Nonisothermal bioreactors in the treatment of vegetation waters from olive oil: laccase versus syringic acid as bioremediation model

Laccase from Trametes versicolor was immobilized by diazotization on a nylon membrane grafted with glycidil methacrylate, using phenylenediamine as spacer and coupling agent. The behavior of these enzyme derivatives was studied under isothermal and nonisothermal conditions by using syringic acid as substrate, in view of the employment of these membranes in processes of detoxification of vegetation waters from olive oil mills. The pH and temperature dependence of catalytic activity under isothermal conditions has shown that these membranes can be usefully employed under extreme pH and temperatures. When employed under nonisothermal conditions, the membranes exhibited an increase of catalytic activity linearly proportional to the applied transmembrane temperature difference. Percentage activity increases ranging from 62% to 18% were found in the range of syringic acid concentration from 0.02 to 0.8 mM, when a difference of 1 degrees C was applied across the catalytic membrane. Because the percentage activity increase is strictly related to the reduction of the production times, the technology of nonisothermal bioreactors has been demonstrated to be an useful tool also in the treatment of vegetation waters from olive oil mills.     

Structure-affinity studies for a novel series of homochiral naphtho and tetrahydronaphtho analogues of alpha 1 antagonist WB-4101

A number of enantiomeric pairs of naphthodioxane, tetrahydronaphthodioxane and naphthoxy analogues of WB-4101 (1) were designed and synthesized in order to improve the selectivity profile of the parent compound, hopefully in favour of the alpha(1a)-AR with respect to the other two alpha(1) subtypes and the 5-HT(1A) receptor. The new compounds 2-8 and, in addition, the two enantiomers of 1 were tested in binding assays on the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR, and the 5-HT(1A) receptor. Two of them, namely the naphtho- and tetrahydronaphthodioxane derivatives (S)-2 and (S)-3, showed lower, but significantly more specific alpha(1a) affinity than (S)-1, while the two enantiomers of the 2-methoxy-1-naphthoxy analogue 6 maintained most of the very high alpha(1a) affinity of (S)-1 and its alpha(1a) versus alpha(1b) selectivity slightly increasing the alpha(1a)/alpha(1d) and alpha(1a)/5HT(1A) affinity ratios. The SAR data were evaluated in the light of known alpha(1) subtype pharmacophores and of the alpha(1a)-AR binding mode of WB-4101 resultant from literature mutagenesis studies disclosing some interesting consonances with these models.     

Synthesis and biological evaluation of 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives as new ligands of cannabinoid receptors

Cannabinoid receptors have been studied extensively in view of their potential functional role in several physiological and pathological processes. For this reason, the search for new potent, selective ligands for subtype CB receptors, CB(1) and CB(2), is still of great importance, in order to investigate their role in various physiological functions. The present study describes the synthesis and the biological properties of a series of 1,8-naphthyridine derivatives, characterised by the presence of some important structural requirements exhibited by other classes of cannabinoid ligands, such as an aliphatic or aromatic carboxamide group in position 3, and an alkyl or arylalkyl substituent in position 1. These compounds were assayed for binding both to the brain and to peripheral cannabinoid receptors (CB(1) and CB(2)). The results obtained indicate that the naphthyridine derivatives examined possess a greater affinity for the CB(2) receptor than for the CB(1) receptor. In particular, derivatives 6a and 7a possess an appreciable affinity for the CB(2) receptor, with K(i) values of 5.5 and 8.0 nM respectively; also compounds 4a, 5a and 8a exhibit a good CB(2) affinity, with K(i) values in the range of 10-44 nM. Furthermore, compounds 3g-i and 18 revealed a good CB(2) selectivity, with a CB(1)/CB(2) ratio >20.     

Stopped-flow fluorescence studies of inhibitor binding to tyrosinase from Streptomyces antibioticus

Tyrosinase (Ty) is a type 3 copper protein involved in the rate-limiting step of melanin synthesis. It is shown that the endogenous Trp fluorescence of tyrosinase from Streptomyces antibioticus is remarkably sensitive to the redox state. The fluorescence emission intensity of the [(Cu(I) Cu(I)] reduced species is more than twice that of the oxygen-bound [Cu(II)-O(2)(2-)-Cu(II)] form. The emission intensity of the oxidized [Cu(II)-OH(-)-Cu(II)] protein (Ty(met)) appears to be dependent on an acid-base equilibrium with a pK(a) value of 4.5 +/- 0.1. The binding of fluoride was studied under pseudo first-order conditions using stopped-flow fluorescence spectroscopy. The kinetic parameters k(on), K(d), and the fraction of fluorescence emission quenched upon fluoride binding show a similar pH dependence as above with an average pK(a) value of 4.62 +/- 0.05. Both observations are related to the dissociation of Cu(2)-bridging hydroxide at low pH. It is further shown that Ty is rapidly inactivated at low pH and that halide protects the enzyme from this inactivation. All results support the hypothesis that halide displaces hydroxide as the Cu(2)-bridging ligand in Ty(met). The relevance of the experimental findings for the catalytic cycle is discussed. The data are consistent with the data obtained from other techniques, validating the use of fluorescence quenching as a sensitive and effective tool in studying ligand binding and substrate conversion.     

Biphasic finite element simulation of the TMJ disc from in vivo kinematic and geometric measurements

Understanding the biomechanical nature of the degeneration of the temporomandibular joint requires a coupling between experimental measurements and numerical simulation. In this study, geometry measured from MRI, and motion obtained from a specially designed optoelectronic system are fed into a three-dimensional biphasic finite element analysis to generate the spatial and temporal mechanical response of the disc. This study demonstrates how this coupling can effectively predict the biomechanical response of the temporomandibular joint disc to physiological loading. For small jaw opening movements, asymmetries in the load of the disc are found, with especially higher shear stresses in the lateral portion.     

Double mechanism for apical tryptophan depletion in polarized human bronchial epithelium

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-gamma in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-gamma. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-gamma caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-gamma treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na(+)-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na(+)-dependent amino acid transporter and an IFN-gamma-inducible degradation by IDO.     

Design of mineral medium for growth of <Emphasis Type="Italic">Actinomadura</Emphasis> sp. ATCC 39727, producer of the glycopeptide A40926: effects of calcium ions and nitrogen sources

Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin, with significant activity against Neisseria gonorrhoeae and precursor of the semi-synthetic antibiotic dalbavancin. In this study the production of A40926 by Actinomadura under a variety of growth conditions was investigated. The use of chemically defined mineral media allowed us to analyze the influence of carbon and nitrogen sources, phosphate, ammonium and calcium on the growth and the antibiotic productivity of Actinomadura. We confirm recent data [Gunnarsson et al. (2003) J Ind Microbiol Biotechnol 30:150–156] that low initial concentrations of phosphate and ammonium are beneficial for growth and A40926 production, and we provide new evidence that the production of A40926 is depressed by calcium, but promoted when l-glutamine or l-asparagine are used as nitrogen sources instead of ammonium salts.     

Mutations of the CK2 phosphorylation site of Sic1 affect cell size and S-Cdk kinase activity in Saccharomyces cerevisiae

By sequence analysis we found an amino acid stretch centred on Serine201 matching a stringent CK2 consensus site within the C-terminal, inhibitory domain of Sic1. Here we show by direct mass spectrometry analysis that Sic1, but not a mutant protein whose CK2 phospho-acceptor site has been mutated to alanine, Sic1S201A, is actually phosphorylated in vitro by CK2 on Serine 201. Mutation of Serine 201 alters the coordination between growth and cell cycle progression. A significant increase of average protein content and of the average protein content at the onset of DNA synthesis is observed for exponentially growing cells harbouring the Sic1S201A protein. A strong reduction of the same parameters is observed in cells harbouring Sic1S201E. The deregulated coordination between cell size and cell cycle is also apparent at the level of S-Cdk activity.