A member of the laccase Iiiultigeiie family in Pletirotins o.rtrecrlus has been cloned and sequenced. The gene structurc has been determined by coinparison with the corresponding cDNA, synthcsizcd by rcverse transcription/PCK amplification.The gene encode a laccase isoenzymc of 533 amino acids which has already been purified and characterized IPalmieri, G., Giardinii, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G. (1993) AppI. Micmbiol. Biotcdanol. 39, 632-6363. More than 92 % of thc protein sequencc, including the N and C termini, has been verified by fast-atom-bornbardmcnl inass spectrometry, thus confirming the correspondence betwccn the gene and its protein product.The protein was N-glycosylated at Asn444. Glycan analysis showed thc presence of only a highmannosc structure containing varying numbers of mannose residucs. The presence of 0-linked oligosaccharides as well as other post-translational modification could be rulcd out by the miss analysis.

Probiotics are live microorganisms that confer health benefits on the host. However, in recent years, several concerns on their use have been raised. In particular, industrial processing and storage of probiotic products are still technological challenges as these could severely impair cell viability. On the other hand, safety of live microorganisms should be taken into account, especially when administered to vulnerable people, such as the elderly and immunodeficient individuals. These drawbacks have enhanced the interest toward new products based on non-viable probiotics such as paraprobiotics and postbiotics. In particular, paraprobiotics, defined as “inactivated microbial cells (non-viable) that confer a health benefit to the consumer,” hold the ability to regulate the adaptive and innate immune systems, exhibit anti-inflammatory, antiproliferative and antioxidant properties and exert antagonistic effect against pathogens. Moreover, paraprobiotics can exhibit enhanced safety, assure technological and practical benefits and can also be used in products suitable for people with weak immunity and the elderly. These features offer an important opportunity to prompt the market with novel functional foods or nutraceuticals that are safer and more stable. This review provides an overview of central issues on paraprobiotics and highlights the urgent need for further studies aimed at assessing safety and efficacy of these products and their mechanisms of action in order to support decisions of regulatory authorities. Finally, a definition is proposed that unambiguously distinguishes paraprobiotics from postbiotics.

Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant α-gliadin (r-α-gliadin) showing the most conserved sequence among the fraction of α-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-α-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-α-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120–139) and p23 (aa 220–239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-α-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-γ secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.

Abstract. Preference rankings virtually appear in all field of science (political sciences, behavioral sciences, machine learning, decision making and so on). The well-know social choice problem consists in trying to find a reasonable procedure to use the aggregate preferences expressed by subjects (usually called judges) to reach a collective decision. This problem turns out to be equivalent to the problem of estimating the consensus (central) ranking from data that is known to be a NP-hard Problem. Emond and Mason in 2002 proposed a branch and bound algorithm to calculate the consensus ranking given n rankings expressed on m objects. Depending on the complexity of the problem, there can be multiple solutions and then the consensus ranking may be not unique. We propose a new algorithm to find the consensus ranking that is equivalent to Emond and Mason's algorithm in terms of at least one of the solutions reached, but permits a really remarkable saving

Mucopolysaccharidosis IIIB (MPS IIIB) is an inherited metabolic disease due to deficiency of α-N-Acetylglucosaminidase (NAGLU) enzyme with subsequent storage of undegraded heparan sulfate (HS). The main clinical manifestations of the disease are profound intellectual disability and neurodegeneration. A label-free quantitative proteomic approach was applied to compare the proteome profile of brains from MPS IIIB and control mice to identify altered neuropathological pathways of MPS IIIB. Proteins were identified through a bottom up analysis and 130 were significantly under-represented and 74 over-represented in MPS IIIB mouse brains compared to wild type (WT). Multiple bioinformatic analyses allowed to identify three major clusters of the differentially abundant proteins: proteins involved in cytoskeletal regulation, synaptic vesicle trafficking, and energy metabolism. The proteome profile of NAGLU−/− mouse brain could pave the way for further studies aimed at identifying novel therapeutic targets for the MPS IIIB. Data are available via ProteomeXchange with the identifier PXD017363.

Probiotics are commensal microorganisms that are present in the intestinal tract and in many fermented foods and positively affect human health, promoting digestion and uptake of dietary nutrients, strengthening intestinal barrier function, modulating immune response, and enhancing antagonism toward pathogens. The proteosurfaceome, i.e., the complex set of proteins present on the bacterial surface, is directly involved as leading actor in the dynamic communication between bacteria and host. In the last decade, the biological relevance of surface-exposed proteins prompted research activities exploiting the potentiality of proteomics to define the complex network of proteins that are involved in the molecular mechanisms at the basis of the adaptation to gastrointestinal environment and the probiotic effects. These studies also took advantages of the recent technological improvements in proteomics, mass spectrometry and bioinformatics that triggered the development of ad hoc designed innovative strategies to characterize the bacterial proteosurfaceome. This mini-review is aimed at describing the key role of proteomics in depicting the cell wall protein architecture and the involvement of surface-exposed proteins in the intimate and dynamic molecular dialogue between probiotics and intestinal epithelial and immune cells.

Polyclonal antibodies raised against a synthetic lactosylated peptide were found to be specifically directed against the carbohydrate moiety of the immunogen, recognizing, in addition to the Amadori compound, lactulose and, to a lesser extent, lactose and galactose. These antibodies were effective in detecting lactosylated proteins both on a model system containing β-casein and on milk subjected to different thermal treatments. Widespread lactosylation of the protein components was found in UHT-treated and sterilized milk, whereas it was very limited in raw and pasteurized milk. The index of lactosylation determined by competitive ELISA tests was linearly correlated to the furosine content of milk samples treated within 60 and 100 °C. This ELISA-based procedure is recommended for rapid monitoring of raw milk processing and extension.