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Biomedical subjects

D Gauthier

Publications and source records attributed to D Gauthier.

At least 19 recordsLinked to original sources
PubMed1999

Distribution of gymnostoma spp. microsymbiotic frankia strains in new caledonia is related to soil type and to host-plant species

The diversity of the Frankia strains that are naturally in symbiosis with plants belonging to the Gymnostoma genus in New Caledonia was investigated. A direct molecular characterization of DNA extracted from nodules was performed, followed by characterization by restriction fragment length polymorphism (RFLP) of the ribosomal rrs-rrl (16S-23S) intergenic spacer (IGS) polymerase chain reaction (PCR)-amplified region. Seventeen different patterns were identified among the 358 microsymbiotic strains studied in the eight species of host plant present in New Caledonia. This genotypical approach permitted us to show that a large diversity existed among the patterns and that these did not exhibit a strict specificity to any host-plant species comparable with that previously found in the Casuarina and Allocasuarina symbioses in Australia. Despite this lack of specificity, a correspondence analysis nevertheless showed that the distribution of these patterns was related to soil type and to host-plant species. Furthermore, several Frankia strains were exclusively associated with the ultramafic soils.

Journal Article↗
PubMed1999

Pulsed-field gel electrophoresis is more efficient than ribotyping and random amplified polymorphic DNA analysis in discrimination of Pasteurella haemolytica strains.

One hundred thirty-three strains of Pasteurella haemolytica of both biotypes (90 and 43 strains of biotypes A and T, respectively) and almost all the serotypes were subjected to ribotyping, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE) analysis for epidemiological purposes. A total of 15 patterns recorded as ribotypes HA to HO were found for the P. haemolytica biotype A strains, with ribotypes HA, HC, and HD being encountered most often (66 strains [74%]); and 20 ribotypes, designated HA' to HT', that were clearly distinct from those observed for biotype A strains were observed for strains of biotype T. RAPD analysis generated a total of 44 (designated Rp1 to Rp44) and 15 (designated Rp1' to Rp 15') unique RAPD patterns for biogroup A and biogroup T, respectively. Analysis of the data indicated that a given combined ribotype-RAPD pattern could be observed for biotype A strains of different serotypes, whatever the zoological or geographic origin, whereas this was not the case for biotype T strains. PFGE appeared to be more efficient in strain discrimination since selected strains from various zoological or geographical origins harboring the same ribotype-RAPD group were further separated into unique entities.

Animals↗
PubMed1998

Proprotein convertase PC1/3-related peptides are potent slow tight-binding inhibitors of murine PC1/3 and Hfurin.

The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like endoprotease family and is synthesized as a preproenzyme. To investigate the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculovirus-infected insect cells, rendered soluble with 6 M guanidine HCl and 20 mM dithiothreitol, and purified by gel filtration and metal-binding affinity chromatography. Two NH2-terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, purified, and chemically characterized. Progress curve kinetic analysis with enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin demonstrated that both fragments were potent slow tight-binding inhibitors of either enzyme with Ki in the low nanomolar range. Additional cleavages at Trp residues yielded fragment9-71, which no longer represents a potent inhibitor. Upon incubation at pH 5.5 in the presence of excess 71-kDa murine PC1/3, NH2-terminal fragment1-98 is cleaved at two sites, as revealed through Western blotting using NH2-terminal-directed PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/31-98 peptide, albeit at a much lesser extent with a micromolar Ki and in a strictly competitive manner. These results suggest that the proregion of PC1/3 is an important feature in regulating its activity.

Amino Acid Sequence↗
PubMed1998

In vitro cleavage of internally quenched fluorogenic human proparathyroid hormone and proparathyroid-related peptide substrates by furin. Generation of a potent inhibitor.

The cleavage of parathyroid hormone (PTH) from its precursor proparathyroid hormone (pro-PTH) is accomplished efficiently by the proprotein convertase furin (Hendy, G. N., Bennett, H. P. J., Gibbs, B. F., Lazure, C., Day, R., and Seidah, N. G. (1995) J. Biol. Chem. 270, 9517-9525). We also showed that a synthetic peptide comprising the -6 to +7 sequence of human pro-PTH is appropriately cleaved by purified furin in vitro. The human pro-PTH processing site Lys-Ser-Val-Lys-Lys-Arg differs from the consensus furin site Arg-Xaa-(Lys/Arg)-Arg that is represented by Arg-Arg-Leu-Lys-Arg in the cleavage site of pro-PTH-related peptide (pro-PTHrP). An earlier study demonstrated that an internally quenched fluorogenic substrate bearing an O-aminobenzoyl fluorescent donor at the NH2 terminus and an acceptor 3-nitrotyrosine near the COOH terminus was appropriately cleaved by the convertases furin and PC1 (Jean, F., Basak, A., DiMaio, J., Seidah, N. G., and Lazure, C. (1995) Biochem. J. 307, 689-695). Here, we have synthesized a series of internally quenched fluorogenic substrates based upon the pro-PTH and pro-PTHrP sequences to determine which residues are important for furin cleavage. Purified recombinant furin and PC1 cleaved the human pro-PTH internally quenched substrate at the appropriate site in an identical manner to that observed with the nonfluorescent peptide. Several substitutions in the P6-P3 sequence were well tolerated; however, replacement of the Lys at the P6 position with Gly and replacement of the P3 Lys by an acidic residue led to markedly compromised cleavage by furin. Furin activity was very sensitive to substitution in P' positions. Replacement of Ser at P1' with Gly and Val at P2' with Ala generated substrates that were less well cleaved. Substitution at the P1' position of Val for Ser in conjunction with Ala for Val at P2', as well as a single substitution of Lys for Val at P2', generated specific inhibitors of furin cleavage. The findings of this study open the way to the rational design of inhibitors of furin with therapeutic potential.

Amino Acid Sequence↗
PubMed1998

Molecular characterization, enzymatic analysis, and purification of murine proprotein convertase-1/3 (PC1/PC3) secreted from recombinant baculovirus-infected insect cells.

A cDNA coding for the murine proprotein convertase-1 (mPC1 also known as mPC3 or mSPC3) was inserted into the Autographa californica nuclear polyhedrosis virus. Following infection of Spodoptera frugiperda cells, the recombinant N-glycosylated protein is secreted into the cell culture medium from which it can be purified to homogeneity as a fully enzymatically active enzyme. Two major secreted molecular forms of mPC1 with apparent molecular weights of 85 and 71 kDa, respectively, and a minor one of 75 kDa are immunodetected in the medium. Automated NH2-terminal sequencing reveals that all three forms result from processing at the predicted zymogen activation site whereas both the 75- and the 71-kDa forms are truncated at their COOH-terminus. Labeling by an active-site titrant demonstrates that the 85-kDa form is optimally labeled at near neutral pH whereas the COOH-truncated forms are optimally labeled at acidic pH. Additionally it is shown that the 85-kDa mPC1 is transformed into the COOH-truncated forms following in vitro incubation at acidic pH levels and in presence of calcium. Concomitantly, the transformation from 85 to 71 kDa is accompanied by a 10- to 40-fold increase in enzymatic activity upon assaying at pH 6.0. The 71-kDa form can be recovered after purification at a level of 1 to 1.5 mg per liter of cell culture medium and is enzymatically stable only in the pH range from 5.0 to 6.5. Cells treated with tunicamycin show a drastically reduced secretion of the convertase in the medium but are not affected by swainsonine and deoxymannojirimycin. Finally, the 85-kDa secreted mPC1 is shown to be sulfated.

1-Deoxynojirimycin↗
PubMed1997

The nodular microsymbionts of Gymnostoma spp. are Elaeagnus-infective Frankia strains.

The phylogenetic relationships of Frankia strains infective on Gymnostoma with other Frankia strains was analyzed. Partial sequencing of the 16S rDNA and use of specific primers showed that the Frankia strains present in Gymnostoma are phylogenetically close to Elaeagnus-infective strains. This finding was confirmed by using the sequences of the hypervariable nifDK intergenic spacer. The strains present in Gymnostoma nodules were close to one another. Clustered with Elaeagnus-infective strains, and distantly related to Casuarina and Alnus-infective strains. Morphological observations of strains and cross-inoculation trials showed that Gymnostoma-infective strains are indistinguishable from Elaeagnus-infective strains. Results of both phenotypic and genotypic approaches indicate that Gymnostoma-infective strains are Elaeagnus infective and not Casuarina infective.

Bacteria↗
PubMed1995

Live cell image segmentation.

A major requirement of an automated, real-time, computer vision-based cell tracking system is an efficient method for segmenting cell images. The usual segmentation algorithms proposed in the literature exhibit weak performance on live unstained cell images, which can be characterized as being of low contrast, intensity-variant, and unevenly illuminated. We propose a two-stage segmentation strategy which involves: 1) extracting an approximate region containing the cell and part of the background near the cell, and 2) segmenting the cell from the background within this region. The approach effectively reduces the influence of peripheral background intensities and texture on the extraction of a cell region. The experimental results show that this approach for segmenting cell images is both fast and robust.

Algorithms↗
PubMed1994

Ultrasonographic detection of abnormal fetal growth with the gestational age-independent, transverse cerebellar diameter/abdominal circumference ratio.

OBJECTIVES: We prospectively evaluated the accuracy of a gestational age-independent method of detecting abnormal growth, the transverse cerebellar diameter/abdominal circumference ratio, and compared this with standard ultrasonographic methods of growth assessment. STUDY DESIGN: We prospectively studied 825 low-risk obstetric patients and 250 patients having risk factors for fetal macrosomia (n = 92) or growth retardation (n = 158). Measured fetal parameters included the biparietal diameter, head circumference, transverse cerebellar diameter, abdominal circumference, and femur length. The estimated fetal weight, head circumference/abdominal circumference, cerebellar diameter/abdominal circumference, and femur length/abdominal circumference ratios were calculated. Reference curves for these parameters were created from a cross-sectional analysis of the low-risk group. Univariate analysis was used to determine the sensitivity, specificity, predictive values, and odds ratios of each individual parameter in identifying a small- or large-for-gestational-age infant. A multivariate logistic regression model with a variable selection procedure was then used to determine whether significance remained when we controlled for other parameters. RESULTS: Within the low-risk group, the transverse cerebellar/abdominal circumference ratio was gestational age independent between 14 and 42 weeks with a mean of 13.68 +/- 0.96. A value exceeding 2 SD of the mean was significantly associated with birth or a small-for-gestational-age infant, being abnormal in 98% and 71% of asymmetrically and symmetrically growth-retarded infants, respectively. Significance was maintained in the multivariate regression model. The ratio was not helpful in detecting the large-for-gestational-age infant. CONCLUSION: The fetal transverse cerebellar diameter/abdominal circumference ratio is an accurate, gestational age-independent method of identifying the small-for-gestational-age but not the large-for-gestational-age infant.

Abdomen↗
PubMed1993

Detection of fetal cocaine exposure by analysis of amniotic fluid.

OBJECTIVE: To determine whether analysis of amniotic fluid (AF) using solid-phase extraction and high-performance liquid chromatography is useful in detecting fetal exposure to cocaine and its metabolites. METHODS: Amniotic fluid and urine samples were obtained from 23 subjects with documented cocaine abuse during pregnancy and five subjects without a history of cocaine abuse. Urine samples were also collected from all newborns. Urine samples were screened using conventional immunoassay techniques and confirmed with thin-layer chromatography. Using a modified solid-phase extraction procedure, we removed cocaine and its metabolites from all AF samples and from the newborn urine samples in which the initial screening was positive. Subsequently, we analyzed the extracts with high-performance liquid chromatography. RESULTS: Cocaine or benzoylecgonine was detected in 74% of AF samples taken from the 23 known cocaine abusers. In these subjects, conventional maternal and neonatal urine toxicology screens were positive in 61 and 35%, respectively. Concentrations in AF ranged from 400 to greater than 5000 ng/mL for benzoylecgonine and from trace to 250 ng/mL for cocaine. Mean benzoylecgonine recovery from AF was significantly greater than from newborn urine (1800 versus 280 ng/mL, respectively; P < .0001). CONCLUSION: Analysis of AF appears to be useful in detecting gestational cocaine exposure.

Amniotic Fluid↗
PubMed1992

Isolation and characterization of hamster brain polyribosome-cytomatrix complexes.

We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins. This method is based on gentle dispersion of brain tissue and low speed centrifugation. This fraction is enriched in typical cytoskeletal proteins as glial fibrillary protein, neurofilament proteins and actin. Messenger RNA did not seem to be involved in the polyribosome association to the cytomatrix as shown by the effect of exposure to micrococcal nuclease. On the other hand, in vivo disruption of protein synthesis by acute experimental phenylketonuria, hypothermia or heat-shock did not cause the release of ribosomes from the cytomatrix.

Actins↗
PubMed1991

In vitro translation in a hamster brain cell-free system.

We have developed a cell-free translation system derived from hamster brain tissue. The optimal incorporation conditions were 160 mM K+, 2 mM Mg2+ and microM spermine. The absence of this latter compound could be compensated only by doubling (to 4 mM) Mg2+ concentration. This system was inhibited by cycloheximide and emetine (elongation inhibitors) as well as NaF and aurintricarboxilic acid (ATA) (initiation inhibitors). The nature of the ATA inhibiting effect was studied by kinetic comparison with cycloheximide and polyribosome sedimentation pattern.

Adenosine Triphosphate↗
PubMed1991

Gastro-intestinal parasitism of cattle in native pasture grazing system in Guadeloupe (French West Indies).

Gastro-intestinal parasitism of creole cows and calves, Limousin x creole cross calves and Brahman x creole cross calves was recorded during seasons of calving in a native grazing system in Guadeloupe, a wet tropical area. Calvings were pooled during the dry season for two herds and during the rainy season for two other herds. For each calving season, calves of one herd were drenched monthly with anthelmintic. A marked periparturient rise in egg excretion was observed in cows. Coccidial infection was always present in calves, but no clinical signs were observed. The main parasites during the first 2 months of life were Strongyloides papillosus and Toxocara vitulorum. Then, Haemonchus placei, Trichostrongylus spp. and Cooperia spp. were the dominant species encountered. Brahman cross calves were the most heavily infested animals. There was no clear relationship between third stage larvae (L3) population size on pasture and worm burdens in calves, except during the second month of life. Despite medium levels of infestation, parasitism inhibited the growth of creole calves: -10.5 kg of bodyweight at weaning (-59 g day-1 of daily bodyweight gain from birth to weaning). The pathological effects of subclinical parasitism were confirmed by a lower packed cell volume and albuminaemia in parasitized calves than in treated calves.

Animals↗
PubMed1989

Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes.

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K+ acetate.

Actins↗
PubMed1989

In vitro localization of the protein synthesis defect associated with experimental phenylketonuria.

We have used a cell-free system derived from hamster brain to investigate protein synthesis during experimental phenylketonuria. In such a system the elongation inhibitor emetine impeded translation in extracts derived from both treated and control animals. On the other hand the initiation inhibitor aurintricarboxylic acid showed no effects on protein synthesis activity of treated hamsters, although it was severely inhibiting in controls. This suggests that initiation is the altered step in brain protein synthesis failure consecutive to phenylketonuria.

Animals↗
PubMed1989

Isolation of hamster brain polyribosomes-cytoskeleton complexes.

We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins. This method is based on gentle homogenization of brain tissue and low speed centrifugation. The mechanism of association of polyribosomes to cytoskeletal structures has been studied by in vitro treatment of this fraction with polyribosome-disaggregating agents. RNase and EDTA, while succeeding in completely disrupting them into monosomes or subunits, did not release them from cytoskeleton. Puromycin showed no noticeable effect.

Animals↗
PubMed1987

Efficacy of RNase inhibitors during brain polysome isolation.

We have investigated the efficiency of heparin, polyvinyl sulfate and yeast RNA (as competitive RNase inhibitors), liver extract (as crude preparation of liver RNase inhibitors) and DEPC (as irreversible non-competitive inhibitor) for the preparation of rat brain polysomes. Sucrose gradient sedimentation profiles, obtained from PMS, were used to determine the optimal concentration of each inhibitor. Diethylpyrocarbonate, whatever the composition of isolation buffer, was found detrimental for brain polysomes. Most of the other inhibitors where found useless or even harmful. A slight positive effect was observed with heparin 0.75 mg/mL both for total yield and sedimentation pattern. It is concluded that the utilisation of most of the widely used RNase inhibitors is of questionnable effectiveness for brain polysome preparation.

Animals↗