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Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the cell surface IGF-I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF-binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 A crystal structure of the complex of IGF-I bound to the N-terminal IGF-binding domain of IGFBP-5 (mini-IGFBP-5), a prototype interaction for all N-terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF-I and the IGFBP-5 fragment and a surrounding network of polar interactions. A solvent-exposed hydrophobic patch is located on the IGF-I pole opposite to the mini-IGFBP-5 binding region and marks the IGF-I receptor binding site.

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Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.

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p19INK4d is a 165 amino acid protein that belongs to the INK4 family of CDK4 and CDK6 inhibitors. Assignments of 1H, 15N and 13C resonances have enabled the determination of the secondary structure of the protein which is largely alpha-helical (residues 14-18, 21-29, 54-62, 77-83, 87-95, 110-116, 120-128, 142-148 and 152-160). The protein comprises five 32-amino acid ankyrin-like repeats; each ankyrin repeat contains a helix-beta-turn-helix core. The exception is the second ankyrin repeat, which lacks the first helix. All beta-turns have a central glycine residue flanked by two residues in beta-conformations. There is also a high conservation of Ala at position 8 in the first helix and Leu-Leu(Val) at positions 17-18 of the second helix in all ankyrin repeats of p19. The location of the helix-turn-helix segments found in p19 should be general for all other members of the INK4 family, including, for example, a homologous tumor suppressor p16INK4a. 1H-15N heteronuclear steady-state NOE measurements on p19 indicate that most of the backbone of p19INK4d exists in a well defined structure of limited conformational flexibility on the nano- to picosecond time scale.

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We examined t-butylhydroquinone (t-BHQ) and t-butylquinone (t-BuQ), two of the major microsomal metabolites of the synthetic antioxidant butylated hydroxyanisole (BHA), for their ability to react with the xenobiotic arylamines aniline and N-methylaniline. A number of substances were isolated by thin-layer chromatography. The main products were quantitatively evaluated and their structures assigned. BHA and t-BHQ yielded reaction products with anilines only in the presence of an oxidant such as iodate (KIO3). We used the Salmonella/microsome mutagenicity assay to test the new compounds for mutagenic activity. The reaction products gave no evidence of mutagenicity in the S. typhimurium strains TA98 and TA100, with or without metabolic activation. In some instances the substituted quinone products are less toxic than t-BuQ alone.

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The reaction mixture and several products arising from the reaction of butylated hydroxyanisole (BHA) and nitrite in anaerobic aqueous acidic solution were separated and tested in the Salmonella mutagenicity test. Among the nine products separable by thin-layer chromatography, 1-hydroxyl-2-tert-butyl-4-methoxy-6-nitrobenzene (BHA-NO2), tert-butyl-substituted para-quinone (t-BuQ) and 3-tert-butyl-5-methoxy-1,2-benzoquinone (t-Bu-o-Q) are dominant. The last compound has not been previously reported in this system. Spot testing indicated at least one further compound of nitroso character and traces of tert-butylhydroquinone (t-BuHQ), which reacts with nitrite to yield t-BuQ. No evidence was found for the formation of the BHA dimer under our conditions. The substances gave no evidence of mutagenicity in the Salmonella typhimurium strains TA 98 and TA 100, either in the standard plate incorporation assay or in the procedure with preincubation with or without S9 mix. In some instances the substances were unstable in the test procedure.

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The influence of added carbohydrate on the decay kinetics of the ascorbic acid free radical generated in deaerated aqueous solutions by the action of potassium ferricyanide on ascorbic acid has been studied. Addition of carbohydrate (less than or equal to 0.5M) generally leads to a small decrease in radical decay rate. In our system this probably arises from reduced reactant diffusion in solutions of increased viscosity rather than the preferential chelation previously suggested to explain inhibition in the copper (II) catalyzed oxidation. The ascorbic acid free radical shows negligible tendency to react with glucose, sorbitol or maltose. The decrease in its decay rate observed on addition of carbohydrate is probably caused by the increase in solvent viscosity.

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42 to 75 pCi cesium-137 and 702 to 903 pCi strontium-90 per kg were found in samples of tobacco of German origin and from commercial cigarette. The presence of these radionuclides in tobacco is attributed to the fallout from nuclear explosions. Taking into consideration mean transfer rates of the radionuclides in the main stream smoke condensate and average consumption of cigarettes, whole body radiation doses for inhalation have been calculated to be in the range 10(-3) to 10(-4) mrem/year and per person. This means, they are in the same order of magnitude as radiation doses by inhalation of ambient air. They are smaller by a factor of 10(-4) than the radiation exposure due to ingestion of these radionuclides from the total diet.

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The rate of free radical formation in aqueous solutions of ascorbic acid in the presence of chelating agents was measured by the ESR-stopped-flow method. Addition of ethylenediamine tetraacetate and trimetaphosphoric acid to the ascorbic acid-oxygen system results in only a minimal change in the rate of ascorbate free radical formation compared to that in absence of inhibitors. Evidence suggests that radical formation arises via secondary reactions involving products.

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It has been proposed that the mutagenic activity associated with the ascorbic acid autoxidation system may involve hydrogen peroxide and peroxide radicals. We report here that the system may involve hydrogen peroxide and peroxide radicals. We report there that the mutagenic effect may also partially reside in as yet unknown secondary products. The observed mutagenicity of 2,3-diketogulonic acid, one of the main oxidation products, reflects its ability to form hydrogen peroxide.

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The participation of semiquinone free radicals during the reaction of ascorbic acid with acidified sodium nitrite has been demonstrated by ESR spectroscopy unambiguously for the first time. Scavenging of the nitrosating agent, reflected by the observed free radical concentration, unexpectedly occurs with scarcely varying efficiency over the pH range 0.1--4.5.

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Apples of the two varieties, Boskoop and Brettacher, were exposed to 35S labeled sulphur dioxide. After storage at several conditions the distribution of 35S-activity among the three fractions, sulphite, sulphate and sulphonate was examined. The major portion of the radioactivity was found in the sulphate fraction. The activity decreased remarkably from the peel towards the core. Appreciable differences in distribution patterns were found between the two apples varieties.

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The relative rate constants for the decay of ascorbate free radical in aqueous solutions in the presence of heavy metal ions, hydrogen peroxide and sulphite were measured the ESR-spectroscopy. The oxidation of ascorbic acid showed a strong pH dependence, reaching a maximum rate at pH 9.6. It was shown that the ascorbic acid radical decay generally follows overall second order kinetics, being however first order in the presence of hydrogen peroxide. The protective effect of sulphur dioxide is proposed to have nutritional implications.

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