Cloning of the <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. FG1 dehalogenase genes and construction of hybrid pathways in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I > F, in the order.     

Morphological and molecular analysis of centropagids from the high Andean plateau (Copepoda: Calanoidea)

In small metazoan invertebrates classical taxonomic analyses can be ambiguous due to the limited number of morphological characters available. This difficulty can yield incorrect estimates of species richness or taxa distribution. The Boeckella genus has been described as the dominant taxon of zooplankton assemblages in the Andean biogeographical region. In this genus, taxonomic classification and delimitation of boundaries between species has long been problematic and controversial. Among South American centropagids Boeckella gracilipes has been regarded as one of the most broadly distributed species, its presence having been reported from Ecuador to Tierra del Fuego. However, in the high Andean plateau some centropagid populations identified as Boeckella gracilipes have also been considered as B. gracilipes titicacae or even identified as a different species, namely Boeckella titicacae. In an attempt to resolve the taxonomic status of the Centropagidae family from the high Andean plateau, we combined traditional and multivariate morphological analyses (integral approach) with the molecular phylogenetic approach. The results obtained allow us to conclude that centropagids collected from the high Andean plateau actually represent a different species, B. titicacae, not B. gracilipes. The phylogenetic reconstruction of the South American Centropagidae family indicated that B. gracilipes represents a sister taxon to B. titicacae. The present study stresses the usefulness of integrating alfa-taxonomy with morphometric and molecular approaches in order to resolve species boundaries, to determine geographical distributions and to investigate evolutionary processes.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

Immunomodulatory effects of bovine colostrum in human peripheral blood mononuclear cells

Human and bovine colostrum (BC) contain a remarkable amount of bioactive substances, including antibodies towards many common pathogens of the intestinal and respiratory tract as well as growth factors, vitamins, cytokines and other proteic, lipidic and glucidic factors. In this study we investigated whether BC had any immunomodulatory effect on human peripheral blood mononuclear cells (PBMC) from healthy donors. To this aim we focused on the production of IL-12 and IFN-gamma, cytokines involved in the Th1 polarization required for a successful immune response towards intracellular pathogens, such as bacteria and viruses. BC induced a dose-dependent production of IL-12 by CD14+ monocytes, but was unable to induce IFN-gamma production. However, BC differentially affected stimuli-induced IFN-gamma production: it enhanced IFN-gamma in response to weak antigenic stimulation and it inhibited IFN-gamma in response to strong antigenic stimulation. These effects were not dose-dependent. We also measured PBMC proliferation, which was substantially unaffected by BC. Our data suggest that the Th1-promoting activity of BC could contribute, together with the antibodies, to the protective effect of BC on the offspring. BC could also represent an inexpensive therapeutic tool in prevention and treatment of several human microbial infections, including influenza.     

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Lycopene prevents 7-ketocholesterol-induced oxidative stress,cell cycle arrest and apoptosis in human macrophages

The present study was undertaken to examine whether lycopene is able to counteract 7-ketocholesterol (7-KC)-induced oxidative stress and apoptosis in human macrophages. Human THP-1 macrophages were exposed to 7-KC (10–25 μM) alone and in combination with lycopene (0.5–2 μM), and we monitored changes in cell oxidative status [reactive oxygen species (ROS) production, NOX-4, hsp70 and hsp90 expressions, 8-OHdG formation] and in cell proliferation and apoptosis. After 24 h of treatment, lycopene significantly reduced the increase in ROS production and in 8-OHdG formation induced by the oxysterol in a dose-dependent manner. Moreover, the carotenoid strongly prevented the increase of NOX-4, hsp70 and hsp90 expressions as well as the phosphorylation of the redox-sensitive p38, JNK and ERK1/2 induced by the oxysterol. The attenuation of 7-KC-induced oxidative stress by lycopene coincided with a normalization of cell growth in human macrophages. Lycopene prevented the arrest in G0/G1 phase of cell cycle induced by the oxysterol and counteracted the increased expression of p53 and p21. Concomitantly, it inhibited 7-KC-induced apoptosis, by limiting caspase-3 activation and the modulatory effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax. Comparing the effects of lycopene, β-carotene and (5Z)-lycopene on ROS production, cell growth and apoptosis show that lycopene and its isomer were more effective than β-carotene in counteracting the dangerous effects of 7-KC in human macrophages. Our study suggests that lycopene may act as a potential antiatherogenic agent by preventing 7-KC-induced oxidative stress and apoptosis in human macrophages.