Fast kinetics analysis of the peroxidatic reaction catalysed by horse liver alcohol dehydrogenase

The kinetics of the enzymatic step of the peroxidatic reaction between NAD and hydrogen peroxide, catalysed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), has been investigated at pH 7 at high enzyme concentration. Under such conditions no burst phase has been observed, thus indicating that the rate-limiting step in the process, which converts NAD into Compound I, either precedes or coincides with the chemical step responsible for the observed spectroscopic change. Kinetic analysis of the data, performed according to a simplified reaction scheme suggests that the rate-limiting step is coincident with the spectroscopic (i.e., chemical) step itself. Furthermore, the absence of a proton burst phase indicates the proton release step does not precede the chemical step, in contrast with the case of ethanol oxidation. A kinetic effect of different premixing conditions on the reaction rate has been observed and attributed to the presence of NADH formed in the 'blank reaction' between NAD and residual ethanol tightly bound to alcohol dehydrogenase. A molecular mechanism for the enzymatic peroxidation step is finally proposed, exploiting the knowledge of the much better known reaction of ethanol oxidation. Inhibition of this reaction by NADH has been investigated with respect to H2O2 (noncompetitive, Ki about 10 microM) and to NAD (competitive, Ki about 0.7 microM). The effect of temperature on the steady-state reaction state (about 65 kJ/mol activation energy) has also been studied.     

Nuclear DNA changes within Helianthus annuus L.: variations in the amount and methylation of repetitive DNA within homozygous progenies

Complex alterations in the redundancy and methylation of repeated DNA sequences were shown to differentiate the nuclear genome of individuals belonging to single progenies of homozygous plants of the sunflower. DNA was extracted from seedlings obtained from seeds collected at the periphery of flowering heads (P DNA) or from seedlings obtained from seeds collected in their middle (M DNA). Three fractions of repeated sequences were isolated from genomic DNA: a highly repetitive fraction (HR), which reassociates within an equivalent Cot of about 2 × 10^-1, and two medium repetitive fractions (MR1 and MR2) having Cot ranges of about 2 × 10^-1-2 and 2-10², respectively. Denaturation kinetics allowed different sequence families to be recognized within each fraction of repetitive DNA, and showed significant differences in sequence redundancy to occur between P and M DNA, particularly as far as the MR2 fraction is concerned. Most DNA sequence families are more represented in P DNA than in M DNA. However, the redundancy of certain sequences is greater in the latter than in the former. Each repetitive DNA fraction was hybridized to Southern blots of genomic P or M DNA which was digested to completion by three pairs of isoschizomeric restriction endonucleases which are either insensitive or sensitive to the methylation of a cytosine in the recognition site. The results obtained showed that the repetitive DNA of H. annuus is highly methylated. Clear-cut differences in the degree of methylation of P and M DNA were found, and these differences were particularly apparent in the MR2 fraction. It is suggested that alterations in the redundancy of given DNA sequences and changes in their methylation patterns are complementary ways to produce continuous genotypic variability within the species which can be exploited in environmental adaptation.Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2     

Assessing the effects of forest management on epiphytic lichens in coppiced forests using different indicators

Abstract

Epiphytic lichens are one of the taxonomic groups most sensitive to forest management. Nevertheless, they have not yet been exhaustively included in the assessment of Sustainable Forest Management. This work aimed at evaluating the effects of forest management on epiphytic lichens in coppiced forests, exploring the spatial patterns of diversity and the composition of communities. Moreover, the goal was to compare the performance of four potential indicators for monitoring the effects of forest management on epiphytic lichens: total lichen diversity, species associated with intensive management, species associated with aged coppiced woodlands and Indicator Species Ratio (ISR). In humid Mediterranean Liguria, 50 sampling units were chosen in Castanea sativa and deciduous Quercus spp. forests subjected to different forest management practices: intensively managed coppice and aged coppice/high forest. The effect of forest management was evident in terms of species composition, since it was possible to find significantly associated species for each of the two management types. At each sampling site, the four indicators were calculated using Indicator Value Analysis and compared through correspondence analysis. The ISR was shown to be a more effective indicator, being independent of floristic composition and the occurrence of rare species.     

Phylogenetic relationships of Italian Bellevalia species (Asparagaceae), inferred from morphology,karyology and molecular systematics

The seven Bellevalia species and subspecies known from Italy, representing about 10% of the genus and three out of six sections, were studied. An integrated morphological, karyological and molecular approach was used to infer phylogenetic and systematic relationships among them. B. romana (the generitype) is the most distinctive species on karyotype asymmetry grounds. B. boissieri and B.dubia, usually considered as subspecies of one species (the latter endemic to Sicily), deserve specific status based on biparental nrDNA markers (internal transcribed spacer, ITS), since they do not form a single clade. The allotetraploid endemic B. pelagica, morphologically similar to B. romana, is sister to the latter under parsimony, both in morphological and ITS trees; it is also related with B. dubia, based on karyotype asymmetry and a uniparental cpDNA marker (trnL^(UAA)–trnF^(GAA) IGS (intergenic spacer)). A second allotetraploid endemic, B. webbiana, is closely related, on morphological, karyological and molecular grounds, with B. boissieri and B. ciliata, and also with B. trifoliata, three species that might all involved in its origin. B. sect. Conicae Feinbr. and sect. Nutantes Feinbr. are here typified, the former (type: B. ciliata) is most likely a synonym of the latter (type: B. trifoliata).     

Effects of sodium dodecyl sulphate on the aquatic macrophytes Azolla and Lemna

Abstract

In this work, we determined the tolerance to sodium dodecyl sulphate (SDS) of Azolla filiculoides and of Lemna minor. The presence of the detergent in the media affected growth more than the chlorophyll content. On the basis of the effect indices, Lemna is more tolerant to SDS than Azolla. The fern had a better capacity, compared with duckweed, to remove and accumulate the pollutant.     

In Vivo and In Vitro Effects of Some Plant Hormones on Rat Erythrocyte Carbonic Anhydrase and Glucose-6-Phosphate Dehydrogenase Activities

The present study was undertaken to determine in vivo and in vitro effects of some plant growth regulators on rat erythrocyte carbonic anhydrase (CA) and glucose-6-phosphate dehydrogenase (G6PD) activities. Both in vivo and in vitro, spermidine and kinetin did not affect enzymatic activities of CA and G6PD, whereas putrescine decreased these activities, and abscisic acid increased them. Since plants use such growth regulators, their effects should be considered on mammals consuming them since they may possess important biological effects.     

A class of pyrrole derivatives endowed with analgesic/anti-inflammatory activity

We report the synthesis and bio-pharmacological evaluation of a class of pyrrole derivatives featuring a small appendage fragment (carbaldehyde, oxime, nitrile) on the central core. Compound 1c proved to be extremely effective in vivo, showing an interesting anti-nociceptic profile that is comparable to reference compounds already marketed, hence representing a great stimulus for a further improvement of this class of molecules.     

HIV-1 Nef induces dendritic cell differentiation: a possible mechanism of uninfected CD4(+) T cell activation

Human immunodeficiency virus (HIV)-1 Nef protein is an essential modulator of AIDS pathogenesis and we have previously demonstrated that rNef enters uninfected human monocytes and induces T cells bystander activation, up-regulating IL-15 production. Since dendritic cells (DCs) play a central role in HIV-1 primary infection we investigated whether rNef affects DCs phenotypic and functional maturation in order to define its role in the immunopathogenesis of AIDS. We found that rNef up-regulates the expression on immature DCs of surface molecules known to be critical for their APC function. These molecules include CD1a, HLA-DR, CD40, CD83, CXCR4, and to a lower extent CD80 and CD86. On the other hand, rNef down-regulates surface expression of HLA-ABC and mannose receptor. The functional consequence of rNef treatment of immature DCs is a decrease in their endocytic and phagocytic activities and an increase in cytokine (IL-1beta, IL-12, IL-15, TNF-alpha) and chemokine (MIP-1alpha, MIP-1beta, IL-8) production as well as in their stimulatory capacity. These results indicate that rNef induces a coordinate series of phenotypic and functional changes promoting DC differentiation and making them more competent APCs. Indeed, Nef induces CD4(+) T cell bystander activation by a novel mechanism involving DCs, thus promoting virus dissemination.