Finding new posttranslational modifications in salivary proline-rich proteins.

Proline-rich proteins (PRPs) are the most complex family of salivary peptides with distinct isoforms and PTMs. Up to date, only the serine phosphorylation at positions 8, 17, and 22 have been experimentally observed on acidic PRP (aPRPs), and at position 8 on basic PRP1 and 2. The presence of a glucoronyl group at Ser17 was also noticed on aPRP.

Proteomic characterization of vanA-containing Enterococcus recovered from Seagulls at the Berlengas Natural Reserve, W Portugal.

Background: Enterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of vanA-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data.

Analysis of low abundance membrane-associated proteins from rat pancreatic zymogen granules.

Zymogen granules (ZG) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging, and regulated apical secretion of digestive enzymes. As there is a critical need for further understanding of the key processes in regulated secretion to develop new therapeutic options in medicine, we applied a suborganellar proteomics approach to identify peripheral membrane-associated ZG proteins. We focused on the analysis of a "basic" group (pH range 6.

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle.

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle.

Lifelong physical activity modulation of the skeletal muscle mitochondrial proteome in mice.

The aim of this study was to investigate the influence of lifestyle on the aging alterations in skeletal muscle mitochondrial proteins. Thirty C57BL/6 strain mice (2 months) were randomly divided into three groups (young, Y; old sedentary, S; and old active, A). The S and A mice were individually placed into standard cages and in cages with running wheels for 25 months.

Oxidative modifications in glycated insulin.

At the present, the term "glycoxidation" is recognized as the synergistic interaction between glycation and oxidative processes which, with the help of redox-active metals, consequently leads to the production of deleterious tissue modifications. The association between glycation and oxidation events is considered one of the major factors in the accumulation of non-functional damaged proteins, enhancing the oxidative damage at the cellular level. Because of the central role of insulin in the biology of diabetes, we investigated the site-specific oxidation of native and glycated insulin (mono, di, and tri-glycated forms), through metal-catalyzed oxidation, with a combination of liquid chromatography and mass spectrometry.

Genomic and proteomic evaluation of antibiotic resistance in Salmonella strains.

Using Salmonella strains identical to those present in the gastrointestinal tract of different animals we aim to determine and compare the proteome of two serotypes, Salmonella Typhimurium and Enteritidis recovered from faecal samples of wild boars and wild rabbits, respectively. The presence of genes responsible for antibiotic resistance was detected by PCR. Proteomes of the two distinct serotypes were determined using 2-DE in order to identify proteins associated with antibiotic resistance or virulence.

Identification of salivary proteins at oil-water interfaces stabilized by lysozyme and beta-lactoglobulin.

In this research, we investigated the interaction occurring between oil-in-water emulsion droplets, stabilized by different emulsifiers, i.e. lysozyme and beta-lactoglobulin (beta-lg), and salivary proteins (SPs) with a molecular mass (M(r)) above about 10kDa.

Binding of epigallocatechin-3-gallate to transthyretin modulates its amyloidogenicity.

More than 100 transthyretin (TTR) variants are associated with hereditary amyloidosis. Approaches for TTR amyloidosis that interfere with any step of the cascade of events leading to fibril formation have therapeutic potential. In this study we tested (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, as an inhibitor of TTR amyloid formation.

Proteolysis activation and proteome alterations in murine skeletal muscle submitted to 1 week of hindlimb suspension.

The aim of this study was to investigate the temporal involvement of different proteolytic systems and muscle proteome changes during experimental disuse atrophy (up to 1 week hindlimb suspension, HS) in murine gastrocnemius muscle. The results showed that proteolysis, cytoprotection mechanisms and signs of cellular infiltration occurred very early. After 1 day of HS, signals of lysosomal activation, rather than programmed cell death (apoptosis), seem to trigger protein breakdown in the whole skeletal muscle.

Oxidation of bovine serum albumin: identification of oxidation products and structural modifications.

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long-lived proteins. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system.

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine.

Adrenaline and reactive oxygen species elicit proteome and energetic metabolism modifications in freshly isolated rat cardiomyocytes.

The sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are well recognized hallmarks of several cardiopathologic conditions, like cardiac ischemia/reperfusion (I/R) and heart failure (HF). The present work aimed to investigate the proteomics and energetic metabolism of cardiomyocytes incubated with ADR and/or ROS. To mimic pathologic conditions, freshly isolated calcium-tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (XXO)].

Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry.

Bovine insulin was glycated under hyperglycemic reducing conditions and in nonreducing conditions. Purification through HPLC allowed isolating glycated forms of insulin and a novel triglycated form (6224.5 Da) was purified.

Effects of olive oil polyphenols on erythrocyte oxidative damage.

Many studies have investigated the protective effects of oleuropein and hydroxytyrosol against cell injury, but few have investigated the protective effects of oleuropein aglycones 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA). The present work studied and compared the capacity of these four compounds, found at high concentrations in olive oil, to protect red blood cells (RBCs) from oxidative injury. The in vitro oxidative stress of RBCs was induced by the water-soluble radical initiator 2,2'-azo-bis(2-amidinopropane) dihydrochloride.

Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure.

Recent studies of chitosan have increased the interest in its conversion to chitooligosaccharides (COSs) because these compounds are water-soluble and have potential use in several biomedical applications. Furthermore, such oligomers may be more advantageous than chitosans because of their much higher absorption profiles at the intestinal level, which permit their facilitated access to systemic circulation and potential distribution throughout the entire human body. In that perspective, it is important to clarify their effect on blood further, namely, on human red blood cells (RBCs).

On-plate digestion using a commercial microfraction collector for nano-HPLC matrix-assisted laser desorption/ionization tandem time-of-flight protein analysis.

A new method for on-plate protein digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis is proposed involving an automated one-step sample separation using nanoflow HPLC followed by nanoliter fraction collection and on-plate digestion with trypsin. This procedure uses a commercial automatic nanoliter fraction collection system for on-line spotting of the eluent onto a MALDI target. After protein digestion, the reaction is stopped by the addition of acidified matrix using the same automated system.

Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis.

We studied 82 Portuguese individuals, 57 with hereditary spherocytosis (HS) and 25 unaffected controls. We performed standardized diagnosis tests, including electrophoretic membrane protein analysis to identify and quantify protein deficiencies underlying HS. Membrane bound hemoglobin (MBH) and band 3 profiles were determined as oxidative stress and aging markers.

Peptide profile of human acquired enamel pellicle using MALDI tandem MS.

The present study proposes a strategy for human in vivo acquired enamel pellicle (AEP) peptidome characterisation based on sequential extraction with guanidine and TFA followed by MALDI-TOF/TOF identification. Three different nanoscale analytical approaches were used: samples were subjected to tryptic digestion followed by nano-HPLC and mass spectrometry (MS and MS/MS) analysis. Undigested samples were analysed by LC-MS (both linear and reflector modes) and LC-MS/MS analysis, and samples were subjected to nano-HPLC followed by on-plate digestion and mass spectrometry (MS and MS/MS) analysis.

Evidences of apoptosis during the early phases of soleus muscle atrophy in hindlimb suspended mice.

The purpose of this study was to investigate the occurrence and time-course of apoptosis in soleus skeletal muscle during the first 48 hours of unloading. Fifty Charles River mice were randomly divided into five groups (n=10 each) according to the time of hindlimb suspension (HS). Mice were suspended for 0 (Control), 6 (6HS), 12 (12HS), 24 (24HS), and 48 hours (48HS).

Salivary clinical data and dental caries susceptibility: is there a relationship?

Caries onset and progression is influenced by diverse bacterial, dietary, environmental, socioeconomic and physiological risk factors. The most significant markers include caries experience, the concentrations of mutans-group streptococci and lactobacilli, as well as protective factors, such as the buffering capacity of saliva. The aim of this study was to evaluate and compare caries risk factors.

Cellular patterns of the atrophic response in murine soleus and gastrocnemius muscles submitted to simulated weightlessness.

The purpose of the present study was to investigate the mechanisms of cell death (apoptosis vs. necrosis) during muscle atrophy induced by 1 week of hindlimb suspension. Biochemical and morphological parameters were examined in murine soleus and gastrocnemius muscles.

Subcellular proteomics of mice gastrocnemius and soleus muscles.

A proteomics characterization of mice soleus and gastrocnemius white portion skeletal muscles was performed using nuclear, mitochondrial/membrane, and cytosolic subcellular fractions. The proposed methodology allowed the elimination of the cytoskeleton proteins from the cytosolic fraction and of basic proteins from the nuclear fraction. The subsequent protein separation by two-dimensional gel electrophoresis prior to mass spectrometry analysis allowed the detection of more than 600 spots in each muscle.

Peptidomic analysis of human acquired enamel pellicle.

Human acquired enamel pellicle is the result of a selective interaction of salivary proteins and peptides with the tooth surface. In the present work, the characterization of the peptides as well as the type of interactions established with the enamel surface was performed. Peptides from in vivo bovine enamel implants in the human oral cavity were sequentially extracted using guanidine and trifluoroacetic acid solutions and the fractions obtained were analysed by LC-MS and LC-MS/MS.

Cytotoxicity and cell signalling induced by continuous mild hyperthermia in freshly isolated mouse hepatocytes.

An increasing body of data has been demonstrating that mammalian cells have elaborate networks of molecular signalling in counteracting heat shock and in developing adaptation to oxidative stress to avoid cell death. However, the precise mechanisms linking heat shock, oxidative stress and cell survival/cell death mechanisms are not yet clearly understood. The purpose of this study was thus to study the time course of hyperthermia-induced oxidative stress and cellular signalling through the activation of heat shock factor 1 (HSF1) and heat shock protein 70 (HSP70), using freshly isolated mouse hepatocytes.