The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O(2)/H(2)O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme-substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.     

From the sequence to the superstructural properties of DNAs

A theoretical model for predicting intrinsic and induced DNA superstructures as well as their thermodynamic properties is presented. Intrinsic sequence-dependent superstructures are evaluated by integrating local deviations from the canonical B-DNA of the different dinucleotide steps. Induced superstructures are obtained by adopting the principle of minimum deformation free energy, evaluated in the Fourier space, in the framework of first-order elasticity. Finally dinucleotide stacking energies and melting temperatures are considered to account for local flexibility. In fact the two scales are strongly correlated. The model works very satisfactorily in predicting the sequence-dependent effects on the DNA experimental behavior, such as the gel electrophoresis retardation, the writhe transitions in topologically constrained domains, the thermodynamic constants of circularization reactions as well as the nucleosome thermodynamic stability constants.     

Perylene diimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase

Four N,N'-disubstituted perylene diimides, having different side chains, have been studied for their ability in inducing G-quadruplex DNA structures. We found that electrostatic interactions between ligands side chains and DNA grooves play a main role not only in the amount of G-quadruplex formed, but also in selecting its topology. Moreover, such compounds show also a different ability to inhibit telomerase. The correlation of these findings suggests the intriguing possibility that different G-quadruplex structures could differently inhibit the enzyme.     

Effects of fasting and hypoxic preconditioning on the hypoxic-reoxygenated ventricular strips of the rat heart

The investigation aimed to assess the effects of hypoxic preconditioning in right ventricle strips of fed and 24-h fasted rats, which display a fast fatty acid catabolism, and to ascertain whether these effects are associated with changes in the tissue levels of long-chain acylCoA and acyl carnitine and glycolytic activity. Strips were mounted isometrically in Krebs-bicarbonate solution with 10 mM dextrose and paced at 1 Hz. Strips were exposed to 30 min hypoxia and 60 min reoxygenation with or without a previous preconditioning cycle of 5 min hypoxia followed by a 10 min reoxygenation. During hypoxia the fasted rat strips underwent a greater contracture with respect to the fed group. Preconditioning reduced the contracture strength and accelerated the post-hypoxic recovery only in the fasted rat strips. Hypoxia evoked an increase in the acylCoA and acyl carnitine tissue-contents of the strips which reached higher levels in the fasted than in the fed rat groups. Preconditioning had no effects on the content of these metabolites. During hypoxia lactate output was lower in the fasted than in the fed rat strips and preconditioning abolished this decrease. These data suggest that the protective effects of hypoxic preconditioning occur in the heart tissue predisposed to the oxidation of fatty acid and can not be ascribed to changes in the accumulation of acylCoA and acyl carnitine but could be due, at least in part, to an activation of glycolysis.     

Sensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot Hybridization

A non‐radioactive dot‐blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)‐labelled minus‐sense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection.     

Agrobacterium rhizogenes—transformed plant roots as a source of grapevine viruses for purification

Agrobacterium rhizogenes-mediated transformation was applied toVitis spp. andNicotiana spp. infected by different grapevine phloem-limited viruses (grapevine fleck virus, grapevine virus A, grapevine virus B) to obtain root cultures for virus purification. All plant species were successfully transformed, and several clones were established in liquid culture. Transformed grapevine roots contained as much virus as non transformed roots and more than leaves, as assessed by ELISA and thin sectioning. Likewise, transformed roots ofNicotiana benthamiana Domin. contained in average more GVA than leaves, especially those at the base and the top of the plant, whereas withNicotiana occidentalis wheel., GVB was apparently less concentrated than in leaves.Nicotiana root grew faster than those ofVitis. All viruses multiplied and persisted in root cultures, which were successfully used for purification. Virus yields were the same (GFkV and GVB) or higher (GVB) than those reported in the literature. Grapevine roots may prove useful for culturing and purifying other non-mechanically transmissible grapevine viruses.     

Aggressive and Foraging Behavioral Interactions Among Ruffe

The ruffe, Gymnocephalus cernuus, is a nonindigenous percid in the Great Lakes. Ruffe are aggressive benthivores and forage over soft substrates. Laboratory studies in pools (100cm diameter, 15cm water depth) were conducted to determine whether fish density (low=2, medium=4, high=6 ruffe per pool) changed foraging and aggressive behaviors with a limited food supply of chironomid larvae. All fish densities demonstrated a hierarchy based on aggressive interactions, but ruffe were most aggressive at low and high fish densities. Time spent in foraging was lowest at the low fish density. The best forager at the low fish density was the most aggressive individual, but the second most aggressive fish at the medium and high fish density was the best forager and also the one chased most frequently. A medium fish density offered the best energetic benefits to ruffe by providing the lowest ratio of time spent in aggression to that spent foraging. Based on our results, ruffe should grow best at an intermediate density. With high ruffe densities, we would also expect disparity in size as the more aggressive fish are able to garner a disproportionate amount of the resources. Alternatively, as the Great Lakes are a fairly open system, ruffe could migrate out of one area to colonize another as populations exceed optimal densities.     

Is there a glycosaminoglycan-related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus.     

Chromatin accessibility to DNA minor groove ligands in vitro: role of linker histones and amino-terminal domains of octamer histones.

Using the circular dichroism spectra, induced in the visible range by the binding of minor groove ligands to DNA, we found that two drugs, DAPI and Hoechst 33258, are able to occupy their specific sites even when these are located inside the nucleosome structure. This high accessibility of the binding sites in the nucleosome is not modified by the removal of the amino-terminal domains of the octamer histones and, surprisingly, it is not reduced by the presence of linker histone. Interesting and reasonable differences were found in the association constants, that reveal the "reluctance" of the ligands to bind the DNA-minor groove when the histones are present.