Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.     

Biocontrol Activity and Plant Growth Promotion Exerted by Aureobasidium pullulans Strains

The most common leguminous plants’ diseases are caused by soil-borne pathogens leading to important economic losses worldwide. Strains L1 and L8, belonging to Aureobasidium pullulans species, were tested in vitro and in vivo as biocontrol agents (BCAs) against Rhizoctonia solani (Rs1) (AG-4) and as plant growth promoters (PGPs). The non-volatile metabolites produced by L1 and L8 strains inhibited the pathogen mycelial growth by 87.9% on average, with no significant differences between the two strains. The lower pathogen diametric growth inhibition was displayed by both yeasts’ volatile metabolites (VOCs) that significantly reduced the colony growth of R. solani, and similarly to the control, with an average of 10.5%. By in vivo assay, L1 and L8 strains showed the ability to control the pathogen virulence probably through the biofilm formation around the bean and soybean plant roots, as confirmed by scanning electron microscope (SEM) analysis. The spectroscopic analysis highlighted the composition of non-volatile compounds: complex carbohydrates (pullulan), degrading enzymes, siderophores and antifungals (aureobasidins). Moreover, the ability of L1 and L8 strains to stimulate the bean and soybean plant roots, stems, and leaves growth was investigated, showing that these yeasts could have an application not only as BCAs but also as plant growth biostimulator.

     

The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

Although acetylated α-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate α-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of α-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of α-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating α-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.     

Pyrano[2,3-e]isoindol-2-ones,new angelicin heteroanalogues

A convenient synthesis of the pyrano[2,3-e]isoindol-2-one ring system, an heteroanalogue of angelicin, is reported. Our synthetic approach consists of the annelation of the pyran ring on the isoindole moiety using 5-dialkylamino- or 5-hydroxymethylene intermediates as building blocks. The photoantiproliferative activity of the new derivatives was studied. Some of them bearing the benzyl group at the 8 position were active with IC₅₀ in the micromolar range. Cell cytotoxicity involves apoptosis, alteration of cell cycle profile and membrane photodamage.     

Archaebacterial lipid membranes as models to study the interaction of 10-N-nonyl acridine orange with phospholipids

The dye 10-N-nonyl acridine orange (NAO) is used to label cardiolipin domains in mitochondria and bacteria. The present work represents the first study on the binding of NAO with archaebacterial lipid membranes. By combining absorption and fluorescence spectroscopy with fluorescence microscopy studies, we investigated the interaction of the dye with (a) authentic standards of archaebacterial cardiolipins, phospholipids and sulfoglycolipids; (b) isolated membranes; (c) living cells of a square-shaped extremely halophilic archaeon. Absorption and fluorescence spectroscopy data indicate that the interaction of NAO with archaebacterial cardiolipin analogues is similar to that occurring with diacidic phospholipids and sulfoglycolipids, suggesting as molecular determinants for NAO binding to archaebacterial lipids the presence of two acidic residues or a combination of acidic and carbohydrate residues. In agreement with absorption spectroscopy data, fluorescence data indicate that NAO fluorescence in archaeal membranes cannot be exclusively attributed to bisphosphatidylglycerol and, therefore, different from mitochondria and bacteria, the dye cannot be used as a cardiolipin specific probe in archaeal microorganisms.     

Volatile emissions and phenolic compound concentrations along a vertical profile of <Emphasis Type="Italic">Populus nigra</Emphasis> leaves exposed to realistic ozone concentrations

Plants are exposed to increasing levels of tropospheric ozone concentrations. This pollutant penetrates in leaves through stomata and quickly reacts inside leaves, thus making plants valuable ozone sinks, but at the same time triggers oxidation processes which lead to leaf injuries. To counteract these negative effects, plants produce an array of antioxidants which react with ozone and reactive molecules which ozone generates in the leaf tissues. In this study, we measured the effect of an ozone concentration which is likely to be attained in many areas of the world in the near future (80 ppb) on leaves of the vertical profile of the widespread agroforestry species Populus nigra. Changes in (1) physiological parameters (photosynthesis and stomatal conductance), (2) ozone uptake, (3) emission of volatile organic compounds (VOCs, i.e. isoprene, methanol and other oxygenated compounds), (4) concentration of antioxidant surface compounds, and (5) concentration of phenolic compounds were assessed. The aim was to assess whether the defensive pathways leading to isoprenoids and phenolics formation were induced when a moderate and chronic increment of ozone is not able to damage photosynthesis. No visual injuries and minor changes in physiology and ozone uptake were observed. The emission of isoprene and oxygenated six-carbon (C6) volatiles were inhibited by ozone, whereas methanol emission was increased, especially in developing leaves. We interpret these results as suggesting an ontogenetic shift in ozone-treated leaves, leading to a slower development and a faster senescence. Most surface and phenolic compounds showed a declining trend in concentration from the youngest to the fully expanded leaves. Ozone reduced the concentrations of chlorogenic acid derivatives at the leaf surface, whereas in total leaf extracts a metabolic shift towards few phenolics with higher antioxidant capacity was observed.     

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.     

Drug-induced expansion and differentiation of V gamma 9V delta 2 T cells in vivo: the role of exogenous IL-2

Human Vgamma9Vdelta2 T cells recognize nonpeptidic Ags generated by the 1-deoxy-d-xylulose 5-phosphate (many eubacteria, algae, plants, and Apicomplexa) and mevalonate (eukaryotes, archaebacteria, and certain eubacteria) pathways of isoprenoid synthesis. The potent Vgamma9Vdelta2 T cell reactivity 1) against certain cancer cells or 2) induced by infectious agents indicates that therapeutic augmentations of Vgamma9Vdelta2 T cell activities may be clinically beneficial. The functional characteristics of Vgamma9Vdelta2 T cells from Macaca fascicularis (cynomolgus monkey) are very similar to those from Homo sapiens. We have found that the i.v. administration of nitrogen-containing bisphosphonate or pyrophosphomonoester drugs into cynomolgus monkeys combined with s.c. low-dose (6 x 10(5) U/animal) IL-2 induces a large pool of CD27+ and CD27- effector/memory T cells in the peripheral blood of treated animals. The administration of these drugs in the absence of IL-2 is substantially less effective, indicating the importance of additional exogenous costimuli. Shortly after the costimulatory IL-2 treatment, only gammadelta (but not alphabeta) T cells expressed the CD69 activation marker, indicating that Vgamma9Vdelta2 T lymphocytes are more responsive to low-dose IL-2 than alphabeta T cells. Up to 100-fold increases in the numbers of peripheral blood Vgamma9Vdelta2 T cells were observed in animals receiving the gammadelta stimulatory drug plus IL-2. Moreover, the expanded Vgamma9Vdelta2 T cells were potent Th1 effectors capable of releasing large amounts of IFN-gamma. These results may be relevant for designing novel (or modifying current) immunotherapeutic trials with nitrogen-containing bisphosphonate or pyrophosphomonoester drugs.     

Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.