RESUMEN
Bacterial superantigens trigger an excessive, Th1-cytokine response leading to toxic shock. We designed a peptide antagonist that inhibits SEB-induced expression of human genes for IL-2, IFN-gamma, and TNF-beta, cytokines that mediate shock. The peptide antagonist shows homology to a beta-strand-hinge-alpha-helix domain that is conserved structurally in superantigens produced by Staphylococcus aureus and Streptococcus pyogenes yet remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. For Th1-cell activation, superantigens depend on this domain. The peptide protected mice against lethal challenge with SEB or SEA. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed broad-spectrum, protective immunity, which rendered them resistant to further lethal challenges with different staphylococcal and streptococcal superantigens. Thus, the lethal effect of superantigens, mediated by Th1 cytokines, can be blocked with a peptide antagonist that inhibits their action at the top of the toxicity cascade, before activation of T cells takes place.
Asunto(s)
Antígenos Bacterianos/toxicidad , Citocinas/genética , Enterotoxinas/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Choque Séptico/prevención & control , Staphylococcus aureus/inmunología , Streptococcus pyogenes/inmunología , Superantígenos/toxicidad , Células TH1/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Citocinas/biosíntesis , Enterotoxinas/química , Enterotoxinas/inmunología , Enterotoxinas/toxicidad , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-2/biosíntesis , Interleucina-2/genética , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Superantígenos/química , Superantígenos/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Nucleoproteínas/metabolismo , Rec A Recombinasas/metabolismo , Daño del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/ultraestructura , Proteínas de Escherichia coli , Modelos Genéticos , Nucleoproteínas/ultraestructuraRESUMEN
Superantigens trigger an excessive cellular immune response, leading to toxic shock. We have designed a peptide antagonist that inhibits superantigen-induced expression of human genes for interleukin-2, gamma interferon and tumor necrosis factor-b, which are cytokines that mediate shock. The peptide shows homology to a b-strand-hinge-a-helix domain that is structurally conserved in superantigens, yet is remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. Superantigens depend on this domain for T-cell activation. The peptide protected mice against lethal challenge with staphylococcal and streptococcal superantigens. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed protective antibodies against superantigen that rendered them resistant to further lethal challenges, even with different superantigens. Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the beginning of the toxicity cascade, before activation of T cells takes place.
Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas , Enterotoxinas/inmunología , Activación de Linfocitos , Proteínas de la Membrana , Choque Séptico/prevención & control , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/inmunología , Sitios de Unión , Células Cultivadas , Secuencia Conservada , Reacciones Cruzadas , Enterotoxinas/antagonistas & inhibidores , Enterotoxinas/química , Enterotoxinas/farmacología , Enterotoxinas/toxicidad , Exotoxinas/inmunología , Exotoxinas/toxicidad , Femenino , Humanos , Inmunización Pasiva , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/química , Oligopéptidos/inmunología , Oligopéptidos/farmacología , Conejos , Choque Séptico/inmunología , Choque Séptico/terapia , Streptococcus pyogenes/inmunología , Superantígenos/química , Superantígenos/farmacología , Linfocitos T/microbiología , Factores de TiempoRESUMEN
Mutations in the human genome are clustered in hot-spot regions, suggesting that some sequences are more prone to accumulate mutations than others. These regions are therefore more likely to lead to the development of cancer. Several pathways leading to the creation of mutations may be influenced by the DNA sequence, including sensitivity to DNA damaging agents, and repair mechanisms. We have analyzed sequence context effects on translesion replication, the error-prone repair of single-stranded DNA regions carrying lesions. By using synthetic oligonucleotides containing systematic variations of sequences flanking a synthetic abasic site, we show that translesion replication by the repair polymerase DNA polymerase beta is stimulated to a moderate extent by low stacking levels of the template nucleotides downstream of the lesion, combined with homopolymeric runs flanking the lesion both upstream and downstream. A strong stimulation of translesion replication by DNA polymerase beta was seen when fork-like flap structures were introduced into the DNA substrate downstream of the lesion. Unlike for gapped substrates, this stimulation was independent of the presence of a phosphate group at the 5' terminus of the flap. These results suggest that DNA polymerase beta may participate in cellular DNA transactions involving higher order structures. The significance of these results for in vivo translesion replication is discussed.
Asunto(s)
ADN Polimerasa beta/química , Replicación del ADN , ADN/química , Sitios de Unión , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Estructura Molecular , Moldes GenéticosRESUMEN
Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD'. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD', RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD', RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10-100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD'-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase eta.
Asunto(s)
Proteínas Bacterianas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Rec A Recombinasas/metabolismo , Secuencia de Bases , ADN Polimerasa III/metabolismo , ADN Polimerasa Dirigida por ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Humanos , Datos de Secuencia Molecular , MutagénesisRESUMEN
Transient expression of IFN-gamma and IL-2 mRNA and its control by post-transcriptional and suppressive mechanisms were analysed in phytohaemagglutinin-induced peripheral blood mononuclear cells (PBMC) from 47 patients with SLE and 31 age-matched normal donors, using quantitative hybridization with antisense RNA probes. In SLE, basal levels of gene expression did not deviate from those of normal donors, but strongly aberrant patterns were obtained upon induction. The ratio of subjects exhibiting highly inducible IFN-gamma gene expression in their PBMC to those showing moderate or low inducibility was increased five-fold in SLE (P = 0.003). High inducibility was observed for 43% of SLE patients and was equally pronounced in partial remission, mild or active disease. Inducibility of IL-2 mRNA, by contrast, remained similar to that for normal donors. However, regulation of IFN-gamma gene expression differed for mild SLE. Patients with mild disease showing high inducibility of IFN-gamma mRNA in their PBMC not only had the highest frequency of responders, but also the highest extent of an individual response, defined by superinduction of mRNA, to agents that relieve suppression (gamma-irradiation) or post-transcriptional down-regulation (cycloheximide). By contrast, patients with active SLE showing high IFN-gamma mRNA inducibility had normal suppressive capacity as well as post-transcriptional control. Hence, both high inducibility of the IFN-gamma gene and its suppression are relevant to disease. Hyperactivation of the IFN-gamma gene may be alleviated in mild SLE by a vigorous, concomitant activation of post-transcriptional control and of cell-mediated suppression.
Asunto(s)
Regulación de la Expresión Génica , Interferón gamma/genética , Interferón gamma/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Adulto , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/efectos de la radiación , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/efectos de la radiación , Cicloheximida/farmacología , Femenino , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/efectos de la radiación , Masculino , Fitohemaglutininas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismoRESUMEN
Human TNF-beta (lymphotoxin) gene expression is down-regulated by immunosuppression. Induction of TNF-beta mRNA in lymphoid cells is greatly enhanced by gamma-irradiation, cyclophosphamide and cimetidine, agents that each inhibit activation of suppressive cells. The level of TNF-beta mRNA expressed in response to stimulation, whether by mitogen or antigen, is reduced strongly by concomitant activation of suppressive cell subsets. Removal of CD8 or CD11b cells leads to a pronounced superinduction of TNF-beta mRNA in the depleted cell population. Induction of TNF-beta mRNA precedes appearance of suppressive cell activity, allowing for temporary expression. The TNF-beta gene is as sensitive as IFN-gamma and IL-2 genes to suppression. Hence, three genes characteristically expressed in Th1 cells, encoding IL-2, IFN-gamma, and TNF-beta, are similarly regulated by cell-mediated suppression. Actual levels of TNF-beta during an immune response are determined by the balance between activities of expressing and suppressing cell subsets, both transiently manifested.
Asunto(s)
Regulación hacia Abajo , Regulación de la Expresión Génica , Linfotoxina-alfa/genética , Antígenos CD8/inmunología , Células Cultivadas , Cimetidina/farmacología , Ciclofosfamida/farmacología , Rayos gamma , Humanos , Inmunosupresores/farmacología , Antígeno de Macrófago-1/inmunologíaRESUMEN
The incidence of positive skin test responses among atopic subjects with suspected respiratory allergy was investigated with commercial and autochthonous pollen extracts of various cultivars of Olea europaea. Pollen was collected from olive trees of well-defined cultivars, extracted, and separated by SDS-PAGE. Immunoblots were used to identify the various IgE-binding proteins of the pollen extracts of the various cultivars. The results revealed six predominant IgE-binding bands, some of which appear in all the cultivars examined. The 18-20-kDa band (Ole e 1) appeared in only eight of the cultivars, but not in the nine others. The presence of specific IgE-binding bands in the various pollen extracts and their correlation with the incidence of positive skin tests are discussed.
Asunto(s)
Polen/inmunología , Hipersensibilidad Respiratoria/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Inmunoglobulina E/inmunología , Pruebas Cutáneas , Árboles/inmunologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) protease (PR) and p6(Pol) are translated as part of the Gag-Pol polyprotein after a ribosomal frameshift. PR is essential to virus replication and is responsible for cleaving Gag and Gag-Pol precursors, but the role of p6(Pol) in HIV-1 infection is poorly understood. Here, we report that (i) PR is present in mature HIV-1 virions primarily as a p6(Pol)-PR fusion protein; (ii) HIV-1 PR cleaves viral precursor proteins expressed in bacterial cells at the Phe-Leu bond (positions 1639 to 1642) located at the junction of the NC and p6(Pol) proteins, releasing the p6(Pol)-PR fusion protein; and (iii) purified p6(Pol)-PR fusion protein undergoes autocleavage in vitro at at least three sites.
Asunto(s)
Proteínas de Fusión gag-pol , VIH-1/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Proteínas Virales de Fusión , ViriónRESUMEN
Linomide (LS-2616, quinoline-3-carboxamide) has strong immunomodulating effects in animal models, inhibiting toxic shock, progressive autoimmune disease and cancer. In humans, linomide strongly reduced the appearance of new lesions in multiple sclerosis yet enhanced immune responses after bone marrow transplantation. In contrast to these clear effects in vivo, attempts to show an effect of linomide in vitro have not been successful and its mode of action remains to be elucidated. Here we show that at concentrations effective in vivo, linomide is active on human peripheral blood mononuclear cells (PBMC), severely inhibiting the induction by Staphylococcus aureus enterotoxin B of mRNA of three cytokine genes expressed in Th1 cells, those for IFN-gamma, IL-2, and tumor necrosis factor-beta. Yet, cell viability was not affected by linomide. The extent of inhibition is dose-dependent on linomide. Linomide also blocked induction of IL-2 and IFN-gamma mRNA by phytohemagglutinin. The inhibitory effect is expressed immediately but can be enhanced significantly by a prolonged exposure of PBMC to linomide, reaching 10-fold. These results support the concept that linomide antagonizes the activation of Th1 cells during a cellular immune response.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Citocinas/biosíntesis , Citocinas/genética , Regulación de la Expresión Génica/genética , Hidroxiquinolinas/farmacología , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Células Cultivadas , Citocinas/antagonistas & inhibidores , Relación Dosis-Respuesta Inmunológica , Enterotoxinas/antagonistas & inhibidores , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Interleucina-2/genética , Leucocitos Mononucleares/efectos de los fármacos , Linfotoxina-alfa/antagonistas & inhibidores , Linfotoxina-alfa/biosíntesis , Linfotoxina-alfa/genética , ARN Mensajero/biosíntesis , Staphylococcus aureus/efectos de los fármacos , Superantígenos/efectos de los fármacosRESUMEN
Histamine is considered to be an activator of cells with suppressive capacity. In agreement with this concept, we show that histamine elicits a strong inhibition of the induced expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes. However, our experiments reveal a novel property of histamine: early in the induction process, it strongly stimulates expression of these two genes in cultured human peripheral blood mononuclear cells (PBMC). The histamine-mediated superinduction of IL-2 mRNA is seen also in a Th cell line, showing that such cells respond directly to histamine. In the course of mitogenic induction, a 20-fold stimulation by histamine is converted into an equally strong inhibition. The response of a PBMC population to histamine thus undergoes a remarkable change following T cell activation. The dual effect of histamine can be blocked by the H2 histamine receptor antagonist cimetidine, while the early activation by histamine is mimicked by the H2 agonist impromidine, showing that both activation and inhibition of IL-2 and IFN-gamma gene expression by histamine are exerted via this receptor. These results support the concept that histamine, released during an immune response, exerts opposite regulatory effects by first activating cells able to express the IL-2 and IFN-gamma genes and only then suppressive cells that become responsive to histamine more slowly, but once activated shut off the expression of these genes.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Histamina/farmacología , Inmunosupresores/farmacología , Interferón gamma/genética , Interleucina-2/genética , Células Cultivadas , Cimetidina/farmacología , Ciclofosfamida/farmacología , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/efectos de los fármacos , Interleucina-2/antagonistas & inhibidores , Receptores Histamínicos H2/efectos de los fármacosRESUMEN
The incidence of skin-tested sensitivity to olive pollen allergens among subjects with suspected atopic respiratory allergy was investigated in various populations of Israelis. This incidence was correlated with the olive cultivars, with the abundance of trees in the patient's neighborhoods, and with the history of exposure of the studied populations to olive pollen. Positive skin reactions to olive pollen, among atopic patients of the Jewish population, is rather high where olive trees are abundant (66%), and lower (29%) where trees are scarce (P < 0.003). Sensitization was significantly lower (P < 0.003) among a population of Israeli atopic Arabs (16%), though these Arabs have lived in an olive-rich area for several generations.
Asunto(s)
Hipersensibilidad a los Alimentos/inmunología , Frutas/inmunología , Polen/inmunología , Árabes , Exposición a Riesgos Ambientales/efectos adversos , Humanos , Pruebas Intradérmicas , Israel/epidemiología , JudíosRESUMEN
The activity of avian sarcoma leukemia virus (ASLV) protease (PR) prior to its release from the precursor protein was determined by introducing mutations at the cleavage site between PR and the adjacent upstream nucleocapsid (NC) protein. Gag DNA fragments containing these mutations were cloned into expression vectors and introduced into Escherichia coli in which the ASLV proteins were expressed. The dipeptide NC-PR containing these mutations did not undergo autoprocessing when expressed in bacterial cells and the fused proteins were devoid of enzymatic activity. However, when the whole Gag polyprotein containing these mutations was expressed in bacterial cells, other PR cleavage sites in the viral Gag polyprotein underwent normal cleavage, indicating that the release of free PR is not a prerequisite for correct processing of the ASLV Gag precursor.
Asunto(s)
Virus del Sarcoma Aviar/enzimología , Endopeptidasas/metabolismo , Productos del Gen gag/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Virus del Sarcoma Aviar/genética , Sitios de Unión , Catálisis , Endopeptidasas/genética , Escherichia coli , Regulación Viral de la Expresión Génica , Productos del Gen gag/genética , Mutación , Péptidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismoRESUMEN
The retrovirus protease (PR), an aspartic PR, is composed of two identical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarcoma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviruses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser and Gly residues to Thr and Ala, respectively. Replacement of Gly with Ala yielded an ASLV PR devoid of proteolytic activity. The Ser to Thr conversion did not alter the substrate specificity of the enzyme. Both wild-type and mutated PRs correctly cleaved viral precursors expressed in bacterial cells, as well as synthetic peptides homologous to ASLV and human immunodeficiency virus type 1 cleavage sites. Bacterially produced ASLV PR with Thr instead of Ser had increased enzymatic activity, as shown by hydrolysis of synthetic peptides. However, this mutation reduced the production of reverse transcriptase-containing particles and infectious virus following transfection of permissive cells with virus DNA.
Asunto(s)
Alpharetrovirus/enzimología , Ácido Aspártico Endopeptidasas/genética , Proteínas de Fusión gag-pol/genética , Productos del Gen gag/genética , Mutación Puntual , Alpharetrovirus/fisiología , Secuencia de Aminoácidos , Ácido Aspártico Endopeptidasas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , Escherichia coli/genética , Proteínas de Fusión gag-pol/metabolismo , Productos del Gen gag/metabolismo , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Virión/enzimología , Replicación ViralRESUMEN
Interleukin-2 (IL-2) regulates the clonal expansion of activated T cells and is produced in limited amounts during an immune response. Mitogenic induction of human IL-2 gene expression elicits a transient wave of unstable mRNA. We show here that transcription continues unabated during and well beyond the time when the wave is subsiding, yet few, if any, new mRNA molecules are generated once the wave has reached its maximum. Instead, IL-2 precursor transcripts accumulate, becoming the majority of expressed IL-2 RNA molecules. The flow of precursor transcripts into mature mRNA becomes inhibited in the course of induction. When translation is blocked (e.g. by cycloheximide), expression of IL-2 mRNA can be superinduced by 2 orders of magnitude. This superinduction is completely dependent upon transcription, yet is not accompanied by any significant increase in the rate of primary transcription or in mRNA stability. Instead, the processing of nuclear IL-2 precursor transcripts is greatly facilitated, resulting in pronounced superinduction of cytoplasmic mRNA. Once its transcription has been induced, therefore, expression of the IL-2 gene is down-regulated extensively at the level of precursor RNA processing.
Asunto(s)
Interleucina-2/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , Células Cultivadas , Cicloheximida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/metabolismo , Precursores de Proteínas/genética , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The Gag and Gag-Pol precursors of avian sarcoma leukemia virus (ASLV) are translated from viral genomic-size mRNA at a molar ratio of about 20:1. Translation of Gag is terminated at the stop codon UAG located at the carboxyl-terminus of the viral protease (PR), whereas a ribosomal frameshift occurring at the carboxyl-terminus of Gag allows translation of the Gag-Pol precursor. To determine how PR is released from the Gag-Pol precursor, a single base (A or T) was inserted at the Gag-Pol junction in order to adjust the translation into a single reading frame. These mutations allow processing of the viral precursor when expressed in bacterial cells, but cause cessation of viral production after transfection of avian cells. The viral PR released from the large precursor is one amino acid longer than PR cleaved from the Gag polyprotein and is terminated by an Ile instead of a Leu residue.
Asunto(s)
Alpharetrovirus/genética , Proteínas de Fusión gag-pol/genética , Biosíntesis de Proteínas , Sistemas de Lectura/genética , Ribosomas/metabolismo , Alpharetrovirus/crecimiento & desarrollo , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Proteínas de Fusión gag-pol/biosíntesis , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteínas RecombinantesRESUMEN
The level of transient expression of human IL-2 and IFN-gamma genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-gamma gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-gamma was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-gamma gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-gamma mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition.
Asunto(s)
Regulación de la Expresión Génica , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Subgrupos Linfocitarios/fisiología , Células Sanguíneas , Antígenos CD11/análisis , Linfocitos T CD8-positivos/fisiología , Moléculas de Adhesión Celular/fisiología , Humanos , Separación Inmunomagnética , Interferón gamma/genética , Interleucina-2/genética , Selectina L , Tonsila Palatina/citología , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Establishment of protective immunity depends critically on IFN-gamma. We show that in human peripheral blood mononuclear cells, low doses of IL-2 greatly potentiate the response of the IFN-gamma gene to mitogen, by over 100-fold. By itself, IL-2 is unable to induce IFN-gamma mRNA to a significant extent. Yet, exposure to IL-2 leads to cellular commitment within a few hours, expressed by greatly enhanced accumulation of IFN-gamma mRNA upon subsequent exposure to phytohemagglutinin. Changes induced by IL-2 do not relieve the requirement for de novo protein synthesis during the early phases of induction of IFN-gamma gene expression. IL-2 may induce a component essential for induction of IFN-gamma mRNA that is utilized during subsequent exposure to a mitogenic signal. Our results demonstrate synergy between IL-2 and mitogen in IFN-gamma gene induction.
Asunto(s)
Regulación de la Expresión Génica , Interferón gamma/biosíntesis , Interleucina-2/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Células Cultivadas , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Interferón gamma/genética , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos , Fitohemaglutininas/farmacología , Biosíntesis de Proteínas , ARN Mensajero/análisis , Factores de Tiempo , Activación TranscripcionalRESUMEN
We have introduced mutations into the region of the genome of human immunodeficiency virus type 1 (HIV-1) that encodes the cleavage sites between the viral protease (PR) and the adjacent upstream region of the polyprotein precursor. Segments containing these mutations were introduced into plasmids, and the retroviral proteins were expressed in Escherichia coli. The mutations prevented cleavage between the PR and the adjacent polypeptide; however, other PR cleavage sites in the polyprotein were cleaved normally, showing that the release of free PR is not a prerequisite for the appropriate processing of HIV-1 precursors.
Asunto(s)
Productos del Gen gag/metabolismo , Proteasa del VIH/metabolismo , VIH-1/enzimología , Procesamiento Proteico-Postraduccional , Escherichia coli/genética , Productos del Gen gag/genética , Proteasa del VIH/genética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Regulated expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes, induced in cultured peripheral blood mononuclear cells from patients with end-stage renal disease on hemodialysis (HD; N = 13) or peritoneal dialysis (PD; N = 13), was compared to that of 32 normal donors. Culture conditions were chosen that measure the transient, phytohemagglutinin-induced expression of IL-2 and IFN-gamma messenger RNA (mRNA), as well the intactness of post-transcriptional and suppressor T cell-dependent mechanisms that control this expression. The latter was achieved by analyzing the superinduction of IL-2 and IFN-gamma mRNA occurring upon culture with cycloheximide or after low-dose gamma-irradiation, respectively. HD subjects showed a complete loss of inducibility of the IL-2 gene, concomitant with decreased inducibility of IFN-gamma mRNA. In PD subjects, by contrast, expression of IL-2 mRNA was as vigorous as in normal donors, while IFN-gamma mRNA was even more strongly inducible. This difference in gene inducibility is caused by a lack of T cell function in HD subjects. The defect in IL-2 gene expression in HD subjects, occurring most likely at transcription, may underly their impaired immune function.