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Biomedical subjects

G Huet

Publications and source records attributed to G Huet.

8 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

A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei.

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.

Adenylyl Cyclases↗
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↗
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↗
PubMedDate not supplied

Serum lipid and lipoprotein abnormalities in human African trypanosomiasis.

We have studied the serum lipoprotein system in human African trypanosomiasis (Trypanosoma brucei gambiense infection). The study was carried out on 74 Congolense patients suffering from sleeping sickness and 34 Congolense control subjects living in the endemic region of Boko Songho. We have determined the serum concentrations of lipids (triglycerides, cholesterol, phospholipids) and apolipoproteins (apolipoprotein A-I and B), and the separation of serum lipoproteins by electrophoresis. For the patients infected with T. b. gambiense, in comparison with control subjects, the results have shown (i) a significant increase in triglyceride concentration and a decrease in cholesterol concentration; (ii) a significant rise in apolipoprotein B concentration and a significant reduction in apolipoprotein A-I concentration; and (iii) an increase in low density lipoproteins and a decrease in high density lipoproteins. We conclude, therefore, that human African trypanosomiasis is associated with marked alterations in the composition and levels of host lipoproteins.

Adolescent↗