
Assuntos
Globinas/biossíntese , Oligonucleotídeos Antissenso/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Alquilação , Sequência de Bases , Colesterol/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/química , Fosfatos/metabolismo , Células Tumorais CultivadasRESUMO
A 34 KD DNA-binding protein fraction from human placenta stimulated endogenous protein synthesis in rabbit reticulocyte and wheat-germ cell-free systems. Though the synthesis of several proteins were stimulated by the 34 KD protein, a dose-dependent increase of two polypeptides of molecular weights 42,000 and 51,000 were distinctly observed in reticulocyte lysates. The synthesis of the major protein (beta-globin) was not affected by the 34 KD protein. In both hemin supplemented and unsupplemented lysates, the ability of 34 KD protein to stimulate the synthesis of high molecular weight (HMW) proteins was drastically reduced by Mg++ and not by dsRNA.
Assuntos
Proteínas de Ligação a DNA/farmacologia , Proteínas da Gravidez/farmacologia , Biossíntese de Proteínas , Animais , Sistema Livre de Células , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Globinas/biossíntese , Humanos , Magnésio/farmacologia , Peso Molecular , Gravidez , RNA de Cadeia Dupla/metabolismo , Coelhos , Reticulócitos/metabolismoRESUMO
Fractions of rabbit reticulocyte lysates retained on heparin-Sepharose 4B catalyze incorporation of labelled amino acids into proteins in the absence of ribosomes, and several characteristics of this reaction are identical with peptide bond formation mediated by aminoacyl-tRNA-protein transferases. At least five different proteins become labelled, as revealed by polyacrylamide gel electrophoresis. Active fractions synthesize aminoacyl-tRNA which is utilized by transferase. Aminoacyl-tRNA-protein transferase activity may be uncoupled from that of aminoacyl-tRNA synthetase by puromycin or lysyl-phenylalanine which both inhibit the transferase activity only. Adenosine and phosphate inhibit aminoacyl-tRNA synthetase as well as the incorporation of labelled amino acids into proteins. This indicates that the incorporation must be preceded by charging of tRNA with amino acids. Presence of three different aminoacyl-tRNA-protein transferases, each of them specific for a group of four amino acids, was demonstrated. Serum albumin stimulates the incorporation of amino acids and a labelling of this protein was demonstrated in mixtures into which it had been added. Addition of both ribosomal subunits and globin messenger ribonucleoprotein significantly changes the pattern of labelled proteins, and synthesis of globin was demonstrated by polyacrylamide gel electrophoresis. Density-gradient analysis revealed formation of a 48 S ribosomal complex and the formation of polyribosomes. Systems composed of active fractions retained on heparin-Sepharose supplemented with ribosomes and globin messengers apparently catalyze the translation of this message, but interactions do exist between the ribosome-mediated peptide synthesis and non-ribosomal incorporation of amino acids into proteins.