RESUMO
Proteinase yscE of the yeast Saccharomyces cerevisiae has been compared with the 20 S cylinder particles of Xenopus laevis. Both proteins are characterized by a similar group of 10-12 polypeptides with molecular masses ranging between 21 and 38 kDa. Antibodies generated against the 20 S Xenopus cylinder particles show cross-reactivity with yeast proteinase yscE subunits. The Xenopus particles and yeast proteinase yscE exhibit an identical image in electron microscopy. Both proteins appear as hollow cylinders mostly composed of four stacked annuli. The Xenopus 20 S particles exhibit proteolytic activity against the three peptide derivatives known to be substrates of proteinase yscE. The pH optimum for activity and the inhibition spectrum of the proteolytic activities of Xenopus 20 S particles and of yeast proteinase yscE are identical. The RNA content of the cylinder particles and of proteinase yscE is below 0.1 RNA chain per molecule. Our data suggest that proteinase yscE from yeast and the 20 S cylinder particles of X. laevis are homologous, highly conserved proteins carrying the catalytic character of a peptidase.
Assuntos
Cisteína Endopeptidases , Proteínas/ultraestrutura , Saccharomyces cerevisiae/enzimologia , Animais , Reações Cruzadas , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Cinética , Microscopia Eletrônica , Inibidores de Proteases/farmacologia , Especificidade por Substrato , Xenopus laevisRESUMO
The KEX2 gene-encoded, membrane-bound Ca2+-dependent thiol endoproteinase, proteinase yscF, responsible for processing of the precursor protein of the sex pheromone alpha-factor of the yeast Saccharomyces cerevisiae was solubilized from the membraneous fraction and partially purified. Gel filtration revealed an apparent Mr of the native protein of around 150,000. Ca2+ concentration for half-maximal activity was in the micromolar range and concentration of the substrate Cbz-Tyr-Lys-Arg-4-nitroanilide for half-maximal velocity was 0.05 mM. The enzyme able to cleave basic amino acids from the carboxy-terminus of peptides and probably involved in final maturation of the alpha-factor peptides generated by proteinase yscF is membrane-associated, active at neutral pH and responds strongly to the serine proteinase inhibitor phenyl-methylsulfonyl fluoride as well as to -SH group blocking agents.
Assuntos
Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Subtilisinas , Cromatografia em Gel , Endopeptidases/isolamento & purificação , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/isolamento & purificação , Fator de Acasalamento , Proteínas de Membrana/isolamento & purificação , Inibidores de Proteases , Processamento de Proteína Pós-TraducionalRESUMO
The potential inhibitory effect of 54 Mexican Indian medicinal plants on the activation of transcription factor NF-kappaB was studied. Band-shift experiments identified the ethanolic leaf extracts of Artemisia ludoviciana ssp. mexicana, Calea zacatechichi, and Polymnia maculata (all rich in sesquiterpene lactones) as inhibitors of NF-kappaB down to a concentration of 25 microg/ml. The sesquiterpene lactones isohelenin and parthenolide prevented NF-kappaB activation completely as low as 5 microM. Treatment of HeLa cells with leaf extract of A. ludoviciana ssp. mexicana, isohelenin and parthenolide prevented the induction of transcription on the IL-6 promoter. These experiments identify the eudesmanolide and germacranolide type of sesquiterpene lactones as potent non-antioxidant inhibitors of NF-kappaB. All plants active in the NF-kappaB assay also showed a delay in the onset of capillary reactions of the allantois membrane in a physiological model for anti-inflammatory activity - the HET-CAM assay.
Assuntos
Lactonas/farmacologia , NF-kappa B/antagonistas & inibidores , Extratos Vegetais/farmacologia , Plantas Medicinais , Sesquiterpenos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Capilares/efeitos dos fármacos , Embrião de Galinha , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Fator C1 de Célula Hospedeira , Humanos , Indígenas Norte-Americanos , Interleucina-6/genética , Medicina Tradicional , México , Fator 1 de Transcrição de Octâmero , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
BACKGROUND AND AIMS: Crew resource management-based operating room team training will be an evident part of future surgical training. Hybrid simulation in the operating room enables the opportunity for trainees to perform higher fidelity training of technical and non-technical skills in a realistic context. We focus on situational motivation and self-efficacy, two important factors for optimal learning in light of a prototype course for teams of residents in surgery and anesthesiology and nurses. MATERIAL AND METHODS: Authentic operating room teams consisting of residents in anesthesia (n = 2), anesthesia nurses (n = 3), residents in surgery (n = 2), and scrub nurses (n = 6) were, during a one-day course, exposed to four different scenarios. Their situational motivation was self-assessed (ranging from 1 = does not correspond at all to 7 = corresponds exactly) immediately after training, and their self-efficacy (graded from 1 to 7) before and after training. Training was performed in a mock-up operating theater equipped with a hybrid patient simulator (SimMan 3G; Laerdal) and a laparoscopic simulator (Lap Mentor Express; Simbionix). The functionality of the systematic hybrid procedure simulation scenario was evaluated by an exit questionnaire (graded from 1 = disagree entirely to 5 = agree completely). RESULTS AND CONCLUSIONS: The trainees were mostly intrinsically motivated, engaged for their own sake, and had a rather great degree of self-determination toward the training situation. Self-efficacy among the team members improved significantly from 4 to 6 (median). Overall evaluation showed very good result with a median grading of 5. We conclude that hybrid simulation is feasible and has the possibility to train an authentic operating team in order to improve individual motivation and confidence.
Assuntos
Competência Clínica/normas , Cirurgia Geral/educação , Motivação , Salas Cirúrgicas , Equipe de Assistência ao Paciente/normas , Currículo , Humanos , Simulação de PacienteRESUMO
Noyes [Noyes, R. M. (1981) Proc. Natl. Acad. Sci. USA 78, 7248-7249] has obtained a result for the Gibbs free energy per unit mass in a continuous flow stirred tank reactor, which states that the steady-state free energy increases monotonically with decreasing residence time for any stable stationary point. We show that his derivation is based on an erroneous stability criterion. We further investigate the dependence of the Gibbs free energy on residence time for the case of mass-action kinetics and ideal mixtures. We then apply this development to Schlögl's trimolecular reaction mechanism [Schlögl, F. (1971) Z. Phys. 248, 446-458] and show that Noyes' conjecture is violated for this simple example.
RESUMO
Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD+ regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD+ regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.
Assuntos
Gluconeogênese/fisiologia , Glioxilatos/metabolismo , Metabolismo dos Lipídeos , Fenômenos Fisiológicos Vegetais , Plantas/enzimologia , Plantas/metabolismoRESUMO
Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic-multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two-dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1-1 are defective in sporulation. Strains carrying the pre1-1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1-1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates.
Assuntos
Cisteína Endopeptidases/genética , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Deleção Cromossômica , Cromossomos Fúngicos , Clonagem Molecular , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Genes Fúngicos , Teste de Complementação Genética , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50.
Assuntos
Ciclosporina/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/efeitos dos fármacos , Precursores de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição , Animais , Transporte Biológico/genética , Transporte Biológico/imunologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta Imunológica , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B , Complexo de Endopeptidases do Proteassoma , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tacrolimo/farmacologia , Fator de Transcrição RelBRESUMO
The promoters of IL-2 and IL-4 genes contain multiple binding sites for octamer factors. In peripheral T lymphocytes and several T cell lines, both the ubiquitous Oct factor Oct-1 and the lymphocyte-specific factor Oct-2 are expressed and bind to the IL-2 and IL-4 promoters. Prominent octamer binding sites of IL-2 and IL-4 promoters are their upstream promoter sites (UPS) which share 14 identical nucleotides. Multiple copies of the IL-2 and IL-4 UPS act as inducible enhancers in T cells, and their induction is inhibited by the immunosuppressant cyclosporin A (CsA). Closely linked to the octamer site, the IL-2 UPS contains a non-canonical AP-1 binding (TRE) site, and mutation in either site to a non-functional factor binding site impairs the induction of the IL-2 promoter. The binding of AP-1 and octamer factors to the IL-2 UPS DNA overlaps, and the tight association and functional cooperation of octamer with AP-1 factors is of crucial importance for the inducible IL-2 UPS activity. Introduction of five or ten spacer nucleotides between both IL-2 UPS sites results in a drastic reduction of inducible UPS activity, both in the loss of suppression by CsA and stimulation by the Ca(2+)-dependent phosphatase calcineurin. Within the IL-4 UPS the Oct and TRE-like motifs are separated by a binding site of nuclear factor of activated T cells (NF-AT). This site shares nine out of ten bp with an IL-2 NF-AT site. The strong binding of NF-ATp to the IL-4 UPS site suppresses the simultaneous binding of Oct factors to the IL-4 UPS. Because the two other Oct binding sites of IL-4 promoter show a similar sequence configuration, the binding of NF-AT seems to prevent the simultaneous binding of Oct factors to the IL-4 promoter. By contrast, both classes of factors bind simultaneously to the IL-2 promoter, and their tight association with AP-1 enhances the IL-2 promoter activity.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Interleucina-2/genética , Interleucina-4/genética , Proteínas Nucleares , Oligodesoxirribonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Fator de Transcrição AP-1/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
The transcription factor NF-ATc is synthesized in three prominent isoforms. These differ in the length of their C terminal peptides and mode of synthesis. Due to a switch from the use of a 3' polyA site to a more proximal polyA site, NF-ATc expression switches from the synthesis of the two longer isoforms in naive T cells to that of short isoform A in T effector cells. The relative low binding affinity of cleavage stimulation factor CstF-64 to the proximal polyA site seems to contribute to its neglect in naive T cells. These alternative polyadenylation events ensure the rapid accumulation of high concentrations of NF-ATc necessary to exceed critical threshold levels of NF-ATc for gene induction in effector T cells.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas Nucleares/metabolismo , Poli A/metabolismo , Linfócitos T Reguladores/metabolismo , Fatores de Transcrição/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Genes Reporter , Humanos , Células Jurkat , Luciferases/genética , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas , Fatores de Poliadenilação e Clivagem de mRNARESUMO
The transcription factor NF-ATc that controls gene expression in T lymphocytes and embryonic cardiac cells is expressed in three prominent isoforms. This is due to alternative splice/polyadenylation events that lead to the predominant synthesis of two long isoforms in naive T cells and a shorter NF-ATc isoform in effector T cells. Whereas the previously described isoform NF-ATc/A contains a relatively short C terminus, the longer isoforms, B and C, span extra C-terminal peptides of 128 and 246 aa, respectively. We show here that in addition to the strong N-terminal trans-activation domain, TAD-A, which is common to all three NF-ATc isoforms, NF-ATc/C contains a second trans-activation domain, TAD-B, in its C-terminal peptide. Various stimuli of T cells that induce the activity of TAD-A also enhance the activity of TAD-B, but, unlike TAD-A, TAD-B remains unphosphorylated by protein from 12-O-tetradecanoyl 12-phorbol 13-acetate-stimulated T cells. The shorter C-terminal peptide of isoform NF-ATc/B exerts a suppressive transcriptional effect. These properties of NF-ATc/B and -C might be of importance for gene regulation in naive T lymphocytes in which NF-ATc/B and -C are predominantly synthesized.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares , Linfócitos T/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/química , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfocinas/biossíntese , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína , Linfócitos T/química , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/química , Ativação Transcricional/imunologiaRESUMO
NF-ATc, an inducibly expressed transcription factor, controls gene expression in T lymphocytes and cardiomyocytes. We show here that the transcriptional co-activators CBP/p300 bind to and control the activity of the inducible N-terminal transactivation domain of NF-ATc, TAD-A. Similar to the N terminal transactivation domain of c-Jun, TAD-A is inducibly phosphorylated, but this phosphorylation is dispensable for the interaction with CBP/p300. Constitutive active versions of c-Raf and Rac synergistically enhance the CBP/p300-mediated increase of TAD-A activity, indicating the important role CBP/p300 plays in the integration of T cell activation signals. Since a mutation of CBP abolishing HAT activity is almost as active as wild-type CBP in T cells, functions of CBP/p300 other than histone acetylation appear to control the NF-AT-dependent transcription in T cells.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Proteínas Nucleares/fisiologia , Linfócitos T/imunologia , Transativadores/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Proteínas de Ligação a DNA/fisiologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais , Fator de Transcrição AP-1/fisiologia , Proteínas rac de Ligação ao GTPRESUMO
Homozygous type I plasminogen deficiency has been identified as a cause of ligneous conjunctivitis. In this study, 5 additional patients with ligneous conjunctivitis are examined. Three unrelated patients (1 boy, 1 elderly woman, and 1 man) had plasminogen antigen levels of less than 0.4, less than 0.4, and 2.4 mg/dL, respectively, but had plasminogen functional residual activity of 17%, 18%, and 17%, respectively. These subjects were compound-heterozygotes for different missense mutations in the plasminogen gene: Lys19 --> Glu/Arg513 --> His, Lys19 --> Glu/Arg216 --> His, and Lys19 --> Glu/Leu128 --> Pro, respectively. The other 2 patients, a 14-year-old boy and his 19-year-old sister, who both presented with a severe course of the disease, exhibited plasminogen antigen and functional activity levels below the detection limit (<0.4 mg/dL and <5%, respectively). These subjects were compound-heterozygotes for a deletion mutation (del Lys212) and a splice site mutation in intron Q (Ex17 + 1del-g) in the plasminogen gene. These findings show that certain compound-heterozygous mutations in the plasminogen gene may be associated with ligneous conjunctivitis. Our findings also suggest that the severity of clinical symptoms of ligneous conjunctivitis and its associated complications may depend on the amount of plasminogen functional residual activity.