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Biomedical subjects

J Mizon

Publications and source records attributed to J Mizon.

At least 37 records · Page 2Linked to original sources
PubMed1994

Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor.

Inter-alpha-trypsin inhibitor (ITI) is a tight complex of three different proteins: bikunin and two heavy chains H1 and H2. In order to demonstrate that the three chains are covalently linked by a chondroitin sulphate chain as previously proposed [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherford, S. and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751], ITI was extensively digested with thermolysin and the glycosaminoglycan-containing fragment was isolated from the digest by ion-exchange chromatography. Its peptide structural determination and mass spectrometry analysis both provide evidence that the different peptide chains constituting ITI are associated by the new cross-link described as the protein-glycosaminoglycan-protein cross-link.

Alpha-Globulins↗
PubMed1994

Preparation and properties of a therapeutic inter-alpha-trypsin inhibitor concentrate from human plasma.

Inter-alpha-trypsin inhibitor (ITI) is a serine protease inhibitor found in human plasma. Its antiprotease activity is due to bikunin which is effective in various types of experimental shock and pancreatitis. Therefore ITI, which releases bikunin by proteolytic cleavage, could be of therapeutic interest. A method for the large-scale isolation of ITI from human plasma is described. ITI was purified from the prothrombin complex concentrate (PCC) by diethylaminoethyl-Sepharose fast-flow chromatography followed by a chromatographic step on immobilized heparin designed to remove C4, factor X and protein C. With this procedure, which was performed under mild conditions, a homogeneous preparation of native ITI was obtained, as demonstrated by electrophoretic and chromatographic analyses. ITI maintained its biological activity, as exhibited by its specific antitryptic activity of 420 +/- 65 IU/g. In order to decrease or eliminate the risk of transmission of viral disease due to lipid-enveloped viruses, the process incorporated a solvent-detergent treatment. Animal studies on the final product revealed no adverse side-effects in terms of toxicity, thrombogenicity or hypotension. This preparation appears suitable for therapeutic evaluation in animal experimental models.

Alpha-Globulins↗
PubMed1993

Inactivation of alpha 1-proteinase inhibitor by alveolar inflammatory cells from smoking patients with or without emphysema.

The aim of this study was to evaluate the ability of alveolar inflammatory cells recovered by bronchoalveolar lavage from the lower respiratory tract of 17 smoking patients with or without emphysema to inactivate alpha 1-proteinase inhibitor (alpha 1-Pl). The presence of emphysema was determined and quantified using CT scan and was evidenced in 8 patients (Group 1), whereas 9 patients exhibited a normal CT scan (Group 2). Patients with emphysema had lower values of FEV1, DLCO, and resting PO2 and higher values of RV/TLC ratio than patients without emphysema. BAL analysis showed a higher percentage of neutrophils and of myeloperoxidase (MPO) in BAL fluid in Group 1 than in Group 2. Alveolar inflammatory cells stimulated or not with phorbol myristate acetate (PMA) were incubated for 45 min with purified alpha 1-Pl, and the results were expressed as a percentage of inactivation of alpha 1-Pl as evaluated by its inhibitory activity against porcine pancreatic elastase or human neutrophil elastase. In Group 2, unstimulated alveolar inflammatory cells inactivated only 3.3 +/- 0.7% alpha 1-Pl and stimulated cells inactivated only 5.4 +/- 1.1% alpha 1-Pl. In marked contrast, in Group 1, a significant loss of the antielastase function of alpha 1-Pl was observed (p < 0.001) when alpha 1-Pl was incubated with unstimulated cells (24.2 +/- 8.9%) or stimulated cells (35 +/- 8.9%) from Group 1. The addition of catalase to the cell suspension was associated with a significant decrease in the inactivation of alpha 1-Pl (from 35 +/- 8.9 to 10.2 +/- 1.2%, Group 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchoalveolar Lavage Fluid↗
PubMed1993

Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter-alpha-trypsin inhibitor (ITI).

Inter-alpha-trypsin inhibitor (ITI) is a complex protein containing two heavy polypeptide chains (H1 and H2) and a light chain, which in the free state is known as bikunin. In vitro cleavage of ITI with different proteases releases bikunin, but does not abolish the antitryptic activity. To study the mechanism of bikunin release, ITI was incubated with human leucocyte elastase (HLE). The resulting ITI fragments were characterized by (i) their electrophoretic and chromatographic behavior. (ii) their immunological reactivity towards antibodies specific for each of the heavy chains H1 and H2, and (iii) their N-terminal sequences. Our results demonstrate that the H2 heavy chain of ITI is particularly sensitive to HLE, and that early cleavage products (M(r)-values 120-150,000) consist of H1 linked to bikunin. A scheme is proposed for the mechanism for ITI degradation.

Alpha-Globulins↗
PubMed1993

[Decrease of fecal beta-galactosidase activity in Crohn disease].

An increased degradation of colonic mucus by bacterial enzymes might participate in the development of mucosal lesions in inflammatory bowel disease. The biodisponibility of drugs used in the treatment of such disease relies upon the metabolic activity of colonic bacterial flora. This activity can be indirectly assessed by measuring fecal enzymatic activities. The aim of this study was to compare fecal beta-galactosidase (beta-gal) activity in controls, in patients suffering from extradigestive inflammatory disease and in patients with Crohn's disease (CD). Three groups were studied including 11 healthy volunteers (6 F, 5 M) mean age 29 years (21-37), 20 patients with rheumatoid arthritis (RA) (17 F, 3 M), mean age 61.5 years, and 34 patients with non operated CD (21 F, 13 M) mean age: 27 years (13-50). The Crohn disease activity index (CDAI) was > 150 in 24 and < 150 in 10. beta-gal activity was measured in fecal extracts by its ability to hydrolyze paranitrophenyl beta-D-galactopyranoside and expressed as units of enzymatic activity/gram of fecal proteins. beta-gal activity was significantly decreased in patients with CD (16 +/- 4.5 U/g) (m +/- sem) as compared with patients with RA (353 +/- 64 U/g) (P < 0.0001) and to controls (263 +/- 40 U/g) (P = 0.002). beta-gal activity was not significantly different in controls and in patients with RA. Patients with active CD had a significantly lower beta-gal activity than patients with quiescent CD (9.5 +/- 3.7 U/g vs 31.4 +/- 11.5 U/g) (P = 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Decreased faecal exoglycosidase activities identify a subset of patients with active Crohn's disease.

1. alpha 1-Proteinase inhibitor (alpha 1-antitrypsin) is excreted in a deglycosylated form (M(r) 38,000) in the faeces of healthy subjects and in patients with quiescent Crohn's disease. By contrast, in most patients with active Crohn's disease, alpha 1-proteinase inhibitor is excreted in a glycosylated form (M(r) 51,000). 2. Faecal extracts containing deglycosylated alpha 1-proteinase inhibitor are able to deglycosylate alpha 1-proteinase inhibitor by an exoglycosidic process. Conversely, we demonstrate that in faecal extracts from patients excreting glycosylated alpha 1-proteinase inhibitor, glycosidase activities, such as N-acetyl-beta-glucosaminidase (EC 3.2.1.30), alpha-mannosidase (EC 3.2.1.24) and particularly beta-galactosidase (EC 3.2.1.23), are strongly decreased. 3. Degradation of glycosidases by proteases could not explain the decreased glycosidase activity in these faecal extracts. 4. Our data suggest that a modification of the bacterial colonic flora (or of its metabolic activity) occurs in most patients with active Crohn's disease and could be responsible for an impaired colonic salvage of carbohydrates.

Acetylglucosaminidase↗
PubMed1992

A simple method for the measurement of different forms of alpha 1 proteinase inhibitor in the faeces of patients with Crohn's disease.

Alpha-1 proteinase inhibitor (alpha 1PI), formerly named alpha 1-antitrypsin, is excreted in the faeces of patients with Crohn's disease as isoforms clearly separated by SDS-PAGE and immunoblot analysis. Relapses in Crohn's disease are generally associated with the appearance in faeces of M(rs) 51,000 and 45,000 glycosylated forms of alpha 1PI, as compared with normal subjects and most of the patients in quiescent phases of their disease who excrete an M(r) 38,000 unglycosylated form of alpha 1PI. We used their differential Concanavalin-A reactivity to design a specific test. The proposed assay is potentially helpful for the follow-up of patients under therapy and for early recognition of attacks of Crohn's disease.

Crohn Disease↗
PubMed1992

The heavy chains of human plasma inter-alpha-trypsin inhibitor: their isolation, their identification by electrophoresis and partial sequencing. Differential reactivity with concanavalin A.

Inter-alpha-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as H1 and H2, respectively. We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H3 inside pre-alpha-trypsin inhibitor (Enghild et al. (1991) J. Biol. Chem. 266, 747-751). Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type N-glycans in both H1 and H2 and that of O-glycans in H2. H1 is eluted from Con-A Sepharose by alpha-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates. The capacity of H2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI.

Alpha-Globulins↗
PubMed1991

A chondroitin-sulfate chain is located on serine-10 of the urinary trypsin inhibitor.

1. The glycopeptide carrying the glycosaminoglycan chain of the urinary trypsin inhibitor (immunologically and structurally related to inter-alpha-trypsin inhibitor) was isolated. 2. The data from amino acid composition and part sequencing of this glycopeptide unambiguously demonstrate that the glycosaminoglycan is covalently linked to serine-10 of the peptide chain of UTI.

Amino Acid Sequence↗
PubMed1991

Deglycosylation of alpha 1-proteinase inhibitor is impaired in the faeces of patients with active inflammatory bowel disease (Crohn's disease).

1. alpha 1-Proteinase inhibitor (alpha 1-antitrypsin) was excreted in the faeces of patients with inflammatory bowel disease in different molecular forms: Mr-51,000 and Mr-45,000 forms were widely found in the stools of patients with active disease, whereas a Mr-38,000 species was frequently recovered from healthy subjects and patients with quiescent disease (Mizon, Becuwe, Balduyck et al. Clin. Chem. 1988; 34, 2268-70). 2. N-Terminal sequencing of the Mr-38,000 form of alpha 1-proteinase inhibitor, after SDS/PAGE and electrotransfer on polyvinyl difluoride membranes, showed that it differed from plasma alpha 1-proteinase inhibitor by the loss of 17 N-terminal amino acids. 3. Carbohydrate analysis of the isolated Mr-38,000 form revealed a total lack of neutral sugars. 4. In contrast, the Mr-51,000 form of alpha 1-proteinase inhibitor is glycosylated and thus could be differentiated by virtue of its reactivity with concanavalin A. The analysis of 25 faecal extracts from patients with Crohn's disease allowed us to confirm that the presence of the glycosylated form of alpha 1-proteinase inhibitor was closely related to the degree of inflammation. 5. From these data, it may be hypothesized that the hydrolytic activity of some glycosidases is greatly reduced in active Crohn's disease.

Adolescent↗
PubMed1991

Oxidative inactivation of alpha 1-proteinase inhibitor by alveolar macrophages from healthy smokers requires the presence of myeloperoxidase.

The aim of this work was to study the ability of human alveolar macrophages (AM) of 10 healthy smokers to inactivate alpha 1-proteinase inhibitor (alpha 1PI). Purified alpha 1PI was incubated for 45 min, with human alveolar macrophages before and after stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. As a positive control, the same experiments were performed in parallel with blood human neutrophils (PMN). Results are expressed as percentage of inactivation of alpha 1PI as evaluated from its inhibitory activity against porcine pancreatic elastase. A strong correlation (r = 0.99) was shown when inhibitory activity of alpha 1PI was evaluated against porcine pancreatic elastase or human neutrophil elastase. Unstimulated AM (1.57 +/- 0.9%) as well as stimulated AM (PMA: 1 +/- 0.4%; zymosan: 3 +/- 0.6%) were unable to inactivate alpha 1PI. Gel electrophoresis of alpha 1PI demonstrated that AM before or after stimulation induced a slight proteolysis of alpha 1PI, whereas both cleaved and complexed alpha 1PI were found when alpha 1PI was incubated with activated PMN. Both unstimulated (22 +/- 2.6%) and activated PMN (PMA: 91.7 +/- 4.7%; zymosan: 90 +/- 5.5%) were responsible for a significant inactivation of alpha 1PI. Catalase, in contrast to superoxide dismutase, was responsible for a near complete protection of alpha 1PI inactivation by PMN. To better determine the role of PMN secretory products, especially myeloperoxidase (MPO), we also investigated the effect of zymosan-activated PMN supernatants or of purified MPO on the alpha 1PI-AM reaction. MPO assay in PMN supernatants demonstrated that activated neutrophils released significant amounts of MPO (16.8 +/- 4.1 U/ml), whereas MPO was undetectable in activated AM supernatants.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

[Inter-alpha-trypsin inhibitor and its plasma and urine derivatives].

Inter-alpha-trypsin inhibitor (ITI) is a serine protease inhibitor present in human plasma at a concentration of 0.4 to 0.5 g/l. Its molecular weight is about 220 kDa. This glycoprotein is a complex of at least three kinds of subunits: two heavy chains and a light one carrying the antiproteasic activity and corresponding to the urinary trypsin inhibitor (UTI). UTI is a proteoglycan containing a chondroitin sulfate side chain; the stability of the ITI complex is clearly dependent upon the integrity of the glycosaminoglycan part of UTI. ITI is mainly synthetized in liver; in case of inflammatory disorders ITI which is very sensitive to numerous proteases, namely elastase and cathepsin G released by stimulated neutrophils, is degraded into lower molecular weight derivatives present in plasma, among which UTI excreted in urine. The physiological role of ITI still remains elusive; ITI may be considered as a precursor of smaller inhibitors. Owing to their small size, they could secondarily diffuse into the extracellular space and reach tissues to protect them from proteolytic destruction. However, their ability to inhibit human granulocytic elastase seems to be too low for their physiological relevance. UTI was recently shown as exhibiting a growth stimulatory activity for human endothelial cells. In addition, UTI exhibits anti-inflammatory effects, the mechanism of which has not yet been elucidated. Owing to its pharmacological properties, UTI might be of therapeutic value in the treatment of inflammatory disorders. At last, the excretion of UTI, which seems to be depending upon neutrophil activation can be considered as a useful test for detecting and monitoring infections.

Biomarkers↗
PubMed1991

[Qualitative study of fecal alpha-1-antitrypsin in patients with inflammatory digestive disease and patients with ileostomy].

Alpha-1-antitrypsin is a glycoprotein which is excreted in feces under three different forms of molecular weight 38, 45 and 51 kDa. The 45 and 38 kDa forms are the result of a partial or total removal of the carbohydrate moiety, respectively. We determined the molecular forms of fecal alpha-1-antitrypsin in 10 controls, 13 patients with protein-losing enteropathy other than inflammatory bowel disease, 70 patients with active (n = 55) (CDAI greater than 150) and inactive (n = 15) (CDAI less than 150) Crohn's disease, 14 patients with active (n = 12) and inactive (n = 2) ulcerative colitis, and 17 patients with ileostomy. Fecal 38 kDa alpha-1-antitrypsin was found in all controls, all patients with protein-losing enteropathy, in 82 percent of patients with inactive inflammatory bowel disease, and in 20 percent of patients with active inflammatory bowel disease. In contrast, the 51 kDa and 45 kDa forms were present in feces of 80 percent of patients with active inflammatory bowel disease, and in only 17 percent of patients with inactive inflammatory bowel disease. Patients with Crohn's disease and the 51 kDa form (n = 39) had significantly higher values of activity index (CDAI) and orosomucoid than patients with Crohn's disease and the 38 kDa form (n = 26) (P less than 0.01). Deglycosylated 38 kDa alpha-1-antitrypsin was never recovered in ileostomy samples. This suggests that deglycosylation of alpha-1-antitrypsin occurred in the colon and is impaired in patients with active inflammatory bowel disease.

Adolescent↗
PubMed1990

Crossed immunoelectrophoresis does not allow accurate determination of inter-alpha-trypsin inhibitor and its derivatives in plasma.

By crossed-immunoelectrophoresis (CIE) of plasma, using anti-inter-alpha-trypsin inhibitor (ITI) immunoglobulins, beside native ITI, related components are visualized as an heterogeneous peak migrating farther than ITI. The area corresponding to this peak is largely increased in case of inflammatory disease. So, quantitative CIE has been previously proposed for discrete evaluation of ITI and its derivatives. We herein present evidence that CIE does not allow a clear separation of ITI from its derivatives: in this system, they are to some extent coprecipitated. Therefore the resulting overestimation of ITI may explain the previously reported absence of inverse relation between relative contents of ITI and derivatives in case of inflammatory disorders. Our data confirm the view that ITI acts as a precursor of smaller immunologically related inhibitors.

Alpha-Globulins↗
PubMed1989

Evidence for the overestimation of molecular masses of proteins after chemical modification and chemical crosslinks on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).

Human trypsin inhibitor (home prepared), lactalbumin, trypsinogen, carbonic anhydrase, and bovine serum albumin were submitted to succinylation and their molecular masses were determined by SDS-PAGE according to the method of Weber and Osborn (1969 J. Biol. Chem. 244, 4406) before and after chemical modification. High estimates of their molecular masses were obtained. The monomer and dimer of arrowhead inhibitor proteinase-B (Chinese vegetable legume) obtained after chemical crosslink(s) were also submitted to SDS-PAGE and their apparent molecular masses were also determined and compared to the native arrowhead inhibitor proteinase-B. Abnormally high estimates of their molecular masses were obtained. Our results agree with those in the literature.

Carbon Radioisotopes↗
PubMed1989

A proteoglycan related to the urinary trypsin inhibitor (UTI) links the two heavy chains of inter-alpha-trypsin inhibitor.

cDNA studies have suggested that inter-alpha-trypsin inhibitor (ITI) is a complex of several different peptide chains; the sequence of the inhibitory part of ITI is in excellent agreement with that of the urinary trypsin inhibitor (UTI). The present report demonstrates that a compound immunologically related to UTI is released by digestion with porcine pancreatic elastase or human leucocyte elastase. Since UTI has been shown to be a proteoglycan, ITI has been treated by chondroitinase. In these conditions, ITI is dissociated and gives rise to two heavy chains (78 and 85 kDa) and one light chain (26 kDa) immunologically related to UTI and which in PAGE moves close to UTIc (produced by chondroitinase treatment of UTI). We suggest that ITI is a non-covalent complex comprising two heavy chains and one light chain immunologically related to UTI and which is also a proteoglycan.

Alpha-Globulins↗