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Biomedical subjects

J L Orsonneau

Publications and source records attributed to J L Orsonneau.

At least 19 recordsLinked to original sources
PubMed1992

Simple and sensitive determination of urea in serum and urine.

In this method for serum and urinary urea determination, the same reagent is used without predilution of urine samples. The method is based on the pH increase resulting from the ammonia released by urease hydrolysis of urea. o-Cresolphthalein complexone is used to monitor the pH change colorimetrically. Urea concentration and absorbance at 570 nm are linearly related for concentrations as great as 600 mmol/L for urine samples and 100 mmol/L for serum. There are no clinically significant interferences from physiological substances or drugs, and precision and accuracy are excellent (CV approximately 2%, except at very low concentrations in serum; analytical recovery was 99% in urine, 100% in serum). Results by this method (y) and by the Astra method (x) for urine correlated well (y = 0.991x - 2.87, Sy/x = 9.21, r = 0.994), as did the results by this method and by the total enzymatic method (x') for serum (y = 1.002x' + 0.192, Sy/x' = 0.598, r = 0.997). This method is applicable to automated as well as manual instruments, and one-reagent or two-reagent formats can be used.

Ammonia↗
PubMed1990

One-step 10(4)-fold purification of transformed glucocorticoid receptor. Method for purifying receptors associated with Mr ca. 90,000 heat-shock protein.

Chromatography of rabbit glucocorticoid-receptor complexes in the absence of sodium molybdate on a Mono Q anion-exchange column induces the transformation of the receptor and allows the resolution of the transformed and non-transformed molecular species. These abilities were used to design a new purification scheme for the glucocorticoid receptor from rabbit liver in its transformed state. Microgram amounts of receptor were obtained using this single-step procedure in less than 2 h. The purification yield was 50-60%. Immunoblot experiments showed that the glucocorticoid receptor was present as an Mr approximately 94,000 polypeptide in these preparations and represented 20-30% of the eluted proteins as determined by densitometric scanning analysis of silver-stained sodium dodecyl sulphate polyacrylamide gels. Finally, the purified receptor was able to interact quantitatively with bulk DNA.

Animals↗
PubMed1990

Reaction of tyrosyl-modifying reagents with the ligand- and DNA-binding domains of the rabbit liver glucocorticoid receptor.

We have studied the effects of p-nitrobenzenesulfonyl fluoride, 4-fluorosulfonyl-1-hydroxy-2-naphtoic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and tetranitromethane on the glucocorticoid receptor from rabbit liver. Our results show that all tyrosine modifying reagents inhibit the binding of [3H]dexamethasone to the receptor. Equilibrium binding experiments revealed that only 4-fluorosulfonyl-1-hydroxy-2-naphtoic acid is a competitive inhibitor while the other chemical probes decrease the concentration of binding sites. Transformation of glucocorticoid-receptor complexes was markedly reduced when heat treatment was performed in the presence of tyrosyl-directed reagents. Taken together, these results indicate for the first time that critical tyrosyl moieties may be involved in both hormone binding and transformation of the glucocorticoid receptor.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Occurrence of glucocorticoid binding sites in solubilized microsomes from rat liver.

Recent studies suggested the presence of specific glucocorticoid binding sites on rat liver microsomal membranes. We report here a new solubilization procedure which allows the physicochemical characterization of the microsomal glucocorticoid binding sites. Solubilization was achieved with 2 mM CHAPS in the presence of 5 mM benzamidine. Binding of [3H]cortisol showed a high affinity (Kd = 5.1 x 10(-9) M) and a limited capacity (0.72 pmol/mg of protein). The binding activity was abolished by elevated temperature and pronase. Competition experiments revealed that natural glucocorticoids and progesterone were highly potent competitors whereas dexamethasone and triamcinolone acetonide did not compete. Chromatography on DEAE Trisacryl and heparin Ultrogel confirmed that the solubilized protein is different from corticosteroid binding globulin and the cytosolic glucocorticoid receptor. Treatment of microsomal fractions with phosphatidyl inositol phospholipase C promoted the release of specific binding activity suggesting a putative glycosylphosphatidyl anchor for this protein. This finding may have interesting implications concerning the mechanism of glucocorticoid hormone action.

Animals↗
PubMed1989

An improved pyrogallol red-molybdate method for determining total urinary protein.

We adapted the pyrogallol red-molybdate method for total urinary protein to the Cobas Bio centrifugal analyzer. The method is simple, rapid, sensitive, and inexpensive. Addition of 25 mg of sodium dodecyl sulfate per liter to the reagent modifies protein reactivities so that the chromogenicity of human gamma globulins is the same as that of albumin. Results by this method and a comparison method that included gel filtration and a modified biuret reaction correlated well (r = 0.951).

Adult↗
PubMed1989

[Microalbuminuria: evaluation of an immunoturbidimetric method and an immunonephelemetric method].

Two methods for measurement microalbuminuria are appraised to take the place of RIA. One is immunonephelemetry, using a "closed" immunochemistry analyzer of modest price. The other one is immunoturbidimetry on an "open" centrifugal analyzer, which is of a middle price. The results which are obtained by IN and IT are compared statistically to the RIA. These methods give satisfaction for practicability, precision and accuracy and can be used in any biological laboratory.

Albuminuria↗
PubMed1986

Interaction of rat liver glucocorticoid receptor with lectins: is the glucocorticoid receptor a glycoprotein?

Although glucocorticoid receptors have been extensively studied in a variety of tissues, the precise nature of the receptor protein still remains unknown. To further characterize this protein we assessed the effects of various lectins on [3H]dexamethasone binding to prepurified preparations of rat liver glucocorticoid receptor. Among the lectins tested only Ulex europeus and Lens culinaris induced a concentration-related decrease in [3H]dexamethasone binding. Following Ulex europeus or Lens culinaris exposure Scatchard analysis showed that these lectins led to a 3-fold reduction in receptor affinity without influencing the concentration of binding sites. These results provide new experimental evidence that rat liver glucocorticoid receptor would possess alpha-L-fucosyl and alpha-D-mannopyranoside residues in close proximity to the glucocorticoid receptor binding domain.

Animals↗
PubMed1985

Nontransformed rabbit liver glucocorticoid receptor: purification, characterization and transformation.

The molybdate-stabilized nontransformed form of the glucocorticoid receptor from rabbit liver has been purified approximately 8,000-fold by a three-step procedure. The first step involved protamine sulfate precipitation which allowed a 5-6-fold purification with 85% yield. The second step, affinity chromatography using a N-(12-dodecyl-amino) 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxamide substituted Sepharose gel, purified the receptor 1,500-2,000-fold as calculated by specific radioactivity. The third step involved high performance liquid chromatography resulting in overall purification near 8,000-fold. The final glucocorticoid receptor appeared about 60% pure. The purified nontransformed glucocorticoid receptor had a sedimentation coefficient of 9 S in 0.16 M phosphate containing 5-20% sucrose gradients and the Stokes radius was 6.1-6.3 nm as determined by low pressure gel filtration and HPLC. Binding specificity of the purified receptor was identical to that previously reported in crude rabbit liver cytosol. Isoelectricfocusing and ion-exchange chromatography showed that the purification procedure affected the net charge of the receptor protein. This phenomenon could be related to interactions between the glucocorticoid receptor and cytosolic factors. SDS polyacrylamide gel electrophoresis showed a major Mr = 94,000 protein band which is in good agreement with previously reported values for glucocorticoid receptors. Transformation of the purified receptor was achieved after removal of molybdate by exposure at 25 degrees C to 0.4 M KCl. Characterization of the molecular forms was performed by means of incorporation into isolated nuclei, affinity towards polyanionic exchangers and high pressure size exclusion chromatography. Results show that about 40% of the receptor is in the transformed state.

Adrenalectomy↗
PubMed1984

Resolution of the molecular forms of rat liver glucocorticoid receptor by affinity chromatography on immobilized heparin.

Rat liver cytosolic glucocorticoid receptor labelled with [3H] dexamethasone and stabilized with molybdate was bound to heparin-ultrogel and eluted with NaCl or heparin as a single peak of radioactivity. After heat exposure of cytosol, two steroid receptor complexes could be separated by NaCl or heparin. Characterization of the two forms was performed by means of affinity towards isolated nuclei, sucrose gradient centrifugation and gel exclusion high performance liquid chromatography. The results presented here suggest that the two forms eluted from heparin-agarose correspond to the untransformed and transformed states of the glucocorticoid receptor complex. Taken together, these observations argue in favor of heparin-ultrogel as a suitable procedure to study the mechanism of glucocorticoid-receptor transformation.

Animals↗
PubMed1984

Influence of molybdate, ionic strength and pH on ligand binding to the glucocorticoid receptor.

The addition of molybdate to rabbit liver cytosol increased significantly the affinity of the glucocorticoid receptor for [3H] dexamethasone without influencing the concentration of binding sites. This effect was concentration dependent. Analysis of the binding data by curve-fitting and Scatchard plot revealed the occurrence of a complex binding process in the presence of molybdate. The pH-dependence curve of the binding was shifted towards alkaline values by the oxyanion. Taken together, these data suggest that molybdate exerts its effects via an interaction with the receptor molecule.

Animals↗
PubMed1983

Physicochemical characterization and transformation of the cytosolic glucocorticoid receptor from rabbit liver.

Characterization of the glucocorticoid receptor from rabbit liver cytosol was studied in vitro. Binding of [3H]-dexamethasone showed a high affinity (KD = 2.4 . 10(-9)M) and a concentration of binding sites of 0.3 . 10(-12) mol/mg proteins. Association and dissociation rate constants for [3H]-dexamethasone were respectively 1.6 . 10(5)M-1 . min-1 and 2.5 . 10(-4) min-1. Competition experiments showed that dexamethasone and triamcinolone acetonide were the most effective competitors while progesterone and aldosterone competed very poorly. The stabilities of bound and unbound forms were investigated with and without molybdate and other oxyanions. Calibrated gel filtration gave a stokes radius of 5.6 nm and an apparent mol. wt of 280,000. The untransformed complex sedimented at 9 s in 5-20% sucrose density gradients and the transformed species sedimented near 4 s. The behaviour of the three forms of receptor, untransformed, transformed and molybdate-stabilized was studied on ionic exchangers. Transformation of the [3H]-dexamethasone--receptor complex was shown to occur both with high ionic strength and elevated temperature. The transformation step was almost completely inhibited by tungstate and molybdate while vanadate revealed a very slight inhibitory effect.

Animals↗
PubMed1977

[Analysis of plasma urea by continuous flow method using enzymes].

An automated continuous flow method is described for the assay of plasmatic urea after hydrolysis by urease followed by NH+4 assay with glutamic-deshydrogenase. Precision and accuracy, comparison with chimical diacetyl-monoxim method are studied. The interaction of endogenous NH+4, the cost of the method are calculated.

Autoanalysis↗