Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.

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Background: Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semisynthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established.

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1. It is well known that neutrophils act as mediators of tissue injury in a variety of inflammatory diseases. Their histotoxic activity is presently thought to involve proteinases and oxidants, primarily hypochlorous acid (HOCl). This oxidant is also capable of inactivating the specific inhibitor of neutrophil elastase (alpha 1-antitrypsin), thereby favouring digestion of the connective matrix. 2. In the present work, we found that sulphanilamide and some sulphanilamide-related anti-inflammatory drugs such as dapsone, nimesulide and sulphapyridine reduce the availability of HOCl in the extracellular microenvironment of activated neutrophils and prevent the inactivation of alpha 1-antitrypsin by these cells in a dose-dependent manner. The ability of each drug to prevent alpha 1-antitrypsin from inactivation by neutrophils correlates significantly with its capacity to reduce the recovery of HOCl from neutrophils. Five other non-steroidal anti-inflammatory drugs were completely ineffective. 3. Therefore, sulphanilamide-related drugs, i.e. dapsone, nimesulide and sulphapyridine, have the potential to reduce the bioavailability of neutrophil-derived HOCl and, in turn, to favour the alpha 1-antitrypsin-dependent control of neutrophil elastolytic activity. These drugs appear as a well-defined group of agents which are particularly prone to attenuate neutrophil histotoxicity. They can also be viewed as a previously unrecognized starting point for the development of new compounds in order to plan rational therapeutic strategies for controlling tissue injury during neutrophilic inflammation.

Paclitaxel is an effective antineoplastic agent originally extracted in low yield from the bark of Taxus brevifolia. Although it was generally considered a particular metabolite of Taxus sp., paclitaxel was recently found in hazel cell cultures. The aim of the present work was to verify whether hazel differentiated tissues could be used as a commercial source of paclitaxel and other taxanes. Thus, shells and leaves of hazel plants were analyzed by ELISA and HPLC-MS. Both shell and leaf extracts contained taxanes. Among these, paclitaxel, 10-deacetylbaccatin III, baccatin III, paclitaxel C, and 7-epipaclitaxel were identified and quantified. Hazel extracts also showed biological activity, inhibiting metaphase to anaphase transition in a human tumor cell line. The level of total taxanes in leaves was higher than in shells collected in the same period from the same plants. However, the finding of these compounds in shells, which are considered discarded material and are mass produced by many food industries, is of interest for the future availability of paclitaxel and other antineoplastic compounds.

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