Lycopene in association with alpha-tocopherol or tomato lipophilic extracts enhances acyl-platelet-activating factor biosynthesis in endothelial cells during oxidative stress.

Lipophilic compounds contained in tomato can prevent cardiovascular diseases by modulating the atherogenic processes in vascular endothelium mediated by oxidized low-density lipoproteins (LDLs). We investigated the effects of lycopene on the metabolism of platelet-activating factor (PAF) and its much less biologically active acyl analog, acyl-PAF, known to prevent LDL oxidation. Lycopene, or lycopene in association with alpha-tocopherol, or whole tomato lipophilic extracts (containing more than 80% lycopene) were used in experiments in which endothelial cells (ECs) are known to synthesize PAF following H(2)O(2)-induced oxidative stress. The results indicated that in each case H(2)O(2)-stimulated PAF biosynthesis in ECs, which is catalyzed by acetyl-CoA acetyltransferase (AT), appeared strongly inhibited. However, acyl-PAF biosynthesis, which also occurs through the PAF-dependent transacetylase (TA), was significantly increased by lycopene only when it was in association with alpha-tocopherol or with the minor compounds present in the whole lipophilic tomato extract. These findings suggest that alpha-tocopherol or lipophilic compounds present in tomato juice potentiate the effects of lycopene on the modulation of PAF and acyl-PAF biosynthesis in ECs during oxidative stress.

Kinase recognition by calmodulin: modeling the interaction with the autoinhibitory region of human cardiac titin kinase.

Calmodulin (CaM)-protein interactions are usually described by studying complexes between synthetic targets of ca 25 amino acids and CaM. To understand the relevance of contacts outside the protein-binding region, we investigated the complex between recombinant human CaM (hCaM) and P7, a 38-residue peptide corresponding to the autoinhibitory domain of human cardiac titin kinase (hTK). To expedite the structure determination of hCaM-P7 we relied upon the high degree of similarity with other CaM-kinase peptide complexes. By using a combined homonuclear NMR spectroscopy and molecular modeling approach, we verified for the bound hCaM similar trends in chemical shifts as well as conservation of NOE patterns, which taken together imply the conservation of CaM secondary structure. P7 was anchored to the protein with 52 experimental intermolecular contacts. The hCaM-P7 structure is very similar to known CaM complexes, but the presence of NOE contacts outside the binding cavity appears to be novel. Comparison with the hTK crystal structure indicates that the P7 charged residues all correspond to accessible side-chains, while the putative anchoring hydrophobic side-chains are partially buried. To test this finding, we also modeled the early steps of the complex formation between Ca(2+)-loaded hCaM and hTK. The calculated trajectories strongly suggest the existence of an "electrostatic funnel", driving the long-range recognition of the two proteins. On the other hand, on a nanosecond time scale, no intermolecular interaction is formed as the P7 hydrophobic residues remain buried inside hTK. These results suggest that charged residues in hTK might be the anchoring points of Ca(2+)/hCaM, favoring the intrasteric regulation of the kinase. Furthermore, our structure, the first of CaM bound to a peptide derived from a kinase whose three-dimensional structure is known, suggests that special care is needed in the choice of template peptides to model protein-protein interactions.

Conformational flexibility in calcitonin: the dynamic properties of human and salmon calcitonin in solution.

We have studied the dynamic properties of human (h) and salmon (s) calcitonin (CT) in solution. For both hormones, distance geometry in torsion-angle space has been used to generate three-dimensional structures consistent with NMR data obtained in sodium dodecyl sulfate micelles. For sCT and hCT we used, respectively, 356 and 275 interproton distances together with hydrogen-bonds as restraints. To better characterize their flexibility and dynamic properties two fully unrestrained 1100-ps molecular dynamics (MD) simulations in methanol were performed on the lowest-energy structures of both hormones. Statistical analyses of average geometric parameters and of their fluctuations performed in the last 1000 ps of the MD run show typical helical values for residues 9-19 of sCT during the whole trajectory. For hCT a shorter helix was observed involving residues 13-21, with a constant helical region in the range 13-19. Angular order parameters S(phi) and S(psi) indicate that hCT exhibits a higher flexibility, distributed along the whole chain, including the helix, while the only flexible amino acid residues in sCT connect three well-defined domains. Finally, our study shows that simulated annealing in torsion-angle space can efficiently be extended to NMR-based three-dimensional structure calculations of helical polypeptides. Furthermore, provided that a sufficient number of NMR restraints describes the system, the method allows the detection of equilibria in solution. This identification occurs through the generation of 'spurious' high-energy structures, which, for right-handed alpha-helices, are likely to be represented by left-handed alpha-helices.

Solution structure of human calcitonin in membrane-mimetic environment: the role of the amphipathic helix.

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHNalpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic alpha-helix from Leu9 to Phel6, followed by a type-I beta-turn between residues Phel6 and Phel9. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure-activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed.

Inhibition mechanisms of HIV-1, Mo-MuLV and AMV reverse transcriptases by Kelletinin A from Buccinulum corneum.

Kelletinin A [ribity pentakis (p-hydroxybenzoate)] (KA), an inhibitor of HTLV-1 replication isolated from Buccinulum corneum, showed a noncompetitive inhibitory activity with respect to the template primer and to dTTP in the poly(rA).oligo(dT)12-18-directed reaction of HIV-1, Mo-MuLV and AMV reverse transcriptases (RT). Analysis of natural and synthetic KA-related compounds showed that the inhibitory activity was strictly related to the structural peculiarities of the molecule. In the presence of DNA as template primer the inhibition mechanism was drastically modified: HIV-1 RT activity was stimulated by low concentrations of KA and was inhibited by increasing the concentration of the compound, while Mo-MuLV and AMV activities were irreversibly inhibited by the formation of a non-reactive complex. The RNase H activities of these RTs were not affected by KA. The results of this study suggest a different mechanism of interaction of Kelletinins with HIV-1 RT compared with other non-nucleoside inhibitors. A possible use of these drugs in combination therapy and in the design of structure-based reverse transcriptase inhibitors is discussed.

Antimitotic and antiviral activities of Kelletinin A in HTLV-1 infected MT2 cells.

Kelletinin A [ribityl-pentakis (p-hydroxybenzoate)] (KA), a natural compound isolated from the marine gastropod Buccinulum corneum, showed antiviral activity on the human T-cell leukemia virus type-1 (HTLV-1) and antimitotic activity on HTLV-1-infected MT2 cells. KA inhibited cellular DNA and RNA synthesis, without influencing protein synthesis, and interfered with viral transcription by reducing the levels of high molecular weight transcripts. Finally, the compound inhibited HTLV-1 reverse transcriptase in vitro.

Effect of avarol, avarone and nine of their natural and synthetic derivatives on microsomal drug-metabolizing enzymes.

Avarol, a sesquiterpenoid hydroquinone, its quinone avarone, both main secondary metabolites from the marine sponge Dysidea avara and nine of their natural and synthetic derivatives were tested for ability to interact selectively with rat liver microsomal phenobarbital (PB)- or 3-methylcholanthrene (3-MC)-induced cytochrome (cyt.) P-450 isoenzymatic forms. Ethoxy- and pentoxyresorufin, aminopyrine and ethoxycoumarin were the specific substrates used for assaying cyt. P-450-dependent mono-oxygenase activities. All compounds were more effective in inhibiting the enzymatic activities measured in microsomes from PB-induced rat liver than those measured in microsomes from 3-MC-treated rats. Avarol and avarone, which were the most active inhibitors, act as mixed-type inhibitors of pentoxyresorufin-O-dealkylase activity. Mono- and diacetyl-derivatives of avarol, being deacetylated by rat liver microsomes, were almost as effective as the parent compound. Conversely, avarone derivatives, where one or two hydrogen atoms of the quinone ring have been substituted, were less effective inhibitors than the parent compound. The selective inhibition of PB-inducible cyt.P-450IIB by avarol and avarone was confirmed by their ability to inhibit the mutagenic activation of cyclophosphamide, as opposed to that of benzo[a]pyrene, which is activated mainly by the 3-MC-inducible cyt.P-450IA.

Kelletinin I and kelletinin A from the marine mollusc Buccinulum corneum are inhibitors of eukaryotic DNA polymerase alpha.

The inhibitory effect of esters of p-hydroxybenzoic acid (kelletinins I and A), extracted from the marine gastropod Buccinulum corneum, have been tested on eukaryotic and prokaryotic enzymes of DNA metabolism such as DNA polymerases alpha and beta, DNA polymerase I, Exo III, pancreatic DNAse I, micrococcal DNAse and E. coli RNA polymerase. Kelletinin I and kelletinin A inhibit preferentially DNA polymerase alpha. The inhibitory effect of kelletinin I involves the hydroxyl group of p-hydroxybenzoic acid.

The use of laboratory methods in contact dermatitis induced by composite materials.

Thin-layer chromatography, column chromatography, mass spectrometry and nuclear magnetic resonance were used to investigate the chemical constituents and haptens responsible in 2 different circumstances where allergic contact dermatitis from composite materials was suspected. In an aircraft factory where epoxy resins were used, tetraglycidyl-4,4'-dimethyl dianiline and phenylglycidyl ether were identified as the haptens responsible for an outbreak of contact dermatitis. The role of abietic acid as the main sensitizer in colophony was confirmed in a case of contact dermatitis occurring in a sportsman with an acute eczematous reaction due to a leg bandage. Identification of the chemical sensitizers was possible only by using the aforementioned laboratory methods.

A new bioactive derivative of avarol from the marine sponge Dysidea avara.

A new derivative of avarol, monoacetyl avarol, has been isolated from the sponge Dysidea avara. The structural elucidation and biological activities are reported.