S100B as a new fecal biomarker of inflammatory bowel diseases.

OBJECTIVE: S100 proteins are demonstrated to exert a protective role in the gastrointestinal tract. In the present study, we investigated whether S100B protein, that is typically expressed by enteroglial cells, is detectable in feces and could be a useful noninvasive indicator of gut chronic inflammation. PATIENTS AND METHODS: This clinical prospective study included n=48 patients suffering Crohn's disease (CD) or ulcerative colitis (UC) and non IBD-controls. The clinical disease activity was evaluated using Harvey-Bradshaw or Mayo Score Index while the diagnosis of IBD was defined based on standard endoscopic and histological criteria. S100B and calprotectin were extracted and analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Unlike calprotectin, S100B was significantly decreased in both CD and UC compared to non IBD-patients. The strongest quantitative alterations of S100B were detected concomitantly with signs of active or quiescent disease, including high/normal expression of fecal calprotectin, mucosal damage/cryptitis, mucin depletion and inflammatory infiltrate, as defined by endoscopic evaluation and histological analysis. At the onset of disease and under no Infliximab-based therapy, the lowest was detected suggesting that S100B in feces could have a potential diagnostic value for IBD. CONCLUSIONS: Testing for S100B and calprotectin could be a useful screening tool to better predict IBD activity.

Identification and purification from the plasma of Type 1 diabetic subjects of a proteolytically active Grp94Evidence that Grp94 is entirely responsible for plasma proteolytic activity.

AIMS/HYPOTHESIS: The overall increase in proteolytic activity in diabetes is known to be associated with the development and progression of vascular complications. Our aim was to investigate in detail the molecular nature of this activity in the plasma of Type 1 diabetic subjects. METHODS: Plasma of both diabetic and control subjects was subjected to various purification procedures (ion exchange and affinity chromatography, HPLC, immunoprecipitation, electrophoresis, immunoblot and mass analyses) to identify the proteins of interest. Biological activities were measured on specific substrates. RESULTS: In diabetic but not normal plasma we identified the presence of two heat shock proteins, Grp94 (Glucose-regulated protein94) and HSP70. The higher-than-normal proteolytic activity of Grp94 was: (i) directed against casein, but not against endogenous plasma proteins; (ii) fully and specifically inhibited only by anti-Grp94 polyclonal antibodies; and (iii) coupled with low-level ATPase activity. In addition, ATP binding to Grp94 was able to modulate proteolytic activity. We found that Grp94 in plasma circulates only as high molecular mass homo- and hetero-complexes, the latter mostly formed with IgG to which Grp94 is also linked by tenacious binding. Proteolytically-active Grp94 was purified by immunoprecipitation, which co-immunoprecipitated alpha(1)antitrypsin. CONCLUSION/INTERPRETATION: Our results show the unexpected extracellular location and characteristic biological function of Grp94 even at a late stage of disease. These findings have physiopathological relevance for predicting activation of both autoimmune and inflammatory processes potentially associated with vascular complications.

The oxidative mechanism of heparin interferes with radical production by glucose and reduces the degree of glycooxidative modifications on human serum albumin.

Among substances which may prove useful in preventing or reducing the progression of glycooxidative modifications of proteins, heparin plays a unique role. To elucidate the mechanism whereby heparin may favourably influence the protein structure during glycation, human serum albumin (HSA) was glycated with both 25 and 50 mM glucose in the absence and presence of 12 microg.mL(-1) low-molecular-mass heparin. Glycation caused: (a) modifications of fluorescence emission and excitation spectra consistent with the covalent attachment of glucose to protein; (b) a significant increase in the esterase activity of HSA on p-nitrophenyl acetate; (c) a reduced susceptibility to tryptic digestion and (d) enhanced formation of high-molecular mass aggregates of HSA. These alterations were accompanied by oxidative reactions, as the EPR spectra showed a clear-cut radical signal, dependent on glucose concentration, further confirmed by measurement of the carbonyl content of HSA, as an indirect proof of oxidative damage. In the presence of heparin all the above alterations, especially at 25 mM glucose, turned out to be antagonized. The effects of heparin were dependent on its specific binding to HSA, which triggered an oxidative mechanism strikingly different from that caused by glucose. In the presence of heparin, only the radical species catalyzed by heparin was detected across all samples of glycated HSA, irrespective of glucose concentration. In addition, at 25 mM glucose, enhancement of the oxidative capacity of heparin was also observed. The results demonstrate that the oxidative mechanism sustained by heparin mediates biological effects that may be beneficial in reducing the extent of glycooxidative damage on HSA.

Role of reducing terminals in unfractionated and low-molecular-mass heparins in causing free radical generation and loss of structure and activity of trypsin.

The role of both the length of saccharide chain and reducing terminals in the heparin molecule in causing oxidative effects on proteins was investigated by employing unfractionated and low-molecular-mass heparins (LMMH), with both intact and reduced reducing terminals on bovine trypsin. The effects of heparin were found to be dependent on both the concentration and time of incubation. Heparins with intact reducing terminals caused significantly higher structural and functional alterations of trypsin compared with heparins with reduced reducing terminals. LMMH was slightly more effective than unfractionated heparin (UNFH) in reducing structural integrity and inhibiting the amidolytic activity of trypsin when used at the same mass, but not molar concentrations. Neither the length of saccharide chains nor the number of intact reducing terminals on the heparin molecule appeared to influence the characteristics of the initial binding of heparin to trypsin, but both these variables crucially affected linkages which in time mediate the inhibition of catalytic activity and the formation of free radicals, ultimately responsible for peptide bond cleavage in trypsin. The results suggest that both a critical number of saccharide units, preferentially lying on shorter chains, and intact reducing terminals in the heparin molecule are involved in setting up the binding which generates radicals and leads to loss of structure and function of the proteinase.

Spin-trapping agent alpha-phenyl N-tert-butylnitrone binds to trypsin and enhances heparin-induced inhibition of amidolytic activity and structural degradation of the enzyme.

The effects of heparin on trypsin have recently been demonstrated to involve inhibition of catalytic activity and degradation of the enzyme by means of an oxidative mechanism. The possibility that alpha-phenyl N-tert-butylnitrone protects heparin-induced radical formation on trypsin was investigated by measuring amidolytic activity and changes in the structure of trypsin in the presence of heparin with and without alpha-phenyl N-tert-butylnitrone. The results show that alpha-phenyl N-tert-butylnitrone does not only prevent, but it even significantly enhances effects of heparin on the enzyme. This is due to the unique property of alpha-phenyl N-tert-butylnitrone, independently of spin-trapping capacity, to modify the trypsin structure by binding irreversibly to the catalytic triad, at sites distinct from those to which heparin binds.

Differential mechanisms for structural and functional alterations of trypsin by heparin, evidence for a specific, radical-generating mechanism at low heparin concentrations.

The oxidative mechanism whereby heparin may interact with various proteins was investigated in detail in this work by addressing the role of doses of heparin on the nature and effects of its binding to bovine trypsin, taken as reference protein. Unfractionated heparin was used at concentrations ranging from 6 to 400 microg/ml with a fixed trypsin concentration (250 microg/ml). At concentrations of up to 60 microg/ml, equivalent to trypsin/heparin molar ratios of between 30 and 3, increasing inhibition of amidolytic activity and radical-dependent peptide bond cleavage of the enzyme was observed, with the appearance in the electrophoretic pattern of new bands of trypsin fragments to which heparin was demonstrated to be bound specifically. Structural modifications were also revealed by increases in fluorescence emission spectra. On the whole, however, the alterations induced by these heparin concentrations only involved a limited number of trypsin molecules. At concentrations from 120 to 400 microg/ml (equivalent trypsin/heparin molar ratios of 1.5-0.46), heparin binding to trypsin appeared to cause more profound and generalized alterations of enzyme structure and function, with dose-dependent quenching of fluorescence emission and almost complete loss of amidolytic activity, although evidence of radical production was lacking. Collectively, the results stress the crucial role of heparin dose on both the nature and effects of its binding to trypsin. The change in heparin effects which reflects distinct underlying molecular mechanisms occurs dramatically at a critical concentration threshold. While a specific, radical-generating mechanism operates at low concentrations, less specific ionic linkages, apparently independent of radical production, best explain the effects of high heparin concentrations.

Albumin contamination of a purified human alpha 1-antitrypsin preparation does not affect either structural conformation or the electrophoretic mobility of the inhibitor.

A partially purified preparation of human alpha 1-antitrypsin (alpha 1-AT) shown to be 60% active as an inhibitor of bovine trypsin, was chosen as starting material to investigate the nature and extent of contamination by human serum albumin (HSA) and to see whether or not such a contamination was responsible for both the reduced inhibitory activity and the slower migratory rate of the proteinase inhibitor in SDS-PAGE. Immunoblotting analysis revealed the presence of HSA in the unprocessed preparation of alpha 1-AT which, both in denaturing and non-denaturing PAGEs, had the same mobility as HSA, appearing as a single band of 65 kDa. By submitting the unprocessed alpha 1-AT preparation to affinity chromatography on an Affi-Gel Blue chromatography column, an apparently highly purified and homogeneous form of alpha 1-AT was obtained, as confirmed by measurement of inhibitory activity and densitometric scanning of SDS-PAGE in non-reducing conditions. However, immunoblotting analysis still revealed the presence of HSA in the most active fractions of the inhibitor eluted from the column, and regardless of purification degree, the molecular mass of the inhibitor was always 65 kDa. Treatment with beta-mercaptoethanol led to separation in SDS-PAGE of HSA as a distinct band of about 10 kDa higher than the alpha 1-AT band, which instead maintained the same mobility as in non-reducing conditions. The results indicate that HSA has not been completely removed from alpha 1-AT, and its presence does not affect the electrophoretic mobility of the inhibitor. The possibility that the structural conformation of the alpha 1-AT, rather than contamination with HSA, was responsible for its abnormal slower migratory rate was therefore tested. For this purpose alpha 1-AT preparations of different degrees of purification were submitted to heat treatment to induce a non-inhibitory conformation such as loop-sheet polymerization. Polymerization was followed both by the appearance in SDS- and non-denaturing PAGEs of high molecular weight bands, which were mostly present in less purified preparations of the inhibitor, and by a decrease in inhibitory activity. A higher degree of polymerization with complete loss of inhibitory activity was observed in the unprocessed alpha 1-AT preparation when dissolved in Na-phosphate buffer at acidic pH, and after dialysis. After heat treatment, the purified alpha 1-AT was shown to run faster in the gel and, in both reducing and non-reducing conditions, the calculated mass of the inhibitor was that expected of about 54 kDa. After reducing treatment, high molecular weight polymers in SDS-PAGEs were reduced, strongly suggesting that disulphide bridges are also involved in the polymerization of alpha 1-AT. Results indicate that the mobility of alpha 1-AT in SDS-PAGE is crucially dependent on its structural conformation which dictates the extent of SDS binding. HSA contaminating the alpha 1-AT preparation does not affect the inhibitor conformation although at a higher degree of contamination and in favourable conditions, it does reduce the inhibitor activity.

Heparin-induced structural modifications and oxidative cleavage of human serum albumin in the absence and presence of glucose--implications for transcapillary leakage of albumin in hyperglycaemia.

Both unfractionated and fractionated, low-molecular-mass heparins were tested on human serum albumin in the absence and presence of glucose at concentrations similar to those frequently found in diabetic hyperglycaemic patients, to ascertain whether heparin and glucose interfered with each other in affecting the conformation of albumin. Reproducible results were obtained with both heparins when used at equal masses, but not when used at equal molar concentrations, suggesting a crucial role of the amount of the saccharide units in determining the observed effects. Spectroscopic studies showed that the binding sites of glucose and heparin on albumin do not overlap and that changes in protein structure depend on complex and mutual interference of glucose and heparin with the protein, although the effects of heparin in modifying the chromophore environment and increasing the ordered structure of the protein also prevailed in the presence of glucose. Heparin binding to albumin rapidly gave rise to oxidative reactions, which were responsible for the increase in the carbonyl content of the protein together with its higher susceptibility to tryptic digestion. Glucose enhanced and prolonged the production of heparin-induced oxidants. Oxidation caused peptide bond cleavage at Lys323 in the primary structure of albumin, yielding two large fragments of 27.5 kDa and 35 kDa which aggregated to form disulphide-linked homodimers visible in SDS/PAGE as two new bands of 54 kDa and 74 kDa, respectively. This was accompanied with a reduction in Val, Glu, and Gly residues, only partially counterbalanced by an increase in Thr and Ser residues. While only a small percentage of albumin molecules underwent fragmentation in the presence of heparin with glucose, albumin turned out to display in an even higher proportion structural modifications consistent with a higher degree of ordered structure. The mechanism(s) underlying this heparin-driven effect and possible physiopathological implications in vivo are discussed.