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991.
Tarugi P Ballarini G Bembi B Battisti C Palmeri S Panzani F Di Leo E Martini C Federico A Calandra S 《Journal of lipid research》2002,43(11):1908-1919
We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene. 相似文献
992.
Bagorda A Guerra L Di Sole F Helmle-Kolb C Favia M Jacobson KA Casavola V Reshkin SJ 《The Journal of membrane biology》2002,188(3):249-259
As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y(1) agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y(1) antagonists, MRS 2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-gamma-S but not by UTP. 2-MeSADP induced a P2Y(1) antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity. 相似文献
993.
Identification of 8-iso-prostaglandin F(2alpha) in rat brain neuronal endings: a possible marker of membrane phospholipid peroxidation 总被引:1,自引:0,他引:1
Brunetti L Orlando G Michelotto B Recinella L Di Nisio C Ciabattoni G Vacca M 《Life sciences》2002,71(20):2447-2455
Isoprostanes are a family of prostaglandin (PG) F and E isomers generated by free-radical attack from membrane bound arachidonic acid. We measured detectable levels of 8-iso-PGF(2alpha) in the perfusates of synaptosomes obtained from different areas of the rat brain cortex. A small but significant release of this isoprostane was found under basal conditions from all the areas explored; being lower in the dorsal cortex in respect to the frontal, parietal and occipital areas. Exposure of synaptosomes to a phospholipase A(2) activator, i.e. calcium-ionophore A23187, an oxidant agent, such as hydrogen peroxide or amyloid beta-peptide did not modify 8-iso-PGF(2alpha) release when these stimuli were applied separately. However, either hydrogen peroxide or amyloid beta-peptide increased 8-iso-PGF(2alpha) release in a dose-dependent manner, when given in the presence of the calcium-ionophore A23187. Synaptosome treatment with a non-selective cyclooxygenase inhibitor (fenoprofen) did not modify 8-iso-PGF(2alpha) release in any way, but treatment with a water soluble antioxidant (Trolox C) completely suppressed isoprostane release under basal conditions, as well as after the oxidant injury induced either by hydrogen peroxide or amyloid beta-peptide. We conclude that, in neuronal endings, 8-iso-PGF(2alpha) is generated under basal conditions and its formation may be increased in a dose-dependent fashion by oxidant stimuli through a cyclooxygenase-independent mechanism involving free radical-catalyzed oxidation of arachidonic acid on membrane phospholipids. 相似文献
994.
The Eps15 homology (EH) domain was originally identified as a motif present in three copies at the NH2-termini of Eps15 and of the related molecule Eps15R. Both of these molecules are substrates for the tyrosine kinase activity of the epidermal growth factor receptor and hence the name 'Eps15 homology' or EH domain [Wong et al. (1994) Oncogene 9, 1591-1597; Wong et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9530-9534; Fazioli et al. (1993) Mol. Cell. Biol. 13, 5814-5828] was derived. The motif was subsequently found in several proteins from yeast to nematode, thus establishing its evolutionary conservation. Initial studies with filter-binding assays and phage-displayed libraries demonstrated its protein:protein interaction abilities and identified specific ligands. Subsequently, structural analyses established the molecular bases of recognition between EH domains and cognate peptides. To date, several EH-containing and EH-binding proteins have been identified, which establish in the cell a network of protein:protein interactions, defined as the EH network. This network coordinates cellular functions connected with endocytosis, actin remodeling and intracellular transduction of signals. 相似文献
995.
Until the present moment, only a scarce number of Latin American domestic cat populations have been studied from a population genetic standpoint. For this reason, the cat populations of La Havana (Cuba), San José (Costa Rica), Bogota and Ibagué (Colombia), Asuncion (Paraguay), Santiago (Chile) and Buenos Aires (Argentina) were sampled for several coat genes. The results obtained were as follows: (1) there was a strong genetic resemblance between several Hispanic American cat populations (especially, those of Buenos Aires, San José and the two Colombian populations studied) and those from South Western United States (California, Texas and Colorado), which adds support to the suspicion that these populations probably have a common origin; (2) The cat population of Santiago (Chile), contrarily to the other Hispanic American populations studied, showed a strong genetic resemblance with some Anglo North American populations; and (3) The l (long hair) and d (dilution) alleles showed systematic higher frequencies in the Hispanic American populations than those observed in Spain. Although the Hispanic American populations were not identical to the current Spanish populations (with the exception of Asuncion), this historic genetic experiment was very different to that found for the British populations and their overseas colonies. 相似文献
996.
Hagen T Di Daniel E Culbert AA Reith AD 《The Journal of biological chemistry》2002,277(26):23330-23335
Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45). 相似文献
997.
Adriamycin-induced senescence in breast tumor cells involves functional p53 and telomere dysfunction 总被引:5,自引:0,他引:5
Elmore LW Rehder CW Di X McChesney PA Jackson-Cook CK Gewirtz DA Holt SE 《The Journal of biological chemistry》2002,277(38):35509-35515
Direct experimental evidence implicates telomere erosion as a primary cause of cellular senescence. Using a well characterized model system for breast cancer, we define here the molecular and cellular consequences of adriamycin treatment in breast tumor cells. Cells acutely exposed to adriamycin exhibited an increase in p53 activity, a decline in telomerase activity, and a dramatic increase in beta-galactosidase, a marker of senescence. Inactivation of wild-type p53 resulted in a transition of the cellular response to adriamycin treatment from replicative senescence to delayed apoptosis, demonstrating that p53 plays an integral role in the fate of breast tumor cells treated with DNA-damaging agents. Stable introduction of hTERT, the catalytic protein component of telomerase, into MCF-7 cells caused an increase in telomerase activity and telomere length. Treatment of MCF-7-hTERT cells with adriamycin produced an identical senescence response as controls without signs of telomere shortening, indicating that the senescence after treatment is telomere length-independent. However, we found that exposure to adriamycin resulted in an overrepresentation of cytogenetic changes involving telomeres, showing an altered telomere state induced by adriamycin is probably a causal factor leading to the senescence phenotype. To our knowledge, these data are the first to demonstrate that the mechanism of adriamycin-induced senescence is dependent on both functional p53 and telomere dysfunction rather than overall shortening. 相似文献
998.
Shainoff JR Smejkal GB DiBello PM Sung SS Bush LA Di Cera E 《The Journal of biological chemistry》2002,277(22):19367-19373
Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form. 相似文献
999.
A method is introduced to identify amino acid residues that dictate the functional diversity acquired during evolution in a protein family. Using over 80 enzymes of the chymotrypsin family, we demonstrate that the general organization of the phylogenetic tree and its functional branch points are fully accounted for by a limited number of residues that cluster around the active site of the protein and define the contact region with the P1-P4 residues of substrate. 相似文献
1000.