Wheat sprout extract-induced apoptosis in human cancer cells by proteasomes modulation

Natural occurring modulators of proteasome functionality are extensively investigated for their implication in cancer therapy. On the basis of our previous evidences both on proteasomal inhibition by monomeric polyphenols, and on the characterization of wheat sprout hydroalcoholic extract, herein we thoroughly report on a comparative study of the effect of wheat sprout extract on both normal and tumour cells. Treatment of isolated 20S proteasomes with wheat sprout extracts induced a gradual inhibition of all proteasome activities. Next, two wheat sprout extract components were separated: a polyphenol and a protein fraction. Both components exerted an in vitro inhibitory effect on proteasome activity. HeLa tumour cells and FHs 74 Int normal cells were exposed to both fractions, resulting in different rates of proteasome inhibition, with tumour cells showing a significantly higher degree of proteasome impairment and apoptosis induction. Furthermore, a decrease in proteasome activities and in cell survival of the human plasmacytoma RPMI 8226 cell line, upon the same treatments, was observed. Collectively, our results provide additional evidences supporting the possible use of natural extracts as coadjuvants in cancer treatments.     

Gel-based proteomics of liver cancer progression in rat

A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.     

Pleistocene to Recent scleractinian deep-water corals and coral facies in the Eastern Mediterranean

Recent investigations of the Eastern Mediterranean Sea carried out during the GECO cruise with RV Urania provided a substantial number of new cold-water coral (CWC) records, including branching and solitary scleractinian species. These new sites are located along steep escarpments and on topographic highs along the margins of Crete, Karpathos, and Rhodes. The majority of the corals represent fossil occurrences, predominantly Late Pleistocene assemblages. Our research documents that the Eastern Mediterranean Basin has been colonized by CWC at favorable times during the Last Glacial, in particular during the Younger Dryas. Schizocyathus fissilis is reported for the first time for the Mediterranean, while the finding of Ceratotrochus magnaghii represents the first record for the Eastern Mediterranean. Various coral facies occur on the southerly island slopes of Crete, Karpathos, and Rhodes, including hardgrounds and loose skeletal sediments. Hardgrounds occur on steep topographies between ca. 500 and 1,700 m, and can conveniently be subdivided as (1) Neopycnodonte-Desmophyllum framestone, (2) Desmophyllum-Caryophyllia framestone, (3) Madrepora-Lophelia rudstone, (4) Pelagic mudstone and wackestone, and (5) Siliciclastic-carbonate conglomerate and breccia. Unconsolidated skeletal sediments containing corals mainly occur on gentler topographic situations between ca. 140 and 600 m and can be subdivided as: (A) Lophelia-Madrepora rubble, (B) Dendrophyllia rubble, (C) Stenocyathus rubble, (D) Caryophyllia calveri rubble, and (E) fine-grained sediment with octocoral axes. Many of these facies types are also present in the western part of the Mediterranean and have fossil representatives from the Pleistocene to the Recent. Radiocarbon dating (AMS-¹⁴C) reveals Younger Dryas ages between 12.4 and 12.0 ka cal BP for Lophelia pertusa and Madrepora oculata. Desmophyllum dianthus occurs during the Last Glacial Maximum (21.8 ka cal BP) and the Younger Dryas (11.7 ka cal BP), as well as during the Late Holocene and subrecent times (4.4?C0.6 ka cal BP). Caryophyllia sarsiae occurs during the Late Glacial (15.5 ka cal BP), while Caryophyllia calveri occurs during the Early Preboreal (10.8 ka cal BP). The ages for the framework-constructing corals L. pertusa and M. oculata are coherent with their temporal predominance during the Younger Dryas in other parts of the Mediterranean.     

Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci

Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, source-specific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy.     

Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.     

N-Terminal Protein Characterization by Mass Spectrometry after Cyanogen Bromide Cleavage using Combined Microscale Liquid- and Solid-Phase Derivatization

A sample preparation method for protein N-terminal peptide isolation from cyanogen bromide (CNBr) protein digests has been developed. In this strategy, the CNBr cleavage was preceded by protein α- and ε-amine acetylation and carboxyamidomethylation in a one-pot reaction scheme. The peptide mixture was adsorbed on ZipTip_C18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate. In the subsequent steps, the peptides were exposed in situ to hydroxylamine for reversal of potential hydroxyl group acylation, followed by reductive release of the disulfide-linked biotinamido moiety from the derivatives. The selectively thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol-sepharose, leaving the N-terminal fragment in the flow-through fraction. The use of the reversed-phase support as a venue for postcleavage serial modification proved instrumental to ensure throughput and completeness of derivatization. By this sequence of solid-phase reactions, the N-terminal peptide could be recognized uniquely in the MALDI-mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-picomole amounts of model protein. The N-terminal CNBr fragments were retrieved selectively from the affinity support. The sample preparation method provides for robustness and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to gel-separated proteins.     

B-Cell Epitopes in NTS-DBL1α of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

Plasmodium falciparum is the most lethal of the human malaria parasites. The virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1α-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1α of IT4_var60) expressed by parasite FCR3S1.2. The plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1α domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. The other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. In conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1α of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. The data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.     

Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study

The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.     

C-Terminal Protein Characterization by Mass Spectrometry using Combined Micro Scale Liquid and Solid-Phase Derivatization

A sample preparation method for protein C-terminal peptide isolation has been developed. In this strategy, protein carboxylate glycinamidation was preceded by carboxyamidomethylation and optional α- and ϵ-amine acetylation in a one-pot reaction, followed by tryptic digestion of the modified protein. The digest was adsorbed on ZipTip_C18 pipette tips for sequential peptide α- and ϵ-amine acetylation and 1-ethyl-(3-dimethylaminopropyl) carbodiimide-mediated carboxylate condensation with ethylenediamine. Amino group-functionalized peptides were scavenged on N-hydroxysuccinimide-activated agarose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were exchanged directly on the support, eliminating sample transfer between the reaction steps. By this sequence of solid-phase reactions, the C-terminal peptide could be uniquely recognized in mass spectra of unfractionated digests of moderate complexity. The use of the sample preparation method was demonstrated with low-level amounts of a model protein. The C-terminal peptides were selectively retrieved from the affinity support and proved highly suitable for structural characterization by collisionally induced dissociation. The sample preparation method provides for robustness and simplicity of operation using standard equipment readily available in most biological laboratories and is expected to be readily expanded to gel-separated proteins.     

Peptidylglycine alpha-amidating monooxygenase (PAM) in Schwann cells and glia as well as neurons

We raised an antiserum against the synthetic peptide FKETTRSFSNECLGTTR corresponding to the amino terminus of the enzyme peptidylglycine alpha-amidating monooxygenase (PAM). Control experiments were performed to determine the specificity of the antiserum and its suitability for the immunohistochemical identification of PAM-containing cells. An immunoaffinity column made with the antibody coupled to Sepharose permitted the isolation of the active enzyme. Peptide-agarose immunoadsorbant removed the antibodies responsible for the characteristic staining patterns in immunohistochemical experiments. As expected from the widespread distribution of amidated peptides in the nervous system, PAM immunoreactivity was detected in perikarya in a variety of locations, including the pituitary, the hypothalamic periventricular and supraoptic nuclei, neocortex, and sensory ganglia. Punctate immunostained fibers, especially around neuronal perikarya, were observed in regions known to receive amidated peptidergic afferents. In addition, PAM immunoreactivity was observed in some neurons not known to produce amidated peptides (e.g., pyramidal cells of the hippocampus). This result suggests that these neurons also produce an amidated peptide. PAM immunoreactivity was also detected in several unexpected cell types, including ependyma, choroid plexus, oligodendroglia, and Schwann cells. The presence of enzymatically active PAM in Schwann cells was confirmed by measurements of amidating activity in ligated and control sciatic nerve. These results suggest that these non-neuronal cells may produce amidated peptides.