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ABSTRACT: Probiotics are living microorganisms that confer beneficial effects to human health when supplied in adequate amounts, by promoting digestion and uptake of dietary nutrients, strengthening intestinal barrier function, modulating immune response and enhancing antagonism towards pathogens. The purpose of the present article is to focus on microbial proteomics, pointing out its usefulness in the investigation of molecular mechanisms underlying probiotic effects. It deals, in particular, with molecular strategies responsible for adaptation to the harsh physical-chemical environment of the gastro-intestinal tract, bacterial adhesion to host epithelial cells and intestinal mucosa and probiotic immunomodulatory properties, as analyzed by proteomics in the past few years.
Current opinion in microbiology 04/2012; 15(3):390-6. · 7.87 Impact Factor
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ABSTRACT: This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in
Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga
neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47kDa having an amino acid sequence quite different from other invertases studied up to now. Its
properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer
inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial
activity during a 6h preincubation period. Tni had a K
m
and k
cat
/K
m
values (at 85°C and pH 6.0) of about 14mM and 5.2×108M−1s−1, respectively.
Extremophiles 04/2012; 13(2):345-354. · 2.94 Impact Factor
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ABSTRACT: Marine invertebrates provide a series of natural products with different biological activities. Several of these compounds and their derivatives showed a potent anticancer effect. Tunicates represent an important source of bioactive agents, leading to the isolation of ecteinascidin-743 (ET-743), a compound isolated from the Caribbean sea squirt Ecteinascidia turbinata with a potent cytotoxic activity against a variety of tumours in vitro and in vivo. Current phase II clinical trials against soft tissue sarcomas in Europe and the United States indicate that ET-743 represents a highly promising anticancer agent. Another example is aplidine from the Mediterranean tunicate Aplidium albicans, with a broad spectrum activity against various types of cancers, such as colorectal, lymphoma, thyroid and renal cancers. In the present work, we reported, for the first time, that a partially purified methanolic extract prepared from the ascidian Ciona intestinalis inhibited cell proliferation in human cell lines of different origin, including Caco2, HPB-ALL, U-937 and HL-60 and induced early apoptotic events, such as caspase-3 activation and internucleosomal DNA degradation. We suggest the presence in the Ciona intestinalis extract of bioactive compounds possessing anticancer activity.
Medicinal Chemistry 04/2008; 4(2):106-9. · 1.50 Impact Factor
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ABSTRACT: We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.
FEBS Journal 06/2007; 274(10):2482-95. · 3.79 Impact Factor
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ABSTRACT: A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.
Applied and Environmental Microbiology 03/2006; 72(2):1180-9. · 3.83 Impact Factor
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Maria Teresa Massucci,
Francesco Giansanti,
Giovanna Di Nino,
Manola Turacchio,
Maria Federica Giardi,
Dario Botti,
Rodolfo Ippoliti,
Beatrice De Giulio,
Barbara De Giulio, Rosa Anna Siciliano,
Rosa Siciliano,
Giovanna Donnarumma,
Piera Valenti,
Alessio Bocedi,
Fabio Polticelli,
Paolo Ascenzi,
Giovanni Antonini
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ABSTRACT: Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.
BioMetals 07/2004; 17(3):249-55. · 2.82 Impact Factor
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ABSTRACT: Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.
Protein Science 12/2003; 12(11):2434-42. · 2.80 Impact Factor
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ABSTRACT: Copper and iron are cofactors of many metallo-proteins that accomplish vital functions, such as oxygen and electron transport. Specific metabolic pathways have been selected through evolution, although still not fully elucidated, to confine the dangerous reactivity of their free ionic forms. Inadequate supply of both metals can severely affect basic physiological functions. A differential analysis of the rat intestinal proteome evidenced the following dietary copper- and iron-deficiencies, i.e., significant changes in the levels of proteins belonging to different functional classes (glucose and fatty acid metabolism, molecular chaperones, cytoskeleton plasticity, vitamin transporters). The presented results bring new perspectives to understand the role of copper and iron in the metabolic pathways and provide novel diagnostic tools to characterize the effects of subclinical deficiencies of both metals in unbalanced nutritional disorders.
Journal of Proteome Research 4(5):1781-8. · 5.11 Impact Factor
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Gabriella Pocsfalvi,
Manuela Cuccurullo,
Gitta Schlosser,
Giuseppina Cacace, Rosa Anna Siciliano,
Maria Fiorella Mazzeo,
Salvatore Scacco,
Tiziana Cocco,
Antonio Gnoni,
Antonio Malorni,
Sergio Papa
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ABSTRACT: Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.
Biochimica et Biophysica Acta 1757(9-10):1438-50. · 4.66 Impact Factor
