Biomedical subjects

P Degand

Publications and source records attributed to P Degand.

At least 19 recordsLinked to original sources

PubMed1992

Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.

Amino Acid Sequence↗

PubMed1992

O-glycosidically linked oligosaccharides from peptidorhamnomannans of Sporothrix schenckii.

beta-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases of Sporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(alpha 1-2)Man-ol, Rha(alpha 1-3)Man(alpha 1-2)Man-ol, Rha(alpha 1-4)GlcA(alpha 1-2)Man(alpha 1-2)Man-ol, and Rha(alpha 1-4)[Rha(alpha 1-2)] GlcA(alpha 1-2)Man(alpha 1-2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.

Amino Acids↗

PubMed1992

Variations in the glycoforms of serum alpha 1-antichymotrypsin in liver diseases and after liver transplantation.

1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the microheterogeneity of alpha 1-antichymotrypsin due to various glycoforms in sera from patients with various liver diseases and after liver transplantation. 2. Studies by isoelectric focusing and immunoblotting and by crossed immunoelectrophoresis without concanavalin A in the first dimension allowed us to show that there is no dramatic variation in electrophoretic heterogeneity of alpha 1-antichymotrypsin in the serum of patients with liver diseases or after liver transplantation when compared with that of normal subjects. Therefore the heterogeneity observed in crossed immunoaffinity electrophoresis is due to various interactions with concanavalin A. 3. The results were expressed as the ratio of concanavalin A non-reactive glycoforms plus concanavalin A weakly reactive glycoforms to concanavalin A reactive glycoforms, called R alpha 1-ACT. R alpha 1-ACT was significantly higher in patients with cirrhosis (n = 53) when compared with normal subjects (n = 30). The median R alpha 1-ACT was 1 (range 0.72-1.25) in normal subjects. It was 1.6 (range 1.18-3.02), 1.45 (range 0.65-4.12) and 2.24 (range 1.03-19) in cirrhosis of Child's grade A, B and C, respectively. There was a dramatic decrease in glycoforms with mostly biantennary glycans in some patients with Child's grade C cirrhosis. Serum levels of alpha 1-antichymotrypsin were lower than normal only in some patients with Child's grade C cirrhosis. 4. Among the patients with acute viral hepatitis studied (n = 17), five were studied longitudinally.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Decrease of testosterone level during an experimental African trypanosomiasis: involvement of a testicular LH receptor desensitization.

To investigate gonadal disorders and changes of the testicular receptors occurring during the sleeping sickness disease (African trypanosomiasis), an experimental model was developed with 10-month-old rats infested by bloodstream forms of two variants of Trypanosoma brucei brucei (AnTat 1.1 A and AnTat 1.8). At the acute phase, three days after inoculation, the animals were sacrificed for estimating the serum levels of LH and testosterone and the number of testicular LH receptors. Considering a possible intervention of the stress during the infestation and to improve our investigations on gonadal imbalance related to trypanosomasis, levels of additional parameters [corticosterone, glucose and transaminases (glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase)] were determined. Stimulation testing with hCG was likewise assessed in infested rats to analyse the testicular testosterone response to gonadotropin. A significant decrease was demonstrated for serum LH and testosterone levels in the infested rats, as well as the loss of: (i) the testicular responsiveness to exogenous gonadotropin; (ii) the number of testicular LH receptors. Moreover, the remaining testicular receptors of infested rats showed an increase in their equilibrium association constant (Ka). Our study suggests that dysfunction of Leydig cells occurring during African trypanosomiasis is in part related to stress induced by the presence of the parasites.

Acute-Phase Reaction↗

PubMed1992

Measurement of elastase and cysteine proteinases in synovial fluid of patients with rheumatoid arthritis, sero-negative spondylarthropathies, and osteoarthritis.

Synovial fluid samples were collected from 45 patients with rheumatoid arthritis, spondylarthropathy, or osteoarthritis, to study their content of elastase (EC 3.4.21.37) and of cysteine proteinases (EC 3.4.22.1, 3.4.22.15). We measured both elastase complexed with alpha 1-proteinase inhibitor and elastase activity toward the substrate L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Cysteine proteinase activities were measured with the substrates N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and Z-Arg-Arg-AMC and the inhibitor E-64 [L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino)-butane]. In all these enzyme assays, higher median values were obtained in inflammatory arthropathies than in osteoarthritis. The concentration of the elastase-alpha 1-proteinase inhibitor complex and of elastase and cysteine proteinase activities were statistically higher in patients with rheumatoid arthritis than in patients with osteoarthritis. The difference in results between patients with spondylarthropathy and patients with osteoarthritis was statistically significant only for the elastase-alpha 1-proteinase inhibitor complex. The median values of the complex and of both enzyme activities were higher in patients with rheumatoid arthritis than in patients with spondylarthropathy; however, the difference was statistically significant only for the cysteine proteinase activity measured with Z-Arg-Arg-AMC substrate. These results suggest that both elastase and cysteine proteinases, which are increased in patients with inflammatory arthritis, are involved in cartilage degradation in these arthropathies.

Adolescent↗

PubMed1992

Human respiratory mucins.

Human respiratory mucins are secreted by goblet cells and mucous glands of the respiratory mucosa. They consist of a broad family of complex glycoproteins with different peptides, or apomucins, corresponding to several genes located on at least three different chromosomes. Glycosylation, the major posttranslational phenomenon, is responsible for about 70-80% of the weight of mucins: it produces an extraordinary diversity of O-glycosidically linked carbohydrate chains which are expressed as several hundreds of different chains in the mucins of a single individual. The variety of mucin peptides and the diversity of carbohydrate chains probably allows many interactions, especially with microorganisms: this may be an essential factor in the defence of the underlying respiratory mucosa.

Animals↗

PubMed1991

Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs.

A lambda gt11 cDNA library constructed from human tracheo-bronchial mucosa was screened with a polyclonal antiserum raised to chemically deglycosylated pronase glycopeptides from human bronchial mucins. Out of 20 positives clones, one partial cDNA clone was isolated and allowed to map a novel human tracheo-bronchial mucin gene. It contains 48 nucleotide tandem repeats quite perfectly identical which encodes a protein containing about 50% of hydroxy amino-acids. This clone hybridized to polydisperse messages produced by human tracheo-bronchial and human colonic mucosae. The gene (proposed name MUC 4) from which cDNA is derived maps to chromosome 3.

Amino Acid Sequence↗

PubMed1991

Structural features of the core proteins of human airway mucins ascertained by cDNA cloning.

Tracheobronchial secretions are one of the most important elements of the mucociliary system that protects the respiratory mucosa. They contain bronchial mucus, which is composed of a group of macromolecules secreted by the goblet cells of the epithelium and the submucosal glands. Bronchial mucins are the most characteristic molecules of this mucus. They form a group of complex, polydispersed O-linked glycoproteins containing sugars, which make up 80% of their weight. The protein core of human airway mucin has been difficult to sequence by traditional technologies because of its high content of serine and threonine residues linked to numerous oligosaccharide chains. We therefore prepared a lambda gt11 cDNA library from one sample of human tracheobronchial mucosa and screened this library with a polyclonal antibody directed against the apopeptides of human bronchial mucins. We obtained 20 positive clones that were sequenced. These sequences were classified into three different types. The use of the nucleotide probes from these clones in Northern blot analysis showed that the RNA messages were extremely polydispersed. At the current time, four of these probes allow us to map human tracheobronchial mucins genes to at least three different chromosomes. These results suggest that the peptide moiety of the human airway mucin is very heterogeneous.

Bronchi↗

PubMed1991

Evidence for different human tracheobronchial mucin peptides deduced from nucleotide cDNA sequences.

Highly glycosylated regions or glycopeptides were obtained by proteolysis of human tracheobronchial mucins. They were chemically deglycosylated and the resulting products were used to raise a rabbit antiserum. This antiserum specifically recognized the superanuclear region of respiratory and colonic goblet cells as areas around and below the nucleus of mucin-secreting cells in tracheobronchial mucous glands. A lambda gt11 cDNA library constructed from human tracheobronchial mucosa was screened with this antiserum. Ten positive clones were obtained from screening half of the library (about 10(6) recombinants). The antibodies were purified by absorption to each positive clone; some purified antibodies were specific for goblet cells and others recognized both goblet and mucous cells, indicating that there is differential cellular expression of mucin peptides. The total or partial amino acid sequences deduced from these cDNA clones could be classified into three groups. The first group contained repetitive sequences of eight amino acid residues, almost perfectly identical, and in different arrangements. The second type exhibited homology at their amino and carboxy-terminal ends. The last group had no distinctive feature except for a high content of hydroxy amino acids typical of mucins. Five different clones could correspond to the carboxy-terminal end of tracheobronchial apomucins. These results indicate that human tracheobronchial apomucins consist of a family of different proteins.

Amino Acid Sequence↗

PubMed1991

Variation in 8-methoxypsoralen profiles during long-term psoralen plus ultraviolet A therapy and correlations between serum 8-methoxypsoralen levels and chromametric parameters.

Three serum 8-methoxypsoralen (8-MOP) kinetics tests were performed on 15 patients undergoing PUVA therapy. The first test was carried out before beginning PUVA treatment, the 2nd one at the 10th session and the 3rd test at the 20th PUVA session. Blood samples were taken every 30 min after the drug was taken and a standard meal ingestion during 3 h to determine the serum 8-MOP peak level and corresponding time. For 14 patients, UVA-induced erythema was measured with a Minolta CR 200 chromameter, 3 (before beginning PUVA treatment, just before 10th session and 48 h later) or 5 times (before beginning PUVA treatment, just before 10th session and 48 h later, just before 20th session and 48 h later). There was a wider distribution of serum 8-MOP peak levels in the 3rd test than in 2nd one and in the 2nd one than in the first one. These facts suggest a heterogeneity in 8-MOP metabolism in the 15 patients: 11 patients showed an earlier serum 8-MOP peak and an earlier appearance of the drug in blood in the 3rd and 2nd test than in the first one. For 9 of these 11 patients, serum 8-MOP peak levels were higher and higher from the first profile to the 3rd one, suggesting an inhibition of 8-MOP metabolism in time. In the remaining 4 patients, the 8-MOP peak time and the appearance of the drug in blood were delayed in the 3rd test compared with the first profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1991

Heterozygous hypobetalipoproteinemia with fasting chylomicronemia.

We describe a disorder in which low-density lipoprotein (LDL)-cholesterol and apolipoprotein B are in low concentration (0.47 mmol/L and 0.28 g/L, respectively) and chylomicrons are still present in plasma after an 18-h fast. The d less than 1.006 fraction was isolated by flotation ultracentrifugation and the apolipoproteins were analyzed by electrophoresis, immunoblotting with anti-apolipoprotein B-100 antiserum, and isoelectric focusing. In the d less than 1.006 fraction of the fasting serum, we found an apolipoprotein B form with the same apparent molecular mass as apolipoprotein B-48 and similar in amount to apolipoprotein B-100 (respective percentages, 46% and 54%). The monosialylated form of the apolipoprotein C-III was severely decreased. After an oral fat load, the repartition of the two species of apolipoprotein B did not change greatly (respective percentages, 60% and 40%), and the concentration of serum triglyceride increased only from 1.20 to 1.65 mmol/L.

Adult↗

PubMed1991

[Thiol-proteolytic activity in rheumatoid polyarthritis. Assay by spectrofluorimetry].

Joint and bone damage in rheumatoid arthritis is thought to be caused primarily by an imbalance between proteolytic proteinases and their specific inhibitors. Matrix destruction can in part result from the activity of lysosomal cystein proteinases such as cathepsin B and L. Cathepsins usually are determined by enzyme immuno-assay. The aim of this study was the direct determination of synovial thiol-proteolytic activity by spectrofluorimetry using a synthetic substrate (Z-Phe-Arg-NMec) and a cystein proteinase specific inhibitor (E64 = L-trans-epoxy-succinyl-leucylamino-(4 guanidino)-butane). 18 rheumatoid synovial fluids were tested compared to 10 osteoarthritic synovial fluids. Thiol-proteolytic activity appeared higher in rheumatoid arthritis compared to osteo-arthrosis (mean value = 1,311 mU/l vs 156 mU/l, p less than 0.01). Synovial thiol-proteolytic activity is well correlated with synovial elastase-alpha1-proteinase-inhibitor complex. The authors found no correlation with synovial polymorphonuclear count (p = 0.38) nor with clinical and biological parameters of disease evolution. The highest values were observed in patient with radiological signs of joint destruction. Synovial thiol-proteolytic activity might represent the potential destructive evolution of rheumatoid arthritis.

Arthritis, Rheumatoid↗

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

Study of proteolytic activities released by incubation of trypanosomes (Trypanosoma brucei brucei) in pH 5.5 and pH 7.0 phosphate/glucose buffers.

Proteolytic activities released by overnight incubation of Antwerpeen Trypanozoon antigenic type (AnTat) 1.1 trypanosomes at 4 degrees C in pH 5.5 and pH 7.0 phosphate/glucose buffers were analyzed in the supernatants obtained after centrifugation of the parasite suspensions. The assays used the fluorogenic substrates N-alpha-benzyloxycarbonyl-L-phenyl-alanyl-L-arginine-7-amido-4- methylcoumarin (Z-Phe-Arg-NMec) and N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine-7- amido-4-methylcoumarin (Z-Arg-Arg-NMec) at two different pHs (6.0 and 8.3). Z-Phe-Arg-NMec hydrolysis was inhibited by 2 microM L-trans-epoxysuccinyllencylamido(4-guanidino)butane (E-64) to a greater extent in the pH 7.0 supernatant than in the pH 5.5 supernatant. Z-Arg-Arg-NMec hydrolysis by the two supernatants was not significantly inhibited by 2 microM E-64. At pH 8.3 this activity was increased more than 2-fold by the addition of dithiothreitol. The hydrolysis activities were analyzed in collected eluates after fractionation of the supernatants by gel permeation high-performance liquid chromatography. Z-Phe-Arg-NMec hydrolytic activity inhibited by 2 microM E-64 was maximal at a retention time of 33 min (approx. Mr 30,000). In addition, a hydrolytic activity against the substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec gave a peak showing a maximum at a retention time of 29 min (approx. Mr 70,000).

Animals↗

PubMed1990

Assignment of human tracheobronchial mucin gene(s) to 11p15 and a tracheobronchial mucin-related sequence to chromosome 13.

Extensive heterogeneity of tracheobronchial mucin RNAs has been described recently. Based on the results of total or partial cDNA sequencing, the mucin cDNAs obtained were classified into three groups. The first group contained 24 bp tandem repeat sequences, the second exhibited homology at their amino- and carboxyl-terminals, and the third group seems to consist of alternative hydrophilic-hydrophobic zones. JER58, JER47 and JER57 probes, representing the first, second, and third tracheobronchial mucin families respectively, were used for chromosome assignment. In human DNAs digested with BamHI, the JER58 probe detected a sequence of 21 kb, the JER47 probe detected a major sequence of 21 kb and a minor sequence of 4 kb, and the JER57 probe detected two sequences of 1.8 kb and 1.3 kb. By somatic hybrid cell analysis, the JER58. JER47, and JER57 major sequences were assigned to chromosome 11 and the JER47 minor sequence to chromosome 13. By in situ hybridization the JER58, JER47 and JER57 probes were assigned to 11p15. Under the experimental conditions used, no specific hybridization to the chromosome 13 region was observed with the JER47 probe. Our results indicate that tracheobronchial mucin gene(s) is/are localized on 11p15. The minor JER47 BamHI sequence localized on chromosome 13 probably corresponds to a tracheal-mucin related sequence. The intestinal mucin gene was also recently localized to the same 11p15 region. Intestinal and tracheobronchial mucins appear different according to their tissue distribution and their cDNA nucleotide sequences. Tracheal mucin probes (JER58, JER47, JER57) and intestinal probes may represent independent genes on 11p15 or else different mRNAs from the same primary transcript produced by differential splicing. Further studies using mucin genomic probes for 11p15 will be required for the elucidation of tracheal and intestinal mucin gene organisation in this region.

Animals↗

PubMed1990

Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat.

Hormonal disorders have been frequently observed in humans and animals infected with tsetse-transmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levels were stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations.

Acute Disease↗

PubMed1990

Variant surface glycoprotein of Trypanosoma brucei brucei AnTat 1.1: influence of the isolation conditions upon the disulfide linked dimer/monomer ratio.

1. Using the variant surface glycoprotein (VSG) isolation procedure described by Baltz et al. ([1976] Ann. Immunol. (Inst. Pasteur) 127 C, 761-774) which involves suspension of the trypanosomes in a pH 5.5 buffer, the Antwerpen trypanozoon antigenic type (AnTat) 1.1 VSG is mainly obtained as a disulfide linked dimeric form with a trace amount of a monomeric form. 2. The use of a parasite suspension buffer at pH 7.0 results in a slight decrease of the VSG dimer/monomer ratio. 3. pH 5.5 and 7.0 supernatants of centrifuged parasite suspensions were submitted to kinetic incubations at different temperatures and pH, and we found conditions involving transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer (shifting the pH 5.5 supernatant to pH 7.0 and incubation at room temperature). 4. This transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer is activated by the addition of 1 mM reduced glutathione, and is inhibited by the addition of 1 mM oxidized glutathione or 0.1 mM N-ethylmaleimide or cadmium acetate.

Animals↗

PubMed1990

Diversity of the peptide moiety of human airway mucins.

A lambda gt11 cDNA library constructed from human tracheal mucosa was screened with rabbit antibodies raised to chemically deglycosylated pronase glycopeptides from bronchial mucins. This library allowed us to select 20 positive clones. The total or partial nucleotide sequences of 14 of them were classified in three different types of organization or families. Antibodies purified from total immuneserum on each positive clone were specific either of goblet cells or goblet cells and mucous cells, showing a differential cellular expression of the peptide axis of mucins. The use of each type of nucleic probe in Northern blot analysis confirmed the existence of a great heterogeneity of the RNAs contributing to a great peptide heterogeneity.

Amino Acid Sequence↗