[Epidemiologic study of urinary calculi in Western France]. / Etude épidémiologique des calculs urinaires dans l'Ouest de la France.

OBJECTIVES: An epidemiologic study of urinary calculi (N = 1843) was conducted in Western France: distribution according to the main chemical compounds, age and sex. Comparison with the results of a study with national recruitment (N = 10,617) and a study with regional recruitment (N = 1774). METHOD: The study involved 1843 stones characterized beforehand by morphological analysis associated with infra-red spectrophotometry (FTIR). If analysis of the composition of the stones was carried out on the totality of calculi, studies related to age and sex included only 1583 cases. Comparison of percentages was made using chi 2 test. RESULTS: The composition in main compounds of calculi was comparable with the results of other studies; minor significant compounds presented great differences, raising the problem of interpretation of the infra-red spectra of the latter. Hence, our work was directed towards the analysis of the major compounds and we showed, like most authors, that monohydrate calcium oxalate is predominant in male (46%) as well as in females (37%). Calculi average sex-ratio was 2.19 but dehydrated calcium oxalate sex-ratio was 4.42, suggesting that this compound is found mainly in men. Conversely, for the majority of phosphate stones, the sex-ratio was lower or equal to one, indicating that they predominate in women. Infectious calculi (particularly struvite calculi) appeared slightly more frequent in our population than in other studies, whereas the number of uric acid calculi was lower. This, however, remains to be confirmed. CONCLUSION: The population studied was not significantly different from the national population regarding lithiasis, except perhaps for uric acid and struvite calculi, despite specific regional differences in diet and the role of nutritional factors in lithogenesis.

Ontogeny of steroid metabolizing enzymes in rat oligodendrocytes.

Rat oligodendroglial cells were isolated from newborn and developing brains and used immediately after, for quantification of steroid metabolizing activities. Oligodendrocytes (Ol) and their progenitor cells were incubated with [(14)C] testosterone, [(14)C] progesterone, [(14)C] pregnenolone or [(14)C] dehydroepiandrosterone (DHEA). Oligodendrocytes and their progenitor cells expressed different steroid metabolizing enzymes. The main activities were 5 alpha reduction of testosterone and progesterone and 3 beta hydroxy steroid dehydrogenase-isomerase which transformed pregnenolone into progesterone and DHEA into Delta 4 androstenedione. 5 alpha reductase activity increased in male and female rats in parallel with testosterone or progesterone. Contrary to this, 3 beta hydroxysteroid dehydrogenase-isomerase activity was found to be high in the young rat and to decrease when testosterone and progesterone plasma concentration increased.

Methylenetetrahydrofolate reductase C677T genotype and venous thromboembolic disease.

BACKGROUND: Many studies have suggested an increased risk of venous thromboembolism (VTE) in patients with mild hyperhomocysteinemia. The C677T mutation in the MTHFR gene has recently been described as a cause of mild hyperhomocysteinemia. OBJECTIVES: To investigate the potential of the C677T mutation in the MTHFR gene in its homozygous state as a risk factor for VTE. METHODS: Case-control study design. The presence of the mutation was determined in all consecutive patients referred from July 1994 to September 1997 and in whom the diagnosis was duly confirmed. Analysis was carried out in a subgroup of VTE patients free from both acquired and genetic risk factors (factor-V mutation and/or prothrombin gene mutation). A control group consisted of 105 volunteer blood donors. RESULTS: In the 366 patients with a confirmed VTE, 253 presented acquired risk factors and 58 were carriers of the factor-V Leiden mutation and/or G20210A mutation of the prothrombin gene. In the remaining 55 patients, VTE was considered as 'unexplained', and the frequency of the C677T mutation MTHFR was 21.8% in its homozygous state and 34.5% in its heterozygous state. In the control group, 9.5% were found homozygous and 34.3% heterozygous. The odds ratio for having VTE in the presence of the mutation in its homozygous state was 2.9 (95% CI 1. 0-8.6). CONCLUSION: This study suggests that the homozygous C677T mutation in the MTHFR gene might be a risk factor of VTE in patients with spontaneous events and without other common genetic risk factors.

Levels of collagen degradation products (telopeptides) in the tear film of patients with keratoconus.

PURPOSE: Levels of collagen degradation products (telopeptides) in the tear film of patients with keratoconus were measured to study the release of telopeptides in the tears. METHODS: Tear samples were collected from 26 keratoconus patients and 36 age-similar human control subjects. Levels of telopeptides were quantified and compared between the two groups. RESULTS: The telopeptide level in tears from keratoconus patients was 2.5-fold higher than in tears from the control group. The telopeptide concentration was age-dependent in both groups. In tears from young people, telopeptide level was 2-fold higher than in tears from the older people. CONCLUSION: The tear film of patients with keratoconus contains higher levels of telopeptides than those of control subjects. Determination of telopeptide levels in tears could be useful for the follow-up of keratoconus development in patients.

Thyroid hormone receptor isoforms are sequentially expressed in oligodendrocyte lineage cells during rat cerebral development.

In the mammalian brain, thyroid hormones regulate myelination. Their actions are mediated by interactions with nuclear receptors that function as ligand-regulated transcription factors. Two genes, alpha and beta, encode different isoforms, of which only the beta and alpha1 isoforms are authentic nuclear triiodothyronine (T3)-receptors (NT3R). In agreement with the important role of T3 on myelination and oligodendrocyte generation, the presence of NT3Rs has been reported in oligodendrocytes and their precursors. We and others have shown that both progenitors and oligodendrocytes in vitro express the alpha1 and alpha2 isoforms, but the expression of the beta1 isoform is confined to differentiated oligodendrocytes, suggesting that they have different functions. To establish if this is the case during development in vivo, we have studied NT3R isoform expression in glial cells isolated by density gradient centrifugation from rat brains of various ages. We report the presence of the alpha1 NT3R and its variant alpha2, but not that of the beta1 isoform, in newborn rat glial progenitors. The pattern of expression of beta1, both at the level of mRNA and protein, parallels the increase in the number of oligodendrocytes. We found a significant change in the kinetic parameters of [125I]-T3 binding to NT3Rs in these cells during the first month of life, consisting of an increase in the binding capacity that peaks with myelination, and a significative decrease in Kd that coincides with the switch from the alpha to the beta1 isoform. Thus, the expression of NT3R isoforms in the rat oligodendrocyte lineage changes radically from the alpha to the beta1 isoform during the period when oligodendrocytes differentiate from progenitors.

[Primary hyperaldosteronism and adenoma of the adrenal cortex. Suppression of aldosterone with dexamethasone]. / Hyperaldostéronisme primaire par adénome de la glomérulée. Freinage de l'aldostérone par la Dexaméthasone.

The aim of the study was to evaluate dexamethazone test in a patient with primary aldosteronism caused by an adrenocortical adenoma. We observed a 50% decrease of plasma aldosterone as in glucocorticoid suppressible aldosteronism (GSA) but absolute value of aldosterone remained higher than 40 pg/ml. Basal plasma and urinary values of 18 OXO and 18 OH cortisol were not significantly elevated as in GSA. Inversely, the evaluation of 11 beta-hydroxylase activity of mineralocorticoids was in favor of a benign adrenal tumor.

[Cushing's disease: role of feeding]. / Maladie de Cushing: Rôle de l'alimentation.

We report a clinical case of a patient with corticotropin dependent Cushing disease. In this patient ingestion of mixed meals is followed by an increase of ACTH and cortisol. This effects seems secondary to ingestion of proteins, and it can be reproduced by intravenous injection of aminoacids. The pattern observed is similar to what is observed in normal subjects. Neurotransmitter substrate from protein meals or after intravenous injection of aminoacids may influence the factors controlling ACTH secretion in the studied patient.

[Polycystic ovary and diazoxide. In vivo study]. / Ovaires polykystiques et diazoxide. Etude in vivo.

The suppression of hyperinsulinism with diazoxide (300 mg/d during 30 days) in a young woman with PCOS and hirsutism, hyperinsulinism and insulinoresistance was followed by a modification of plasma androgens. Testosterone (T) and free testosterone (fT) were reduced after ten days and then increased but always remained below the baseline level. DHEAS had increased 200% by day 10, and 3 alpha-adiol G to three times its basal value by day 20. These modifications were constant during the treatment. fT decrease was secondary to reduction of hyperinsulinism which was followed by an increase of TeBG and a modest and transient reduction of androgen theca cells production. DHEAS increase was due to hyperinsulinism suppression which stimulated adrenal 17-20 lyase activity. 3 alpha-adiol G increase was concomittant, and can be considered as an index of adrenal androgen secretion.

Cloning, sequencing and expression of Pseudomonas testosteroni gene encoding 3 alpha-hydroxysteroid dehydrogenase.

We describe the cloning, sequencing and expression of the 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) gene of Pseudomonas testosteroni. A genomic library of P. testosteroni total DNA constructed from SauIIIA digests ligated to an lambda gt11 vector was probed with a polyclonal antibody raised against purified enzyme. Subclones derived from a recombinant phage containing a 1746 bp insert were sequenced and found to contain an open reading frame of 696 bp that corresponds to a protein of 231 amino acid residues. A search for homologous proteins was performed. No similarity was observed when comparing 3 alpha-HSD with known members of the short-chain dehydrogenase family. However a small proteic fragment (80 amino acids) shows homology with the N-terminal sequence of bacterial L7/L12 ribosomal proteins.

Cytochrome P-450 2E1 in rat liver, kidney and lung microsomes after chronic administration of ethanol either orally or by inhalation.

In this study, microsomal cytochrome P-450 2E1 (CYP2E1) contents and activities were tested in liver, kidney and lung from Wistar rats after the following treatments (1) oral administration of a 10% ethanol solution for 4 weeks; (2) pair fed controls; (3) oral administration of a 5% acetone solution for 1 week; (4) inhalation of ethanol vapour for 4 weeks. CYP2E1 activity was measured using chlorzoxazone as substrate and CYP2E1 content was measured using Western blot analysis. In addition, the cellular distribution of CYP2E1 was studied in liver, lung and kidney by immunohistochemistry. Basal liver CYP2E1 was 10-20 times lower in lung and kidney than in liver. Inhalation was clearly the most efficient way of inducing CYP2E1, probably due to the continuous and high alcohol exposure. Among the organs tested, lung appeared to be the tissue least sensitive to induction even after ethanol inhalation, suggesting the absence of local induction. After ethanol intoxication, immunostaining was increased in the centrilobular region of the liver, in the alveolar cells of the lung and in the proximal convoluted tube of the kidney. The CYP2E1 activities decreased to control values in the three tissues tested, within 24 h after cessation of intoxication.

Gene amplifications in advanced-stage human prostate cancer.

Gene amplification is a model of proto-oncogene alterations occasionally observed in human tumors. This amplification can, in some cases, have prognostic value (N-myc in neuroblastoma, c-erbB2 and int-2 in breast cancer, etc.). Amplifications of the proto-oncogenes c-myc, c-erbB2 and int-2 have not yet been report in prostate adenocarcinoma, which, like breast cancer, is hormone dependent. We sought amplifications of these three proto-oncogenes by means of Southern blotting in 15 human prostate adenocarcinoma specimens, most of which were advanced (7 stage C and 6 stage D1 or D2). We confirmed the lack of c-myc and c-erbB2 amplification, regardless of the stage, in contrast to the case of breast cancer. Int-2 amplification was observed in one advanced tumor with bone metastases, out of a total of six stage D tumors. The precise frequency of int-2 amplification and its role in prostate carcinogenesis remain to be determined.

[Cloning of genes coding for 3-alpha-hydroxysteroid dehydrogenase and for (3-17)-beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni]. / Clonage des gènes codant pour la 3 alpha-hydroxystéroïde déshydrogénase et pour la (3-17)beta-hydroxystéroïde déshydrogénase de Pseudomonas testosteroni.

A genomic library of Pseudomonas testosteroi total DNA constructed from SauIIIA digests ligated to a lambda gt11 vector was probed with different polyclonal antibodies raised against purified 3 alpha-HSD and (3 beta-17 beta)-HSD. Two different clones reacting with one antibody were selected. The clone reacting with (3-17)beta-HSD antibody contained a 2,661-base pair insert and was found to contained an open reading frame of 765 base pair that corresponds to a protein of 254 amino-acid residues. A 1,492-base pair was inserte in pBR 322 plasmid vector; the recombinant bacterie over expressed the (3-17)beta-HSD gene. The clone reacting with 3 alpha-HSD antibody contained a 1746 base pair insert which contained an open reading frame of 696 base pairs that corresponds to a protein of 231 amino-acid residues. A search for homologous proteins was performed. Distant similarities were found between (3-17)beta-HSD and members of the short-chain alcool dehydrogenase (SCAD) family but no similarity was observed between 3 alpha-HSD and proteins of this family.

Purification of testosterone 5 alpha-reductase from human prostate by a four-step chromatographic procedure.

Nuclear membrane bound testosterone 5 alpha-reductase solubilized in active form from human prostatic tissue by 0.5% n-octyl beta-D-glucopyranoside was purified by a four-step chromatographic procedure including DEAE-Trisacryl ion exchange, hydroxylapatite adsorption, testosterone-Sepharose affinity and Sepharose 4B gel filtration. A purification of approximately 30-fold was achieved judging from the increase in the specific enzymatic activity. We have purified the acidic pH-optimum 5 alpha-reductase type 2 isoenzyme. The apparent molecular weight of the purified enzyme was estimated as 42,000 by SDS-PAGE. At the same time we isolated a 38 kDa protein characterized by a real affinity for testosterone and by a possible association to the 5 alpha-reductase enzyme.

Glycosylated nature of testosterone 5 alpha-reductase 2 purified from human prostate.

5 alpha-reductase 2 from human prostate solubilized into an active and stable form using a non-ionic detergent octyl glucoside was successfully purified using a four-step chromatographic procedure. The enzyme was obtained as an apparently homogeneous protein exhibiting an apparent molecular weight of 42 kDa upon SDS-PAGE. Con A, DBA, UEA-I, and RCA60 lectins recognized this protein. After treatment with O-glycosidase and neuraminidase, a protein of an apparent molecular weight about 30 kDa appeared. On the other hand, N-glycosidase treatment of this enzyme had no effect. These results indicate that the human prostate testosterone 5 alpha-reductase 2 is an O-glycosylated sialoglycoprotein with a peptide moiety of about 30 kDa; the oligosaccharide side chains contain mannose, N-acetyl galactosamine, fucose, galactose and sialic acids.

Characterization and solubilization of testosterone 17 beta-hydroxysteroid dehydrogenase in human hyperplastic prostate.

17 beta-Hydroxysteroid dehydrogenase is a membrane-bound enzyme in human prostate. Solubilization of this enzyme can only be obtained in the presence of detergents. The optimal solubilization mixture contained 50 mM Tris-HCl buffer pH 9.0, 20% glycerol, 0.1 M KCl and 5 mg/ml of the non-ionic detergent N-octyl glucoside. In these conditions, the soluble fraction contained more than 90% of the enzymatic activity. A 2.5-fold increase of specific activity was obtained during solubilization under optimal conditions.

Cloning, DNA sequencing and expression of (3-17)beta hydroxysteroid dehydrogenase from Pseudomonas testosteroni.

We describe the cloning, sequencing and overexpression of the (3-17)beta hydroxysteroid dehydrogenase gene of Pseudomonas testosteroni. A genomic library of Ps. testosteroni total DNA constructed from SauIIIA digests ligated to a lambda gt11 vector was probed with polyclonal antibody raised against purified enzyme. Subclones derived from a recombinant phage containing a 2661-base-pair insert were sequenced and found to contain an open reading frame of 765 base pairs that corresponds to a protein of 254 amino acid residues. A 1492-base-pair fragment was inserted into pBR322 plasmid vector and used to construct a strain of E. coli HB101 that overexpressed the steroid dehydrogenase gene.

Nature of cytochromes P450 involved in the 2-/4-hydroxylations of estradiol in human liver microsomes.

Kinetics of the 2- and 4-hydroxylations of estradiol (E2) by human liver microsomal samples were studied to determine the major P450 isoform involved in these endogenous reactions. Thirty human liver microsomal samples were analysed. Metabolism of 25 microM [14C]E2 produced 2-hydroxy and 4-hydroxy derivatives with a ratio of 3.2 +/- 1.5 and a great inter-individual variation. Kinetic analysis of the 2- and 4-hydroxylations of E2 exhibited a curvilinear double reciprocal plot with an apparent Km of 15 microM. Further experiments demonstrated that alpha-naphthoflavone, testosterone and progesterone increased the 2-hydroxylation activity, suggesting the involvement of a substrate activation mechanism. These two hydroxylations of E2 were shown to be catalysed by cytochrome P450 with an apparent dissociation constant Ks of 0.8 microM. These 2- and 4-hydroxylations inter-correlated significantly (r = 0.93; N = 30). The 2-hydroxylation of E2 correlated with four monooxygenase activities known to be supported by P450 3A4/3A5, namely nifedipine oxidation (r = 0.78; N = 29); erythromycin N-demethylation (r = 0.69; N = 27), testosterone 6 beta-hydroxylation (r = 0.66; N = 25) and tamoxifen N-demethylation (r = 0.64; N = 29). On the other hand, E2-hydroxylations did not correlate with activities supported by P450 1A2 and P450 2E1. Furthermore, drugs as cyclosporin, diltiazem, triacetyl-oleandomycin and 17 alpha-ethynylestradiol inhibited more than 90% of the E2-hydroxylations at concentrations < 250 microM, while weak inhibition was shown with 500 microM cimetidine and no significant inhibition with caffeine, phenacetin and omeprazole. Finally, 2- and 4-hydroxylations of E2 correlated significantly with the content of P450 3A4/3A5 immunodetected by a monoclonal antibody anti-human P450-nifedipine (r = 0.84; N = 28). E2-hydroxylation activities were inhibited by more than 80% with polyclonal anti-human anti-P450-nifedipine. Preincubation of human liver microsomes with 100 microM gestodene (a suicide substrate of P450 3A4) inactivated this P450 isoform and accordingly allowed evaluation of the contribution of other P450 isoforms to the E2 metabolism to about 21% (+/- 17%, N = 29). All these results taken together suggest that P450 3A4/3A5 are the major forms involved in the formation of catecholestrogens in the human liver microsomes.

Partial purification of 3 alpha- and 3 beta-hydroxysteroid dehydrogenases from human hyperplastic prostate. Comparison between the two enzymes.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) activity has been purified to homogeneity, the enzyme is a monomer with a Mw of 32,000 Da. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) activity has been partially purified and has an apparent Mw of 30,000 Da. Both enzymes have the same cofactor requirements, optimal pH. However, 3 beta-HSD appeared to be an integral protein dependent on protein environment for its activity while 3 alpha-HSD activity is a protein more loosely associated to membranes.

The effects of chronic ethanol administration on rat liver and erythrocyte lipid composition: modulatory role of 'evening primrose oil'.

The effects of chronic administration of ethanol on the lipid composition of erythrocytes and liver of rats were investigated. Ethanol was chronically administered alone or in combination with Evening Primrose Oil (containing 10% v/v gamma-linolenic acid) in a nutritionally balanced milk diet. Chronic administration of ethanol alone significantly decreased the content of arachidonic acid in erythrocyte membranes, whereas the concomitant administration of Evening Primrose Oil reversed this effect. The triacylglycerol content was significantly increased in the microsomal fraction of the liver after chronic ethanol administration. Ethanol also significantly increased the ratio of cholesterol:cholesteryl esters in the microsomal fraction. The arachidonic acid content of the whole liver fraction was significantly reduced after chronic administration of ethanol, whereas concomitant administration of Evening Primrose Oil did not reverse this effect. The administration of Evening Primrose Oil during chronic ethanol intake may have beneficial effects, as it reverses some of the effects of ethanol on erythrocyte and hepatocyte membrane lipids which may be detrimental to health.

Changes in whole brain membranes of rats following pre- and post-natal exposure to ethanol.

The chronic effects of ethanol on the fatty acid composition of rats that have been exposed to ethanol in utero were examined. Ten female Wistar rats were fed a nutritionally adequate liquid diet for 3 weeks before mating, throughout gestation and until the offspring reached the 10th or 20th post-natal day. Whole brain lipid changes were examined at these 2 time points. On day 10, a decrease in 18:1 lipid content was found, indicating tolerance development. However, by day 20 an increase in polyunsaturated fatty acid content (20:4) was detected, indicating that ethanol may be causing an increase in membrane fluidity. Although these results are contrary to those found in adult rats following chronic ethanol administration, it seems likely that, in the immature animal, the brain is still undergoing rapid development and therefore may be affected differentially by ethanol.