50 Hz extremely low frequency electromagnetic fields enhance protein carbonyl groups content in cancer cells: effects on proteasomal systems.

Electromagnetic fields are an assessed cause of prolonging free radicals lifespan. This study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein oxidation and on the 20S proteasome functionality, the complex responsible for the degradation of oxidized proteins. Caco 2 cells were exposed, for 24-72 hours, to 1 mT, 50 Hz electromagnetic fields. The treatment induced a time-dependent increase both in cell growth and in protein oxidation, more evident in the presence of TPA, while no changes in cell viability were detected. Exposing the cells to 50 Hz electromagnetic fields caused a global activation of the 20S proteasome catalytic components, particularly evident at 72 hours exposure and in the presence of TPA. The finding that EGCG, a natural antioxidant compound, counteracted the field-related pro-oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals.

Pomegranate fruit components modulate human thrombin.

Pomegranate (Punica granatum) is an important source of polyphenols with assessed antioxidant properties. The aims of this study were: (i) the characterization of the monomeric phenolic variability on each isolated fruit component (endocarp, mesocarp, aril); (ii) the study on the effect of pomegranate fruit components on human thrombin amidolytic activity. Collectively, our data show that pomegranate components contain bioactive metabolites (mainly ellagic acid) and suggest a potential role for the pomegranate extract in the regulation of a number of physio-pathological processes involving thrombin (or thrombin-like proteinase).

Wheat sprout extract induces changes on 20S proteasomes functionality.

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. beta-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins.

Flavonoids inhibit the amidolytic activity of human thrombin.

The effect of a group of natural flavonoids on human thrombin amidolytic activity was investigated using a spectrophotometric inhibition assay while information on the kinetics and thermodynamics was obtained using optical biosensor techniques. All the flavonoids tested acted as reversible inhibitors, and the quercetin-thrombin complex was found to be most stable at pH=7.5. Docking analysis indicated that quercetin's inhibitory behavior could be related to its planar structure and low steric hindrance, and to its ability to form a critical H-bond with thrombin His57.

Aspirin modulates LPS-induced nitric oxide release in rat glial cells.

Nitric oxide and prostaglandins are among the numerous substances released by activated glial cells. The aim of this study was to evaluate the effect of high-level aspirin on iNOS expression in cultured rat glial cells treated with lipopolysaccharide (LPS) as pathological stimulator. Using Western Blotting, we verified that aspirin enhanced LPS-induced iNOS expression and the presence of 15-deoxy-Delta(12,14)-prostaglandin (15d-PGJ2) suppressed this aspirin effect. However, the exposure of LPS-treated glial cells to aspirin resulted in a decrease of NO production. These results suggest that aspirin interferes with the cross-talk of prostaglandins and NO, blocking the endogenous negative control exerted by COX products on iNOS expression. On the other side, aspirin seems to act directly on iNOS reducing its activity, even if it does not completely block NO release by LPS-stimulated glial cells. Then aspirin could maintain homeostatic functions of NO, while it prevents toxic effects, corresponding to high NO concentrations.

Sindrome de zollinger-ellison: análisis de tres casos / Zollinger-ellison syndrome: analysis of three case

Los gastrinomas están entre los tumores neuroendocrinos pancreáticos más frecuentes, y el 60 por ciento son malignos. Al momento del diagnóstico el 50 por ciento ya presentan matástasis aunque se trata de lesiones de bajo grado de malignidad, y el 60 por ciento de estas lesiones se originan en lugares extrapancreáticos. Los tres casos que se analizaron demostraron la utilidad de los niveles de gastrina seriados, la ecoendoscopia y la sintigrafía para receptores de somatostatina marcados con Indio 111 en la detección de lesiones metastásicas

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain.

The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.

Interaction of Hsp90 with 20S proteasome: thermodynamic and kinetic characterization.

The proteasome and heat shock proteins have been found in the centrosome. The evidence of their copurification reported by several studies suggests that they form stable complex. In addition, Hsp90 is involved in the loading of proteasome-generated antigenic peptides to the class I major histocompatibility complex. In this article, we report a detailed thermodynamic and kinetic characterization of the Hsp90-20S proteasome interaction, using a surface plasmon resonance technique. The modulation exerted by protons in solution has been investigated, and the results have been discussed, taking into account structural motifs characterizing the binding interface between the two macromolecules.

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.

Different functional modulation by heterotropic ligands (2,3-diphosphoglycerate and chlorides) of the two haemoglobins from fallow-deer (Dama dama).

Two haemoglobin components have been identified and purified from fallow-deer (Dama dama) erythrocytes. They are present in similar amounts and the two tetrameric molecules share the same alpha chain, while two different beta chains are detected in the two components. The beta chains differ by 14 residues, even though they both have 145 amino-acid residues, which account for a molecular mass of 16,023 and 16,064 Da, respectively, while alpha chain has 141 residues, yielding a molecular mass of 15,142 Da. Compared with human Hb, the N-terminal region of both beta chains shows deletion of Val beta 1 and the replacement of His beta 2 by a methionyl residue, a modification which is common to most ruminant haemoglobins. Although both isolated components show a low intrinsic affinity for oxygen, meaningful differences between the two haemoglobins have been found with respect to the effect of heterotropic effectors, such as 2,3-diphosphoglycerate and chloride ions. In view of the high sequence homology between the two components, the different effect of heterotropic ligands has been tentatively correlated to possible localized structural variations between beta chains of the two haemoglobin components.

Isolation and characterization of bovine thymus multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC or proteasome) from bovine thymus was isolated and purified to homogeneity applying a protocol utilizing ion exchange and gel permeation chromatography as major purification tools. The purified complex shows molecular properties that are common for proteasomal molecules (high molecular mass, multisubunit organization, and multiple proteolytic activities) even though a peculiar subunit composition and the presence of specific regulatory mechanisms affecting the assembled proteolytic activities suggest a specialized function for this complex. Thymus proteasome is characterized by the presence of LMP2, LMP7, and LMP10 (MECL1) subunits, which replace the X, Y, and Z subunits. Since a similar complex was previously isolated in bovine spleen, it appears that the proteasomal population containing the LMP subunits is characteristic for organs involved in immune response. Both the thymus and spleen proteasomes are characterized by a marked efficiency in cleaving peptide bonds after branched-chain and aromatic amino acids, indicating that this proteasomal population is most likely involved in intracellular processing of class I antigenic peptides and is an example of an "in vivo" functioning immunoproteasome. However, in spite of several similarities, the complexes isolated from the two lymphoid organs do not show superimposable functional properties, which suggests the presence of organ-specific regulatory mechanisms affecting each of the proteolytic components assembled in the complex.

Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study.

The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.

Peroxynitrite-mediated oxidation of fibrinogen inhibits clot formation.

The clotting activity of human fibrinogen was fully inhibited in vitro by peroxynitrite. The decrease of activity followed an exponential function and the concentration of peroxynitrite needed to inhibit 50% of fibrinogen clotting was 22 microM at 25 degrees C. The oxidative modification(s) induced by the peroxynitrite system (i.e. ONOO-, ONOOH and ONOOH*) appeared specifically to affect fibrin clot formation (through the inhibition of fibrinogen polymerization) since the interaction of peroxynitrite-modified fibrinogen with thrombin appeared to be unaffected. The addition of NaHCO3 decreased the peroxynitrite effect on fibrinogen clotting, suggesting that the reactive species formed by the reaction of CO2 with peroxynitrite are less efficient oxidants of peroxynitrite itself. Similar effects were observed after addition of bilirubin, which also exerted a significant protection against peroxynitrite-mediated modification of fibrinogen.

Isolation and N-terminal sequence of multiple forms of granulins in human urine.

Three molecular forms of granulins (also known as epithelins) were isolated, for the first time, in human urine. Their N-terminal sequences, which have also been determined, are identical to those of granulins A and B, previously isolated from human leukocytes, and of granulin F, never isolated before but whose primary structure is known on the basis of the cDNA sequence. The urinary molecules, which show a molecular weight of about 6.5 kDa, are most likely produced by a posttranslational proteolytic processing occurring at the level of the kidney, which appears to be the organ richest in granulin precursor mRNA. The molecular events underlying the precursor processing are unknown, even though the involvement of the protease kallikrein, an enzyme thought to be responsible for the processing of several polypeptidic growth factor precursors, could be hypothesized. Granulins, however, do not show antikallikrein activity. The presence in human urine of isoform F, previously not identified from other human sources, seems to support the hypothesis that mature forms of granulins are generated by an organ-specific precursor processing, on the basis of particular physiological requirements, and to suggest also that this isoform may play "in vivo" an important and specific role in the epithelial cells of the human kidney.

Acid-stable serine proteinase inhibitors in the urine of Alzheimer disease subjects.

A comparative study of the levels of acid-stable proteinase inhibitors (kallikrein and trypsin inhibitors) in the urine of healthy and Alzheimer subjects, of both sexes, has been performed. A preliminary characterization of the purified inhibitors indicates that the urinary antitryptic activity is accounted for by the presence of the well known Urinary Trypsin Inhibitor (UTI) while an apparently new molecule appears to be responsible for the antikallikrein activity. The urinary levels of kallikrein inhibitors are very similar in healthy and sick subjects while the levels of trypsin inhibitors appear significatively increased in Alzheimer subjects of both sexes. The data presented here support the hypothesis that unpaired proteolytic processes could be involved in the pathogenesis of Alzheimer's disease and suggest that the levels of urinary acid-stable inhibitors may prove to be useful markers of the disease.

Heterotropic modulation of the protease-inhibitor-recognition process. Cations effect the binding properties of alpha-chymotrypsin.

The effect of calcium and lanthanide ions (e.g. terbium) on the binding properties of alpha-chymotrypsin has been studied focussing on the modulation exerted by cations on the interaction of the enzyme with the bovine pancreatic trypsin inhibitor (BPTI or Kunitz inhibitor). The results obtained indicate that the cation binding induces conformational transitions, on the enzyme molecule, which destabilize the enzyme-inhibitor complex formation affecting the interaction of the inhibitor with the secondary specificity site of the proteolytic enzyme. This negative heterotropic effect can be observed only with macromolecular inhibitors (or substrates), displaying an extended interacting surface with the enzyme, and it seems linked to the number of positive charges carried by the cations. Thus, owing to the large conformational changes induced by the binding of trivalent cations, the divalent ones (e.g. calcium) appear to be more suitable for a fine regulation of the enzyme activity. The mutual correlation between inhibitors binding to (and calcium release by) the proteolytic enzymes (and vice versa) could assume an important physiological significance linking parameters, such as calcium concentration and the activity levels of proteolytic enzymes, which are both of great importance for the cell life.

Proteinase inhibitors of the Kunitz family in fallow deer organs: a comparative study.

Protein proteinase inhibitors belonging to the Kunitz family have been isolated and characterized in several fallow deer organs. In all the organs studied we found the basic pancreatic trypsin inhibitor (BPTI) while its isoforms, previously isolated and characterized in organs of other ruminant species (bovids and ovids), were absent. In the kidney, in addition to BPTI, active fragments of the inter-alpha-trypsin inhibitor were also isolated. The distribution of Kunitz-type inhibitors in different species of ruminants is compared and discussed on the basis of the expression of their encoding genes.

High-performance liquid chromatographic separation of aprotinin-like inhibitors and their determination in very small amounts.

A reversed-phase high-performance liquid chromatographic method for the determination and quantitative recovery of fully active aprotinin (the basic pancreatic trypsin inhibitor or Kunitz inhibitor) and aprotinin-like inhibitors in amounts down to 0.5 micrograms is reported. The method, which allows separation of aprotinin isoinhibitors characterized by small differences in the primary structure with respect to aprotinin itself, appears to be suitable for the quantitation and identification of aprotinin-like inhibitors in human biological fluids, in which they appear to be present at very low levels.