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51.
Kurbanyan K Nguyen KL To P Rivas EV Lueras AM Kosinski C Steryo M González A Mah DA Stemp ED 《Biochemistry》2003,42(34):10269-10281
DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA. 相似文献
52.
Diana Moreira Vasco Rodrigues Maria Abengozar Luis Rivas Eduardo Rial Mireille Laforge Xiaoling Li Marc Foretz Benoit Viollet Jér?me Estaquier Anabela Cordeiro da Silva Ricardo Silvestre 《PLoS pathogens》2015,11(3)
Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis. 相似文献
53.
Cristina Elena Canteros María Julia Madariaga William Lee María Cristina Rivas Graciela Davel Ricardo Iachini 《Revista iberoamericana de micología》2010,27(1):14-19
BackgroundThree fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country.AimTo investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province.MethodsWe applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48 kDa antigen bands of Coccidioides sp.ResultsSpecific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested.ConclusionsThis is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease. 相似文献
54.
Alexandre Elbaz Xiying Wu Daniel Rivas Jeffrey M. Gimble Gustavo Duque 《Journal of cellular and molecular medicine》2010,14(4):982-991
Although increased bone marrow fat in age‐related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two‐chamber system to co‐culture normal human osteoblasts (NHOst) with differentiating pre‐adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co‐culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS‐formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte‐conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age‐related changes in bone mass and can be prevented by the inhibition of FA synthase. 相似文献
55.
56.
Interest in the use of low-copy nuclear genes for phylogenetic analyses of
plants has grown rapidly, because highly repetitive genes such as those
commonly used are limited in number. Furthermore, because low- copy genes
are subject to different evolutionary processes than are plastid genes or
highly repetitive nuclear markers, they provide a valuable source of
independent phylogenetic evidence. The gene for granule-bound starch
synthase (GBSSI or waxy) exists in a single copy in nearly all plants
examined so far. Our study of GBSSI had three parts: (1) Amino acid
sequences were compared across a broad taxonomic range, including grasses,
four dicotyledons, and the microbial homologs of GBSSI. Inferred structural
information was used to aid in the alignment of these very divergent
sequences. The informed alignments highlight amino acids that are conserved
across all sequences, and demonstrate that structural motifs can be highly
conserved in spite of marked divergence in amino acid sequence. (2)
Maximum-likelihood (ML) analyses were used to examine exon sequence
evolution throughout grasses. Differences in probabilities among
substitution types and marked among-site rate variation contributed to the
observed pattern of variation. Of the parameters examined in our set of
likelihood models, the inclusion of among-site rate variation following a
gamma distribution caused the greatest improvement in likelihood score. (3)
We performed cladistic parsimony analyses of GBSSI sequences throughout
grasses, within tribes, and within genera to examine the phylogenetic
utility of the gene. Introns provide useful information among very closely
related species, but quickly become difficult to align among more divergent
taxa. Exons are variable enough to provide extensive resolution within the
family, but with low bootstrap support. The combined results of amino acid
sequence comparisons, maximum-likelihood analyses, and phylogenetic studies
underscore factors that might affect phylogenetic reconstruction. In this
case, accommodation of the variable rate of evolution among sites might be
the first step in maximizing the phylogenetic utility of GBSSI.
相似文献
57.
Maritza Martínez Gladys Len De Pinto Sofía Alvrez Nola Gonzlez De Troconis Edgar Ocando Carlos Rivas 《Biochemical Systematics and Ecology》1995,23(7-8):843-848
An analytical study has been made of six gum specimens from Albizia lebbeck, Leguminosae. Galactose, mannose, arabinose, glucuronic acid and its 4-O--metyl analogue are present in all the specimens studied. Rhamnose was not detected according to sugar analysis and spectral data. The absence of this sugar, the high acidity and the relatively low limit viscosity number contrast with the values reported previously for one African sample of A. lebbeck. The lead content is much higher than that recorded for other Albizia gums. 13C-NMR spectrum of this gum, in deuterium oxide, shows good resolution. 相似文献
58.
Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation. 下载免费PDF全文
J J Calvete G Rivas M Maruri M V Alvarez J L McGregor C L Hew J Gonzalez-Rodriguez 《The Biochemical journal》1988,250(3):697-704
Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated. 相似文献
59.
Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3′ UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches. 相似文献
60.