Sequence dependence of translational positioning of core nucleosomes

The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modifications of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is specified by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wild-type and a mutant sequence differ by +2 bp, corresponding to an overall helical periodicity of approximately 10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The DNA immediately flanking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.     

Syringicin, a new alpha-elicitin from an isolate of Phytophthora syringae, pathogenic to citrus fruit

The primary structure of syringicin (syr), a new acidic alpha-elicitin, isolated from culture filtrates of Phytophthora syringae, causal agent of citrus fruit rot, has been determined using a combined approch based on Edman degradation and MALDI-MS (TTCTT TQQTA AYVAL VSILS DSSFN QCATD SGYSM LTATA LPTTA QYKLM CASTA CKTMI TKIVS LNAPD CELTV PTSGL VLNVY SYANG FSSTC ASL). Syr has 98 amino acids with a M(r) of 10194.6+/-0.2, which was determined by electrospray ionisation-mass spectrometry (ES-MS) and in agreement with three disulphide bridges, located between Cys3-Cys71, Cys27-Cys56 and Cys51-Cys95. Syr induces a hypersensitive response and electrolyte leakage in tobacco. These are characteristic elicitor properties of the group and in agreement with the molecular mechanism recently proposed for this kind of protein. Finally, its possible applications in biological agriculture and biomedicine are briefly discussed.     

Maroteaux-lamy syndrome: five novel mutations and their structural localization

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.     

Synthesis and biological activity of 4-alkoxy chalcones: potential hydrophobic modulators of P-glycoprotein-mediated multidrug resistance

A series of 4-alkoxy-2′,4′,6′-trihydroxychalcones have been synthesized and evaluated for their ability to inhibit P-glycoprotein-mediated multidrug resistance (MDR) by direct binding to a purified protein domain containing an ATP-binding site and a modulator-interacting region. The introduction of hydrophobic alkoxy goups at position 4 led to much more active compounds as compared to the parent chalcone. The binding affinity increased as a function of the chain length, up to the octyloxy derivative for which a K_D of 20 nM was obtained.     

Effects of TNF-alpha/colchicine combined treatment on Burkitt lymphoma cells: molecular and ultrastructural changes

Tumour necrosis factor alpha (TNF-alpha) kills Daudi cells (Human Burkitt Lymphoma), inducing either necrosis or apoptosis without DNA fragmentation. Therefore, we were interested in studying the molecular and ultrastructural events occurring when the nucleus is more accessible and cells are blocked in mitosis, following colchicine treatment. In fact, as early as after 1 h treatment a typical ladder pattern was shown by means of DNA gel electrophoresis. In parallel the quantitative analysis of the different morphological patterns observed gave evidence of an increased percentage of primary necrosis after 6 h treatment, and a higher incidence of cells in late apoptosis as well as in secondary necrosis after 24 h treatment. Our findings show that Daudi cells respond to the combined treatment with an increased formation of micronuclei and nuclear alterations which follow a number of early mitochondrial changes and result in enhanced cell death. These data imply that TNF-alpha-induced apoptosis of Daudi cells can be triggered by mitochondrial changes and is somehow related to microtubule organization.     

Purification and characterization of amine oxidase from pea seedlings

A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.     

Vascular non-protein thiols: prooxidants or antioxidants in atherogenesis?

Abstract-cell-mediated lipoprotein oxidation may be due to generation of non-protein thiols (NP-SH) from cystine with formation of oxidizing species. However, NP-SH, especially GSH, may also exert antioxidant effects in vitro and in vivo. To further investigate whether vascular NP-SH could be prooxidants or antioxidants in atherosclerosis, we have correlated the aortic content of NP-SH with that of lipoperoxides in 10 rabbits fed on a fat-enriched atherogenic diet for 9 weeks. As compared to 7 control rabbits, aortic NP-SH and lipoperoxides were significantly increased in the fat-fed animals. The levels of NP-SH were strongly and inversely correlated with those of lipid peroxidation in the atherosclerotic aorta (r(s) -0.92, P < 0.0001 for thiobarbituric acid reactive substances, and r(s) -0.80, P < 0.01 for fluorescent damage products of lipid peroxidation). A similar trend was evident also in the control rabbits (r(s) -0.60 for both indices of lipid peroxidation). Thus, the present data suggest that vascular NP-SH exert significant antioxidant-antilipoperoxidative effects in vivo especially in fat diet-related atherogenic conditions.     

Study of eight cases of coccidioidomycosis in a hospital of Buenos Aires.

Some clinical, epidemiological and diagnostic aspects from eight patients with chronic coccidioidomycosis (five pulmonary and three disseminated), diagnosed in the Mu?iz Hospital, were retrospectively analyzed. At diagnosis, lung cavitation and hemoptysis were present in five and four patients, respectively. Smoking (three cases) and alcoholism (two cases) were the most frequent predisposing factors. Diagnosis was achieved by microscopy and cultures from sputum (five cases), tongue and lymph node biopsies and scraping of cutaneous lesion achieved diagnosis. At diagnosis, most patients had positive coccidioidin skin test and serology. Four patients were born within the endemic area and two worked in contact with the soil of the same area.     

Docking of flexible ligands to flexible receptors in solution by molecular dynamics simulation

In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of an algorithm previously reported by Di Nola et al. (Di Nola et al., Proteins 1994;19:174-182). The method allows a fast exploration of the receptor surface, using a high temperature of the center of mass translational motion, while the ligand internal motions, the solvent, and the receptor are simulated at room temperature. In addition, the method allows a fast center of mass motion of the ligand, even in solution. The dampening effect of the solvent can be overcome by applying different weights to the interactions between system subsets (solvent, receptor, and ligand). Specific ligand-receptor distances have been used to compare the results of the simulations with the crystal structure. The method is applied, as a test system, to the docking of the phosphocholine to the immunoglobulin McPC603. The results show the similarity of structure between the complex in solution and in the crystal.     

The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.