Alberto Grossi

Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages

Podosomes, adhesion structures capable of matrix degradation, have been linked with the ability of cells to perform chemotaxis and invade tissues. Wiskott-Aldrich Syndrome protein (WASp), an effector of the RhoGTPase Cdc42 and a Src family kinase substrate, regulates macrophage podosome formation. In this study, we demonstrate that WASp is active in podosomes by using TIRF-FRET microscopy. Pharmacological and RNA interference approaches suggested that continuous WASp activity is required for podosome formation and function. Rescue experiments using point mutations demonstrate an absolute requirement for Cdc42 binding to WASp in podosome formation. Although tyrosine phosphorylation was not absolutely required for podosome formation, phosphorylation did regulate the rate of podosome nucleation and actin filament stability. Importantly, WASp tyrosine phosphorylation does not alter WASp activation, instead phosphorylation appears to be important for the restriction of WASp activity to podosomes. In addition, the matrix-degrading ability of cells requires WASp phosphorylation. Chemotactic responses to CSF-1 were also attenuated in the absence of endogenous WASp, which could not be rescued with either tyrosine mutation. These results suggest a more complex role for tyrosine phosphorylation than simply in the regulation of WASp activity, and suggest a link between podosome dynamics and macrophage migration.

Synergistic cooperation of high pressure and carrot dietary fibre on texture and colour of pork sausages.

In order to investigate the synergistic cooperation between high pressure treatment (HP) and carrot dietary fibre, two formulations of pork sausages containing different percentage of carrot dietary fibre were pressurized at 500 and 600 MPa, for 1 second, 3, 6, and 9 min at 40, 50, and 60 °C. HP treatments significantly increase Youngs Modulus and affect Hencky strain values. We conclude that HP processing and carrot dietary fibre markedly improved emulsion strength resulting in firm sausages. Colour changes were investigated and significant increase in L* value and decrease in a* value were found, indicating that HP, temperature, and dietary fibre can affect physico-chemical properties of the meat matrix altering the intrinsic ability to absorb or reflect light. The sensory evaluation showed that HP treatment synergistically cooperate with carrot dietary fibre improving sensorial attributes like homogeneity, creaminess, fattiness, and firmness as detected by Napping in combination with Ultra-Flash Profile.

Reduction of salt in pork sausages by the addition of carrot fibre or potato starch and high pressure treatment

The combined effect of high pressure processing (HPP) (400, 600 and 800 MPa) and carrot fibre (CF) and potato starch (PS) on low salt (1.2%) pork sausages was investigated and compared with high (1.8%) salt sausages. Sausages had a marked increase in whitening with increasing content of fibre or starch, pressure level, and process temperature. The degree of redness was mainly affected by pressure level and heat treatment. An important finding regarding salt reduction was that the use of starch or fibre had more impact on textural properties than the level of salt since Youngs modulus and strain at fracture were mainly affected by formulation and HPP. Water binding capacity of low salt sausages was improved to the same level as high salt sausages with HPP and addition of CF or PS particularly by the addition of PS which produced sausages with better sensory properties than CF. The sensory analysis showed that this approach is promising for producing low salt sausages.

Evidence for post-mortem m-calpain autolysis in porcine muscle

The objective of the present work was to characterize changes in calpain activity in pork post-mortem. Samples from pig M. longissimus dorsi and M. semimembranosus were collected three days post-mortem from 75 animals and analyzed with casein zymography. The results indicated post-mortem autolysis of m-calpain as two m-calpain bands were observed on the zymogram gel. Use of M. longissimus dorsi from three pigs collected at different times during storage further confirmed post-mortem autolysis of m-calpain. The activity of the autolyzed form of m-calpain was detectable at day 3 and further increased at day 6. The results also showed a decrease in the non-autolyzed m-calpain activity during post-mortem storage. Collectively, these results suggest that m-calpain is active post-mortem in porcine muscles.

Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides

Bovine collagen was pre-treated (boiled or high pressure (HP)-treated) and then hydrolysed by 6 proteases. The degree of hydrolysis (DH) and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates were measured. All enzymes used were able to partly degrade collagen and release ACE-inhibitory peptides. The highest ACE-inhibitory activity was obtained with Alcalase. Pretreatment significantly influenced the DH and ACE-inhibition. For most enzymes, boiling for 5 min resulted in a significantly higher DH and ACE-inhibitory activity. With Alcalase and collagenase, hydrolysis and release of ACE-inhibitory peptides occurred without any pretreatment, but HP-treatment significantly improved the DH and ACE-inhibitory activity. HP did not markedly affect the hydrolysis with the other enzymes. The major peptides obtained with Alcalase were identified; all were released from the triple helix structure of collagen. Many of these peptides had C-terminal sequences similar to known ACE-inhibitory peptides. The present results suggest that collagen-rich food materials are good substrates for the release of potent ACE-inhibitory peptides, when proper pre-treatment and enzymatic treatment is applied.

Water properties and structure of pork sausages as affected by high-pressure processing and addition of carrot fibre.

The effects of high-pressure processing (HPP) and addition of carrot fibre on pork sausages have been studied using NMR T₂ relaxometry and measurements of water-binding capacity (WBC) by centrifugation. Significant effects of temperature (raw, 40, 50, or 60 °C), holding time (1s, 3, 6, or 9 min), and addition of carrot fibre on the distribution and mobility of water were found. However, the effect of carrot fibre could not be explained by structural changes in the sausages when examined by confocal laser scanning microscopy (CLSM). Correlations between T₂ relaxation measurements and WBC determined by centrifugation revealed that T₂ relaxation times were able to explain more than 90% of the variation in WBC for both non-pressure and pressure-treated sausages. However, only 49% of the variation was explained for pressure-treated sausages with carrot fibre, indicating that combining addition of fibre and high pressure treatment causes non-coherent changes in T₂ NMR relaxation times.

The effect of high pressure on the functional properties of pork myofibrillar proteins

Complementary methodologies were used to analyse the pressure-induced modification and functionality of myofibrillar proteins from pork meat pressurised at 200, 400, 600, or 800 MPa (10 min, 5 or 20 °C). Pressure at 400 MPa was found to be the threshold for loss of solubility, and the structural proteins, myosin and actin, lost their native solubility due to aggregation. The results from the extraction of proteins with different reagents targeting the disruption of specific molecular interactions suggested that pressure-induced aggregation was caused mainly by hydrogen bonding during pressurisation and not hydrophobic interactions nor disulphide cross-links. Furthermore, the soluble proteins were exposed to remarkable structural changes already at 200 MPa and lost their native functionality. The modification of the proteins in pressurised meat affected the water binding sites of the myofibrillar proteins and, thereby, the interactions between proteins and water molecules, and distribution between myofibrillar and extra-myofibrillar compartments.

High pressure treatment of brine enhanced pork affects endopeptidase activity, protein solubility, and peptide formation

In order to study the effect of high-pressure (HP) treatment and two different methods of brine addition (important for lysosomal membrane destabilisation) on lysosomal enzymes activity and protein degradation, pork semitendinosus muscle was brine enhanced by injection or tumbling, and HP treated at 600 MPa following storage at 2 °C for up to 8 weeks. In this report a novel protocol for SDS gelatin zymography was established, and an increase of cathepsin B and L activity after HP treatment was shown followed by a decrease during storage. No calpain activity was detected following HP treatment. HP treatment was shown to induce a decrease in protein solubility in both myofibrillar and sarcoplasmic fractions. LC-MS analysis of these fractions showed changes in the peptide pattern during storage. Western blot analysis showed that troponin-T was indeed degraded during storage after HP treatment. The results therefore suggest that HP treatment induced an increase in cathepsin activity, which subsequently affected the myofibrillar protein degradation pattern in pork meat.

Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression: a proteomic approach

Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study, we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress to specific receptors on the cell surface. We showed that mechanical stimuli promote an increase in FAK (focal adhesion kinase) phosphorylation. In order to further shed light in the process of myoblast‐induced differentiation by mechanical stimuli, we performed a proteomic analysis. Thirteen proteins were found to be affected by mechanical stimulation including galectin‐1, annexin III and RhoGDI (Rho guanine‐nucleotide‐dissociation inhibitor). In this study, we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of galectin‐1 in mechanically stimulated myoblasts. A potential involvement of galectin‐1 in myoblast differentiation is presented.

Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation‐competent myoblast stage into fused, multinucleated myotubes. During embryonic development, myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch‐ or load‐induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ influx in myocytes. Evidence of the involvement of Ca2+‐dependent activity in myoblast fusion, cell membrane and cytoskeleton component reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch‐ or load‐induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated that mechanical stimulation via laminin receptors leads to an increase in m‐calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m‐calpain relocates into focal adhesion complexes and is followed by a breakdown of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m‐calpain in the control of muscle precursor cell differentiation and thus strengthen the idea of its implication during the initial events of muscle development.