Aluminum modulation of proteolytic activities

The effect of aluminum ions on the activity of proteolyic activities, mainly serine proteases and calpains, has been examined. Aluminum affects the biological activity of proteolytic activities either through a direct effect on the enzyme molecule or through a deregulation of the control mechanisms acting on them. Binding of the ion, most likely results in molecular rearrangements affecting both the substrates binding site and the site involved in the recognition of macromolecular inhibitors. As whole, the data reported clearly indicate that aluminum significatively affects the intracellular protein turnover, most likely triggering catastrophic events for the cellular life. The physiopathological significance of these effects has been discussed, in particular for neurological disorders (the Alzheimer's disease included) where an imbalance of proteolytic as well as antiproteolytic systems appears a crucial event both for the formation of neuritic plaques and neurofibrillary tangles which are the major hallmarks of the disease. © 2002 Elsevier Science B.V. All rights reserved

Optical coherence tomography features of the repair tissue following RPE tear and their correlation with visual outcomes

To assess the optical coherence tomography (OCT) features of the repair tissue after retinal pigment epithelial (RPE) tear in neovascular age-related macular degeneration. Retrospective, observational study. Medical and imaging records of patients that developed tears after starting anti-VEGF treatment and with at least 12 months of follow-up were reviewed. OCT reflectivity of the RPE-subretinal hyperreflective tissue (SHT) complex was measured at 6, 12 and 18 months (when available). Reflectivity of the adjacent unaffected RPE-Bruch’s membrane was taken as internal reference. Other variables: grade and rip occurrence (early/late); number of intravitreal injections; type of macular neovascularization; sub-macular hemorrhage (SMH) at onset. Forty-nine eyes (age: 76.1 ± 7.0 years; VA: 0.54 ± 0.27 LogMAR) were included. Thirty-eight eyes had OCT signs of healing during the follow-up, with 21 showing SMH at baseline. Final VA positively correlated with the number of injections and negatively correlated with the RPE-SHT reflectivity and the presence of SMH (p < 0.001). Reflectivity of the RPE-SHT complex was positively associated with time and SMH at baseline (p < 0.05). In our study, most eyes showed signs of tissue repair after RPE tear. The reflectivity of repair tissue, the SMH presence and the number of anti-VEGF injections appeared to be major predictors of visual outcomes

Structure-function relationships in bovine thymus 20S proteasome: a fluorimetric study

The structure-function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle. (C) 2001 Elsevier Science B.V. All rights reserved

Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects

Peroxynitrite is a potent reactive oxygen species that is believed to mediate deleterious protein modifications in a wide variety of neurodegenerative disorders. In this study, we have analysed the effects of oxidative damage induced by peroxynitrite on a cysteine-free mutant of dihydrofolate reductase (SE-DHFR), from a functional and a structural point of view. The peroxynitrite-mediated oxidation results in the inhibition, concentration-dependent, of the catalytic activity. This effect is strongly influenced by the HCO(3)(-)/CO(2) buffering system, that we observed to significantly affect the yield of protein oxidation by modulating the peroxynitrite-induced modification of aromatic residues. Because of this effect, in presence of bicarbonate system, we have observed a protection of enzymatic activity of SE-DHFR with regard to peroxynitrite. The thermodynamic stability of the oxidized protein has been studied in comparison with the non-oxidized protein by differential scanning calorimetry. The thermodynamic parameters obtained showed a decrease of stability of SE-DHFR upon oxidation, evaluated in terms of Gibbs free energy of about 1.25 kcal/mol at 25 degrees C, with respect to the non-oxidized protein. Together, these data indicate that structural and functional alterations induced by peroxynitrite may play a direct role in compromising DHFR function in multiple pathological conditions

Wide-field choroidal thickness profile in healthy eyes

The introduction of wide field optical coherence tomography (WF-OCT) has provided newer insights in the imaging of peripheral choroid. We evaluated choroidal thickness (CT) and large choroidal vessel thickness (LCVT) of 20 eyes in horizontal and vertical meridians using WF-OCT. A high-definition line scan through the fovea in both horizontal and vertical meridian was captured in primary and extremes of gaze to obtain scans up to mid-equator. CT and LCVT measurements were done across predefined points in macular area and all quadrants. LCVT was calculated after identifying a large choroidal vessel near choroidoscleral interface. The main outcome measures were differences in CT and LCVT in macular and four quadrants. Mean CT (331.23\u2009\ub1\u200976.34\u2009\ub5) and LCVT (201.46\u2009\ub1\u200954.31\u2009\ub5) in vertical macular segment were significantly more than CT (245.79\u2009\ub1\u200955.38\u2009\ub5; p\u2009=\u20090.0002) and LCVT (150.48\u2009\ub1\u200952.58\u2009\ub5; p\u2009=\u20090.004) in horizontal macular segment. CT at peripheral points in all quadrants was significantly reduced as compared to subfoveal CT (all p values\u2009&lt;\u20090.05) with maximum reduction in inferior quadrant (64.5%). Using linear regression, only quadrant had a significant effect on CT and LCVT (both p\u2009&lt;\u20090.001). CT and LCVT are highest at the macular area with reduction towards the periphery. The contribution of LCVT to CT is less at the fovea compared to other peripheral points

Physico-Chemical Characterization of Keratin from Wool and Chicken Feathers Extracted Using Refined Chemical Methods

In this work, the characteristic structure of keratin extracted from two different kinds of industrial waste, namely sheep wool and chicken feathers, using the sulfitolysis method to allow film deposition, has been investigated. The structural and microscopic properties have been studied by means of scanning electron microscopy (SEM), Raman spectroscopy, atomic force microscopy (AFM), and infrared (IR) spectroscopy. Following this, small-angle X-ray scattering (SAXS) analysis for intermediate filaments has been performed. The results indicate that the assembly character of the fiber can be obtained by using the most suitable extraction method, to respond to hydration, thermal, and redox agents. The amorphous part of the fiber and medium range structure is variously affected by the competition between polar bonds (reversible hydrogen bonds) and disulfide bonds (DB), the covalent irreversible ones, and has been investigated by using fine structural methods such as Raman and SAXS, which have depicted in detail the intermediate filaments of keratin from the two different animal origins. The preservation of the secondary structure of the protein obtained does offer a potential for further application of the waste-obtained keratin in polymer films and, possibly, biocomposites

Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen

The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37 degrees C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the beta-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation inter-mediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour, which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism

Characterization and Biological Activities of In Vitro Digested Olive Pomace Polyphenols Evaluated on Ex Vivo Human Immune Blood Cells

Olive pomace (OP) represents one of the main by-products of olive oil production, which still contains high quantities of health-promoting bioactive compounds. In the present study, three batches of sun-dried OP were characterized for their profile in phenolic compounds (by HPLC-DAD) and in vitro antioxidant properties (ABTS, FRAP and DPPH assays) before (methanolic extracts) and after (aqueous extracts) their simulated in vitro digestion and dialysis. Phenolic profiles, and, accordingly, the antioxidant activities, showed significant differences among the three OP batches, and most compounds showed good bioaccessibility after simulated digestion. Based on these preliminary screenings, the best OP aqueous extract (OP-W) was further characterized for its peptide composition and subdivided into seven fractions (OP-F). The most promising OP-F (characterized for its metabolome) and OP-W samples were then assessed for their potential anti-inflammatory properties in ex vivo human peripheral mononuclear cells (PBMCs) triggered or not with lipopolysaccharide (LPS). The levels of 16 pro-and anti-inflammatory cytokines were measured in PBMC culture media by multiplex ELISA assay, whereas the gene expressions of interleukin-6 (IL-6), IL-10 and TNF-alpha were measured by real time RT-qPCR. Interestingly, OP-W and PO-F samples had a similar effect in reducing the expressions of IL-6 and TNF-alpha, but only OP-W was able to reduce the release of these inflammatory mediators, suggesting that the anti-inflammatory activity of OP-W is different from that of OP-F

Hairy garlic (Allium subhirsutum) from Sicily (Italy): LC-DAD-MSn analysis of secondary metabolites and in vitro biological properties

Allium subhirsutum, known as hairy garlic, is a bulbous plant widespread in the Mediterranean area and locally used as a food and spice. In the present study, the chemical profile of the ethanolic extracts from bulbs (BE) and aerial parts (APE) were analyzed by HPLC-ESI-MSn, and antioxidant properties were evaluated by DPPH, ABTS and TEAC assays. The traditional use in the diet, and the well documented biological activity of Allium species suggest a potential as a new nutraceutical. For this reason, the potential usefulness of this food can be considered in the treatment and prevention of degenerative Alzheimer disease. For this reason, acetylcholinesterase inhibitory property was investigated. Furthermore, due to the observed presence of sulfur-containing and phenolic constituents, the cytotoxicity on tumor cells line was investigated. Results revealed significant AChE inhibitory activity for BE and APE. Both extracts exhibited also moderate antioxidant properties in the in vitro assays. Finally, limited cytotoxic activity was observed towards Human colon carcinoma and adenocarcinoma cell line, with differences between the individual parts tested. HPLC-ESI-MSn analysis showed that hairy garlic is a good source of sulphur compounds, flavonoids and phenylpropanoids derivatives, thus being a valid alternative to the common garlic (A. sativum). This work opens new opportunities for the application of A. subhirsutum as a health-promoting food