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991.
Caspase cleavage of the nuclear autoantigen LEDGF/p75 abrogates its pro-survival function: implications for autoimmunity in atopic disorders 总被引:3,自引:0,他引:3
Lens epithelium-derived growth factor p75 (LEDGF/p75) is a nuclear autoantigen in atopic disorders implicated in cellular protection against stress-induced apoptosis. We observed that LEDGF/p75 was cleaved during apoptosis into fragments of 65 and 58 kD generated by caspases-3 and -7 cleaving at three sites: DEVPD30/G, DAQD486/G and WEID85/N. Sequence analysis revealed that the DEVPD30/G and WEID85/N sites lie within the highly conserved HATH (homologous to amino terminus of hepatoma-derived growth factor) region, also known as PWWP domain. Alignment of proteins containing this domain failed to reveal conservation of the DEVPD30/G and WEID85/N sites, suggesting that the HATH/PWWP domain of LEDGF/p75 may be specifically targeted by caspases. Overexpression of LEDGF/p75 protected HepG2 cells from serum starvation-induced cell death, whereas expression of the 65 kD fragment failed to protect. The apoptotic cleavage of LEDGF/p75 may contribute to the pathogenesis of atopic disorders by abrogating its pro-survival function and enhancing its immunogenicity. 相似文献
992.
993.
alphaPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms 总被引:10,自引:0,他引:10
Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms. 相似文献
994.
In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented. 相似文献
995.
Daniels CB Wood PG Loptako OV Codd JR Johnston SD Orgeig S 《Physiological and biochemical zoology : PBZ》1999,72(6):691-698
Surfactant occurs in cyclically inflating and deflating, gas-holding structures of vertebrates to reduce the surface tension of the inner fluid lining, thereby preventing collapse and decreasing the work of inflation. Here we determined the presence of surfactant in material lavaged from the airspace in the gas mantle of the pulmonate snail Helix aspersa. Surfactant is characterized by the presence of disaturated phospholipid (DSP), especially disaturated phosphatidylcholine (PC), lavaged from the airspace, by the presence of lamellated osmiophilic bodies (LBs) in the airspaces and epithelial tissue, and by the ability of the lavage to reduce surface tension of fluid in a surface balance. Lavage had a DSP/phospholipid (PL) ratio of 0.085, compared to 0.011 in membranes, with the major PL being PC (45.3%). Cholesterol, the primary fluidizer for pulmonary surfactant, was similar in lavage and in lipids extracted from cell homogenates (cholesterol/PL: 0.04 and 0. 03, respectively). LBs were found in the tissues and airspaces. The surface activity of the lavage material is defined as the ability to reduce surface tension under compression to values much lower than that of water. In addition, surface-active lipids will vary surface tension, increasing it upon inspiration as the surface area expands. By these criteria, the surface activity of lavaged material was poor and most similar to that shown by pulmonary lavage of fish and toads. Snail surfactant displays structures, a biochemical PL profile, and biophysical properties similar to surfactant obtained from primitive fish, teleost swim bladders, the lung of the Dipnoan Neoceratodus forsteri, and the amphibian Bufo marinus. However, the cholesterol/PL and cholesterol/DSP ratios are more similar to the amphibian B. marinus than to the fish, and this similarity may indicate a crucial physicochemical relationship for these lipids. 相似文献
996.
Production of coenzyme F420 and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F420-5,6. M. smegmatis produced 1 to 3 μmol of intracellular F420 per liter of culture, which was more than the 0.85 to 1.0 μmol of F420-2 per liter usually obtained with Methanobacterium thermoautotrophicum and ~10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F420 than with previous methods. 相似文献
997.
Malcolm Daniels Lucas P Hart P Shing Ho Jean-Pierre Ballini Paul Vigny Jean-Claude Brochon 《Photochemical & photobiological sciences》2007,6(8):883-893
A study has been made of the fluorescence of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA, in buffered neutral aqueous solution at room temperature, excited by synchrotron radiation at 280 nm and 250 nm and by a frequency-doubled pulse dye laser at 290 nm. Exciting at 280 nm, the B form shows a uni-modal UV spectrum with lambdaf(max) approximately 340 nm. The Z form has in addition a visible emission lambdaf(max) at 450 nm. The spectral positions remain unchanged on exciting at 250 nm but the relative intensities change considerably. Decay profiles have been obtained at 360 nm and 450 nm for both the B and Z forms and have been analyzed by fitting to a pseudo-continuous distribution of 100 (and occasionally 200) exponentials, ranging from 10 ps to 20 ns, by optimizing the 'entropy' of the signal (the method of maximum entropy). We find the mean lifetimes for both wavelengths of emission and for both structural forms fall into three well-separated regions in the ranges indicated tau1 approximately 0.04-0.21 ns, tau2 approximately 0.9-1.26 ns, and tau3 approximately 5.1-6.5 ns. The UV emission, from its spectral position and half-width, correlates with monomeric emission from m5C (and from C for poly d(G-C)). However the lifetime tau1 is approximately 2 orders of magnitude longer than the monomers and points to an involvement of protonated guanosine (GH+, tauf approximately 200 ps) in the overall absorption/emission sequence. In the UV the tau3 emission is predominant, with fractional time-integrated emission approximately 86% for B DNA and approximately 64% for Z. We suggest it results from exciton (stacked) absorption followed by dissociative emission. For Z DNA the visible (450 nm) emission is dominated by a tau3 species (approximately 91%) with a lifetime of 6.5 ns and we suggest it represents a hetero-excimer emission consequent upon absorption by the strongly overlapped base-stacking, which differs from that in B DNA. The weak emission corresponding to tau2 is made more apparent by scanned gated detection of the emission from laser excitation (290 nm) of single-crystal d(m5C-G)3. A central role is attributed to the tight stacking of the bases in the Z form which correlates with enhanced hypochromism at 250 nm vs. 280 nm and with the reversal of the fluorescence intensity ratios UV-visible between these wavelengths. 相似文献
998.
Evolution of alcohol dehydrogenase genes in peonies (Paeonia): phylogenetic relationships of putative nonhybrid species 总被引:11,自引:0,他引:11
Alcohol dehydrogenase genes were amplified by PCR, cloned, and sequenced
from 11 putative nonhybrid species of the angiosperm genus Paeonia.
Sequences of five exons and six intron regions of the Adh gene were used to
reconstruct the phylogeny of these species. Two paralogous genes, Adh1A,
and Adh2, were found; an additional gene, Adh1B, is also present in section
Moutan. Phylogenetic analyses of exon sequences of the Adh genes of Paeonia
and a variety of other angiosperms imply that duplication of Adh1 and Adh2
occurred prior to the divergence of Paeonia species and was followed by a
duplication resulting in Adh1A and Adh1B. Concerted evolution appears to be
absent between these paralogous loci. Phylogenetic analysis of only the
Paeonia Adh exon sequences, positioning the root of the tree between the
paralogous genes Adh1 and Adh2, suggests that the first evolutionary split
within the genus occurred between the shrubby section Moutan and the other
two herbaceous sections Oneapia and Paeonia. Restriction of Adh1B genes to
section Moutan may have resulted from deletion of Adh1B from the common
ancestor of sections Oneapia and Paeonia. A relative-rate test was designed
to compare rates of molecular change among lineages based on the divergence
of paralogous genes, and the results indicate a slower rate of evolution
within the shrubby section Moutan than in section Oneapia. This may be
responsible for the relatively long branch length of section Oneapia and
the short branch length between section Moutan and the other two sections
found on the Adh, ITS (nrDNA), and matK (cpDNA) phylogenies of the genus.
Adh1 and Adh2 intron sequences cannot be aligned, and we therefore carried
out separate analyses of Adh1A and Adh2 genes using exon and intron
sequences together. The Templeton test suggested that there is not
significant incongruence among Adh1A, ITS, and matK data sets, but that
these three data sets conflict significantly with Adh2 sequence data. A
combined analysis of Adh1A, ITS, and matK sequences produced a tree that is
better resolved than that of any individual gene, and congruent with
morphology and the results of artificial hybridization. It is therefore
considered to be the current best estimate of the species phylogeny.
Paraphyly of section Paeonia in the Adh2 gene tree may be caused by longer
coalescence times and random sorting of ancestral alleles.
相似文献
999.
A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule 总被引:10,自引:5,他引:5
C. E. Barber J. L. Tang J. X. Feng M. Q. Pan T. J. G. Wilson H. Slater J. M. Dow P. Williams & M. J. Daniels 《Molecular microbiology》1997,24(3):555-566
Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris ( Xcc ). The phenotype of mutants in one of the genes, rpfF , can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipopolysaccharide and lipid A from Xcc , and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes. 相似文献
1000.
Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells 总被引:20,自引:1,他引:19 下载免费PDF全文
Suranganie Dharmawardhane Luraynne C. Sanders Stuart S. Martin R. Hugh Daniels Gary M. Bokoch 《The Journal of cell biology》1997,138(6):1265-1278
Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes. 相似文献