RESUMEN
The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.
Asunto(s)
ADN Ribosómico/efectos de la radiación , ADN Polimerasa Dirigida por ADN , Nucleosomas/efectos de la radiación , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/efectos de la radiación , Rayos Ultravioleta , Animales , Cromatina/efectos de la radiación , ADN Ribosómico/genética , Relación Dosis-Respuesta en la Radiación , Histonas/metabolismo , Histonas/efectos de la radiación , Radical Hidroxilo/análisis , Nucleosomas/genética , Dímeros de Pirimidina , Proteínas Virales/metabolismo , XenopusRESUMEN
We examined repair of DNA strand breaks induced by the anti-cancer drug bleomycin in both Pol I and Pol II transcribed genes in permeabilized human fibroblasts. The majority of these breaks (>80%) are single strand breaks (SSBs) thought to be repaired by base excision repair enzymes. Repair was examined in each strand of a 7. 2-kilobase fragment, completely within the Pol I transcribed region of ribosomal DNA (rDNA) and an 8.3-kilobase fragment completely within the Pol II transcribed region of the dihydrofolate reductase (DHFR) gene. Bleomycin dose-response studies revealed no bias for SSBs in either strand of the rDNA fragment. Furthermore, repair of SSBs is rapid (approximately 80% resealed in 60 min) in both the transcribed and nontranscribed strands of rDNA. Rapid repair of SSBs is also observed in both strands of the DHFR gene (approximately 60% resealed in 60 min). In contrast, little (or no) repair of UV photodimers occurs in either strand of human rDNA, regardless of whether cells are confluent or actively growing. Thus, DNA lesions in human ribosomal genes may be more accessible to base excision repair enzymes than those involved in nucleotide excision repair.
Asunto(s)
Reparación del ADN , ADN Ribosómico/genética , Bleomicina/administración & dosificación , Bleomicina/farmacología , Células Cultivadas , Daño del ADN , ADN Ribosómico/efectos de la radiación , ADN de Cadena Simple/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Dímeros de Pirimidina , Tetrahidrofolato Deshidrogenasa/genética , Rayos UltravioletaRESUMEN
Using radioimmunoassays, we examined rates of removal of UV-induced pyrimidine-pyrimidone (6-4) photoproducts ((6-4)PDs) and cyclobutane pyrimidine dimers (CPDs) from 146-base pair nucleosome core DNA (and 166-base pair chromatosome DNA) of confluent human diploid fibroblasts. Dose-response experiments indicate that the yield of (6-4)PDs in core DNA is about 30% that of CPDs in the UV dose range of 0-200 J/m2. Repair experiments indicate that, at 40 J/m2, (6-4)PDs are removed much faster (approximately 75% in 2 h) from nucleosome core (and chromatosome) DNA than CPDs (10-15% in 2 h). A slow rate of removal of CPDs is also observed when the UV dose is reduced to 10 J/m2 (i.e. even when the level of CPDs is less than that of (6-4)PDs at 40 J/m2). These results indicate that (a) the accessibility of repair proteins to (6-4)PDs in nucleosomes is markedly different than their accessibility to CPDs and/or (b) repair enzymes are much more efficient at incising and removing (6-4)PDs than CPDs in human chromatin.
Asunto(s)
Reparación del ADN , ADN/efectos de la radiación , Nucleosomas/metabolismo , Dímeros de Pirimidina/metabolismo , Células Cultivadas , Humanos , Rayos UltravioletaRESUMEN
The major photoproduct formed in DNA, following absorption of ultraviolet (UV) light, is the cis-syn cyclobutane pyrimidine dimer (CPD). Formation of CPDs in DNA packaged into chromatin prior to UV irradiation results in a striking preference for these photoproducts to be oriented away from the histone surface in nucleosomes (Gale, J. M., Nissen, K. A., and Smerdon, M. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6644-6648). In this report, we show that formation of nucleosomes onto UV-irradiated DNA results in a similar distribution of these photoproducts, indicating that preexisting CPDs in the DNA molecule can influence its rotational setting on the histone surface during nucleosome formation. This bias is less pronounced in the central three helical turns encompassing the dyad axis of nucleosomes, where the helix is overwound (10.7 bases/turn) relative to the "outer" portion of core DNA (10.0 bases/turn). Such a change in the rotational setting of DNA on the surface of newly formed nucleosomes could expose normally inaccessible DNA sequences to factors which control DNA processing.
Asunto(s)
Daño del ADN , ADN/química , ADN/efectos de la radiación , Histonas/química , Nucleosomas/química , Rayos Ultravioleta , Animales , Bovinos , Pollos , Conformación de Ácido Nucleico , Dímeros de Pirimidina/químicaRESUMEN
Nucleosome core particles undergo a conformational change at ionic strengths below 0.2 mM; the fluorescence anisotropy decay of bound ethidium indicates that under these conditions the particle adopts a highly extended structure. We have measured the distribution of UV-induced DNA damage (primarily cyclobutane-pyrimidine dimers) through a process termed photofootprinting. As the core particle is exposed to ionic strengths below 0.2 mM, the photofootprint pattern changes from that observed for native cores, with a characteristic 10.3 base repeat pattern presumably derived largely from the bending of DNA around the histone octamer, to a more evenly distributed pattern resembling that of free DNA. These results provide clear evidence that the DNA in the core particle at these very low ionic strengths, although still tightly bound to histones, is no longer bent to a significant degree.
Asunto(s)
Daño del ADN , ADN/efectos de la radiación , Nucleosomas/ultraestructura , Rayos Ultravioleta , Animales , Pollos , ADN/ultraestructura , Eritrocitos/ultraestructura , Modelos Estructurales , Conformación de Ácido Nucleico/efectos de la radiación , Nucleosomas/efectos de la radiación , Concentración OsmolarRESUMEN
This review focuses on parasite enzymes which are involved in the detoxification of oxygen radicals, and covers the following enzymes: superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase. These enzymes are crucial for parasites to evade oxygen-mediated attack by host leukocytes, both intracellularly and extracellularly. In addition, the newly defined parasite system involving trypanothione, trypanothione peroxidase and trypanothione reductase is discussed.
Asunto(s)
Oxígeno/metabolismo , Parásitos/enzimología , Animales , Catalasa/metabolismo , Radicales Libres , Glutatión/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
This review covers some of the basic mechanisms whereby parasites evade host responses. These mechanisms include; antigenic variation, repeated antigenic determinants, induction of suppressor cells, acquisition of host proteins or molecular mimicry, proteinase destruction of host effector molecules, proteinase inhibitor-mediated inhibition of humoral and cellular immune effector arms and immunosuppressive products of parasite arachidonic acid metabolism. Vet. Parasitol.
Asunto(s)
Interacciones Huésped-Parásitos , Enfermedades Parasitarias en Animales , Animales , Gatos , Enfermedades Parasitarias/inmunología , Enfermedades Parasitarias/parasitología , Enfermedades Parasitarias/fisiopatología , Ratas , Teniasis/inmunología , Teniasis/parasitología , Teniasis/fisiopatología , Teniasis/veterinariaRESUMEN
Taeniaestatin, a recently isolated Taenia taeniaeformis proteinase inhibitor, was used to inhibit equine neutrophil migration. Taeniaestatin itself was not chemotactic when used as a chemotactic factor but taeniaestatin did inhibit neutrophil chemokinesis when tested in a Zigmond-Hirsch checkerboard assay. A dose-dependent inhibition of both chemokinesis and chemotaxis was observed when zymosan activated bovine sera (ZABS) was used as the chemotactic factor. This inhibition was greater than 95% when 5 mu of taeniaestatin was present on both the cell and chemotactic factor side of the chambers. Equine neutrophils gave dose- and time-dependent migration responses to purified bovine C5a with an ED50 of 1.04 X 10(-7)M. Taeniaestatin inhibited the C5a-mediated chemotactic and chemokinetic neutrophil responses (51% using 1 mu and greater than 95% with 5 mu of taeniaestatin). The inhibition of leucocyte motility by taeniaestatin was reversible and without cytotoxicity at the highest doses of taeniaestatin tested.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Proteínas del Helminto , Hormonas de Invertebrados/farmacología , Neutrófilos/efectos de los fármacos , Animales , Factores Quimiotácticos/farmacología , Relación Dosis-Respuesta a Droga , Caballos , L-Lactato Deshidrogenasa/metabolismo , Neutrófilos/metabolismo , Taenia/fisiologíaRESUMEN
A proteinase inhibitor, taeniaestatin, isolated from the larval stage of the cestode Taenia taeniaeformis inhibits endogenous IL 2 generation in murine lymphocytes and IL 1 induced proliferation of murine thymocytes in a dose-dependent manner. However, taeniaestatin does not inhibit exogenous IL 2-induced proliferation of an IL 2-dependent cell line at any dose tested. These data indicate that the lack of IL 2 generation may be due in part to inhibition of a crucial cell-associated proteinase subsequent to cellular activation, or the lack of an effective IL 2 signal for differentiation. Our results are novel findings concerning molecular pathways for parasite inhibition of host immune responses, and suggest that selected proteinase inhibitors may be useful in clinical situations in which IL 1 or IL 2 are elevated.
Asunto(s)
Proteínas del Helminto , Interleucina-1/antagonistas & inhibidores , Interleucina-2/antagonistas & inhibidores , Hormonas de Invertebrados/farmacología , Activación de Linfocitos , Linfocitos/inmunología , Inhibidores de Proteasas/farmacología , Animales , Interleucina-1/inmunología , Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Endogámicas Lew , Bazo/inmunología , Taenia , Timo/inmunologíaRESUMEN
Superoxide dismutase was purified from Taenia taeniaeformis metacestodes by sequential ion exchange chromatography on quaternary-amino-ethyl-cellulose, gel filtration chromatography on ACA 44 and ion exchange chromatography on DEAE-cellulose, followed by chromatofocusing on polybuffer exchanger 94. This isolation procedure resulted in the detection of a single protein-staining band on alkaline gels, coincident with enzyme activity. We have, however, detected what appear to be two peaks of enzyme activity within this single protein-staining band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using gradient slab gels and analysis under reducing conditions, resulted in the detection of only one protein at an apparent Mr of 16,600, while analysis under non-reducing conditions, gave a single protein of an apparent Mr of 64,000. The isoelectric point of the purified protein is 6.6. Boiling for 3 min completely destroyed the enzyme, whereas incubation for 2 h at 37 degrees C resulted in the loss of 56% of the enzymic activity. Incubation with 10 mM KCN resulted in 83% inhibition of the enzyme. We have detected up to 168 U ml-1 of enzyme activity in the cyst fluid surrounding the parasite in situ. This is the first instance in which any parasite superoxide dismutase has been purified to apparent homogeneity. Parasite-mediated enzymic destruction of superoxide anion can not only protect against oxygen toxicity as a result of normal parasite respiratory processes but also may serve as yet another mechanism used by tissue-dwelling parasites to evade host immunologic attack.
Asunto(s)
Superóxido Dismutasa/metabolismo , Taenia/enzimología , Animales , Cromatografía , Electroforesis , Calor , Concentración de Iones de Hidrógeno , Peso Molecular , Cianuro de Potasio/farmacología , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/aislamiento & purificación , Taenia/crecimiento & desarrolloRESUMEN
Rat splenic lymphocytes, cultured in vitro for 3 days in the presence of a larval cestode proteinase inhibitor, exhibited a marked suppression of proliferation when stimulated with Con A, PHA, PWM and ovalbumin. Reduced responsiveness was observed over a full range of concentrations of Con A (16-fold), PHA (50-fold), PWM (four-fold) and ovalbumin (16-fold). These results indicated that the inhibitory action could not be overcome by increasing the mitogen or antigen doses beyond optimal levels. This suppressive effect disappeared when the Taenia taeniaeformis proteinase inhibitor was added 20 h after the initiation of culture, suggesting that the inhibitor affects lymphocyte blastogenesis during the early stages of lymphocyte activation.
Asunto(s)
Activación de Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Inhibidores de Proteasas/farmacología , Bazo/inmunología , Taenia/enzimología , Animales , Antígenos/inmunología , División Celular , Células Cultivadas , Cinética , Lectinas/farmacología , Ovalbúmina/inmunología , Ratas , Factores de TiempoRESUMEN
Platelet-activating factor (PAF), a lipid released as a result of immediate allergic reactions from basophils and mast cells as well as by a variety of other cell types and stimuli, is one of the most potent platelet agonists and hypotensive agents known. Equine platelets stimulated over a wide range of PAF concentrations aggregated in a time- and dose-dependent manner. Maximum aggregation was observed at concentrations of PAF as low as 3.58 x 10(-14) M with platelet-rich plasma (PRP) and 3.58 x 10(-16) M with washed platelets. Furthermore, the aggregation observed did not appear to be breed-dependent. Finally, the platelet arachidonate pathway appeared to play no role in PAF-induced aggregation as exogenous arachidonate did not enhance the reaction, nor were equine platelets pretreated with 38 microM aspirin inhibited in their response to PAF. This level of aspirin totally inhibited the equine platelet aggregation response to arachidonate.