Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver,kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.     

Structural and biochemical characterization of toad liver fatty acid-binding protein

Two paralogous groups of fatty acid-binding proteins (FABPs) have been described in vertebrate liver: liver FABP (L-FABP) type, extensively characterized in mammals, and liver basic FABP (Lb-FABP) found in fish, amphibians, reptiles, and birds. We describe here the toad Lb-FABP complete amino acid sequence, its X-ray structure to 2.5 A resolution, ligand-binding properties, and mechanism of fatty acid transfer to phospholipid membranes. Alignment of the amino acid sequence of toad Lb-FABP with known L-FABPs and Lb-FABPs shows that it is more closely related to the other Lb-FABPs. Toad Lb-FABP conserves the 12 characteristic residues present in all Lb-FABPs and absent in L-FABPs and presents the canonical fold characteristic of all the members of this protein family. Eight out of the 12 conserved residues point to the lipid-binding cavity of the molecule. In contrast, most of the 25 L-FABP conserved residues are in clusters on the surface of the molecule. The helix-turn-helix motif shows both a negative and positive electrostatic potential surface as in rat L-FABP, and in contrast with the other FABP types. The mechanism of anthroyloxy-labeled fatty acids transfer from Lb-FABP to phospholipid membranes occurs by a diffusion-mediated process, as previously shown for L-FABP, but the rate of transfer is 1 order of magnitude faster. Toad Lb-FABP can bind two cis-parinaric acid molecules but only one trans-parinaric acid molecule while L-FABP binds two molecules of both parinaric acid isomers. Although toad Lb-FABP shares with L-FABP a broad ligand-binding specificity, the relative affinity is different.     

The effect of protein conformational flexibility on the electronic properties of a chromophore

In this paper we address the question of how a protein environment can modulate the absorption spectrum of a chromophore during a molecular dynamics simulation. The effect of the protein is modeled as an external field acting on the unperturbed eigenstates of the chromophore. Using a first-principles method recently developed in our group, we calculated the perturbed electronic energies for each frame and the corresponding wavelength absorption during the simulation. We apply this method to a nanosencond timescale molecular dynamics simulation of the light-harvesting peridinin-chlorophyll-protein complex from Amphidinium carterae, where chlorophyll was selected among the chromophores of the complex for the calculation. The combination of this quantum-classical calculation with the analysis of the large amplitude motions of the protein makes it possible to point out the relationship between the conformational flexibility of the environment and the excitation wavelength of the chromophore. Results support the idea of the existence of a correlation between protein conformational flexibility and chlorophyll electronic transitions induced by light.     

An interface model for the periodontal ligament

A nonlinear interface constitutive law is formulated for modeling the mechanical behavior of the periodontal ligament. This gives an accurate interpolation of the few available experimental results and provides a reasonably simple model for mechanical applications. The model is analyzed from the viewpoints of both mathematical consistency and effectiveness in numerical calculations. In order to demonstrate the latter, suitable two- and three-dimensional nonlinear interface finite elements have been implemented.     

Eukaryotic cell signaling and transcriptional activation induced by bacterial porins

The protein composition of the outer membrane of Gram-negative bacteria consists of about 20 immunochemically distinct proteins, termed outer membrane proteins (OMPs). Apart from their structural role, OMPs have been shown to have other functions, particularly with regard to transport, and have been classified as permeases and porins. Porins, during their interaction with the host, are immunogenic and also directly stimulate several cellular functions. Porins work both as molecules present on the bacterial surface and as molecules released by bacteria. Lipopolysaccharide and OMPs, the major structural macromolecular constituents of the outer membrane, carry out a fundamental role in the pathogenesis of Gram-negative infections. This brief review describes the multiple facets of the biological activities of porins both in vitro and in vivo, particularly focusing on their ability to induce the expression of cytokines and other factors that modulate cellular activities with either pathological or adaptive consequences. This brief discussion will focus on the signal transmission mechanisms induced by bacterial porins.     

Role for mannose-sensitive hemagglutinin in promoting interactions between Vibrio cholerae El Tor and mussel hemolymph

The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18 degrees C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type V. cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.     

Submerged and solid-state production of laccase and Mn-peroxidase by Panus tigrinus on olive mill wastewater-based media

The possible use of olive-mill wastewater (OMW) as a growth medium for the production of extracellular laccase and manganese peroxidase (MnP) from the white-rot fungus Panus tigrinus (P. tigrinus) CBS 577.79 was studied using a properly formulated OMW-based medium (2-fold diluted OMW supplemented with 0.5% sucrose and 0.1% yeast extract) either in a stirred-tank or an air-lift reactor. Solid-state fermentation (SSF) was also performed in a rotary drum reactor using maize stalks moistened with the OMW-based medium. Highest levels of laccase and manganese peroxidase activity were obtained in the stirred-tank reactor (4600+/-98 U l(-1) on day 13) and in the air-lift reactor (410+/-22 on day 7), respectively. Based on total enzyme activities, SSF appears to be more suitable than LSF but the latter exhibits better volumetric productivities.     

Antioxidant activity of galloyl quinic derivatives isolated from P. lentiscus leaves

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5- O -galloyl, 3,5- O -digalloyl, 3,4,5- O -trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide ( O 2 - ) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC 50 ) of di- and tri-galloyl derivatives did not exceed 30 μM for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.     

Acute ischemia/hypoxia in rat hippocampal neurons activates nuclear ubiquitin and alters both chromatin and DNA

We investigated early alterations in rat neurons after experimental ischemic stress. Transient ischemia was generated by bilateral occlusion of the carotids after hypoxia. Data show a relevant increase of the nuclear level of ubiquitin 2 h post-stress as evaluated by immuno-cytolocalization. Ubiquitin returns to normal levels after 6 h. The increase in ischemic/hypoxic rats was localized preferentially in nuclei of hippocampal neurons, although some augmentation was also shown essentially in dendrites. The activation of ubiquitin system is related to a defective homeostasis and might trigger different degenerative processes. With respect to this, we observed chromatin alterations by densitometric analysis. The shown extensive DNA degeneration is consistent with the occurrence of necrotic phenomena at an early stage. However the parallel internucleosomal specific DNA fragmentation, strongly suggests that apoptotic events also occur. In any case both necrosis and apoptosis are likely to occur at same time, although apoptosis is less extensive, and two phenomena take place in different neural cells.     

Structure elucidation of clavilactone D: an inhibitor of protein tyrosine kinases

Clavilactones D and E were isolated from an agar culture of the Basidiomycetous fungus Clitocybe clavipes, and their structure was elucidated by 1H- and 13C-NMR studies. Clavilactone D is an inhibitor of tyrosine kinases.