Biomedical subjects
Publications and source records attributed to A Lefèvre.
For 17 eyes consecutively operated on because of posterior luxation of the crystalline lens, the nucleus and the intraocular lens, the surgical complications and results are discussed in relationship with the techniques used.
Treatment of K9 mouse Leydig cells with 3 x 10(-6) M retinol (R) and retinoic acid (RA) resulted in 75% and 65% reduction of 125I-labeled hCG binding respectively, when assayed at 35 degrees C. This effect was dose-dependent and was first detected 12 h after initiation of treatment: it was maximal at 48 h for RA. R and RA had no significant effect on the rate of internalization and degradation of 125I-hCG as measured by disappearance of acid-releasable (i.e. surface-bound) radioactivity from the cells and by the appearance of trichloracetic acid-soluble label in the medium. When exposed to increasing concentrations of hCG for 24 h, both retinoid-treated and control cells 'down-regulated' their gonadotropin receptors with the same dose-dependent pattern. The kinetics of reappearance of the receptors was similar for retinoid-treated and control cells, but for treated cells the maximal number of receptors reinitiated at 24 h never exceeded 40% of the values observed with control cells. Scatchard plot analysis confirmed a decrease in hCG receptor number from approximately 26,000 to approximately 6400 and approximately 3500 sites per cell after R and RA treatment. Kd values for 125I-hCG binding were 2 x 10(-10) M, 7.3 x 10(-11) M and 6.9 x 10(-11) M for control, R- and RA-treated cells respectively. On the basis of our data it is likely that retinoid-induced reduction in 125I-hCG binding to K9 Leydig cells is due to decreased receptor synthesis.
A method for the purification of epithelial cells from the three anatomical regions of the rat epididymis (corpus, caput and cauda) is described. An enzymic digestion followed by sedimentation of crude cell suspension on discontinuous Percoll gradient yielded quite pure active epithelial cell population as judged by morphological and functional studies. Electron microscopy analysis showed that cells from bands corresponding to densities 1.055 and 1.06 g/ml the gradient preserved a morphology compatible with their epithelial origin and their absorptive and secretory functions. Moreover, they stained positively with anticytokeratin antibody (95-97%) and were negative for antidesmin antibody. They selectively bound L-carnitine through a time-dependent and saturable system and differences in the rate of binding were apparent according to the three anatomical regions of the epididymis.
Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from a carcinoma of the uterine cervix and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT media; some experiments were carried out in medium with delipidized serum. The hybrid cells were characterized by indirect immunofluorescence with a biotin-streptavidin system using a panel of nine monoclonal antibodies specific for membrane and cytoplasmic antigens of parental cells: intermediate filaments (keratins and vimentin), HLA class 1 (beta 2-microglobulin), cell activation (EGF and transferrin receptors) and cellular adhesion (fibronectin and laminin). All of these antigens were expressed in HeLa cells cultured in conventional medium or with delipidized serum. Conversely mouse fibroblasts contained only vimentin, fibronectin and laminin. All the parental antigens were present in first passage hybrid cells cultured in conventional medium. Vimentin, fibronectin and laminin were maintained in fourth passage hybrids whereas keratins, beta 2-microglobulin, EGF and transferrin receptors were no longer detected. When propagated in medium with delipidized serum, hybrid cells re-expressed these antigens after 5 days of culture. These findings suggest that the reexpression of HeLa cell antigens in hybrid cells was related to deficiency in vitamin A.
Hybrids constructed by fusing mouse Leydig cells with mouse adrenal Y1 cells were able to randomly express all the parental specific traits but for the response to gonadotropin (hCG) and corticotropin (ACTH): three of them, YDYL 14, 17 and 19, metabolized both progesterone and dehydroepiandrosterone into testosterone accounting for 17 alpha-hydroxylase, 17-20-lyase, 17-ketoreductase and 3 beta-hydroxysteroid dehydrogenase activities. Under basal conditions, 17 alpha-hydroxylase and 17-20-lyase activities were high in the three clones as compared to parental Leydig cells, and were no longer stimulated by cAMP in YDYL 17 and 19. The hybrids responded to various hormones such as prostaglandin E2 (PGE2), vasoactive intestinal peptide (VIP) and prolactin (PRL) which are not directly implicated in the expression of steroidogenesis; they generally retained the Y1 morphological response to 8-bromo cAMP. On extended culture, reexpression of ACTH sensitivity occurred in one clone, YDYL 9. This reexpression was correlated with a Robertsonian translocation between mouse chromosomes 2 and 11, while extinction required the presence of an intact mouse chromosome 11.
Hybridization between a mouse Leydig tumor cell line, MA-10, which produces cyclic AMP and progesterone under human chorionic gonadotropin (hCG) stimulation, and freshly isolated mouse Leydig cells gave rise to 54 hybrid clones, one of which, LK17, was capable of hCG-stimulated testosterone production. Subcloning of this hybrid resulted in the emergence of a subclone, K9, whose testosterone production is more than 10 times that of parent clone LK17, after hCG stimulation, with an ED50 of 37 pM. Testosterone synthesis by K9 cells was multiplied by 25 after gonadotropin stimulation, and binding of hCG declined after prolonged exposure to the hormone. These similarities with murine Leydig cells in primary culture make the K9 clone an attractive alternative for physiological studies.
The diterpene, forskolin, stimulated cAMP accumulation about 15-fold over basal levels in purified mouse Leydig cells; however, it remained far less potent than hCG. Simultaneous addition of forskolin and hCG resulted in a striking synergistic stimulation of cAMP production. In contrast, forskolin-enhanced testosterone accumulation was never synergistic with that produced by maximal concentrations of hCG. hCG (3 X 10(-9) M) lowered about 6-fold the ED50 for forskolin-elicited cAMP accumulation and increased the maximal response to forskolin about 16-fold. Conversely, forskolin 10(-6) M) reduced the ED50 for hCG 2-fold but had a much smaller effect (2-3-fold) on maximal response. Moreover, pretreatment with hCG induced only a homologous desensitization of adenylate cyclase, whereas the enzyme became partially resistant to both hCG and forskolin in cells pretreated with forskolin. The homologous hCG-induced desensitization and the partial heterologous one induced by forskolin suggest that more than the catalytic unit of the cyclase is required for the diterpene activation.
Three different techniques (electro-immunodiffusion: EIA, laser nephelemetry and competitive enzymo-assay: CELIA) were compared in order to select an accurate and reproducible assay of plasma fibronectin that could be used in testing large numbers of samples. Nephelemetry, gave results similar to the EIA and CELIA in assays performed with samples from 40 healthy subjects and was used to determine the plasma concentration of fibronectin in 182 male and female blood donors, ranging in age from 18 to 60. The results demonstrate a great variability in plasma fibronectin levels and show that the values are significantly lower in women, 322.9 +/- 94.7 mg/l, compared to 360.8 +/- 97.6 mg/l for men and increase with age. We conclude that the plasma fibronectin levels in various diseases must be interpreted with caution and that it seems more interesting to study the variations of values in comparison with the clinical evolution of the patients.
Collagenase dispersed cells from immature or mature rat testis were separated into 11 bands on a discontinuous Percoll gradient. Erythrocytes sedimented at the bottom. Only three bands (8, 9 and 10) bind efficiently [125I]hCG. In immature rats, cells in bands 8, 9 and 10 bind hCG with the same affinity, but differ slightly in their total number of sites (average 65,000 +/- 4,000 sites per Leydig cell) and in their cAMP and testosterone production in response to hCG. They present 8% of the total number of dispersed cells. In mature rat testis, populations 8, 9 and 10 differed markedly in their capacity to bind [125I]hCG and in their response to hCG as regards an increase in cAMP and testosterone production. Testosterone production was increased 2- and 17-fold in bands 8 and 10, respectively. In mature rats only 1.2% of the total cells are Leydig cells. In both mature and immature rats, band 10 is made of 100% Leydig cells, as all cells stained positively for 3 beta-hydroxysteroid dehydrogenase.
After describing the clinical picture of Parkinsonism, other Parkinson and depressive syndromes, the authors clarify ascending dopaminergic and serotoninergic pathways in the central nervous system as well as current knowledge on the nigro-striatal pathway. They also underline the interest of the probenecid test in the diagnosis of Parkinsonsism with or without depression. Neurochemical criteria of dopa-sensititivy and dopa-resistance are proposed as well as criteria aimed at serotoninergic therapy. Finally, the use of this neurochemical test in treatment and follow up is illustrated by a clinical case report.
Characterization of a cytosolic protein of adrenal origin that binds cholesterol. Adrenal cytosol contains a thermostable protein which binds cholesterol specifically and non-covalently. The sedimentation coefficient of the complex is close to 3 S. The side chain of the cholesterol molecule is responsible for the binding specificity.
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Ketonuria has been observed in alcoholics. To study the mechanism of this effect, healthy, volunteers were given adequate diets (36% of calories as lipid and 15% as protein) for 18 days, with isocaloric replacement of carbohydrate (46% of calories) by either ethanol or additional fat. The latter resulted in a high fat diet, with 82% of calories as lipid. After about 1 wk of alcohol, massive and persistent ketonuria developed. Compared with the control period, there was a 30-fold increase in fasting blood acetoacetate and beta-hydroxybutyrate (P < 0.001). With the high fat diet, acetoacetate and beta-hydroxybutyrate increased 8- to 10-fold (P < 0.001). In the postprandial state, ethanol also induced hyperketonemia, but less markedly than when ethanol followed an overnight fast. With low fat diets (5% of calories), alcohol (46% of total calories) did not induce ketonuria or hyperketonemia, suggesting that a combination of alcohol and dietary fat is necessary. The addition of alcohol to rat liver slices did not affect ketogenesis. In rats pretreated with alcohol for 3 days, however, ketonemia developed, hepatic glycogen was decreased, and liver slices (incubated with palmitate-(14)C and glucose) had a significant increase in acetoacetate production, when compared to carbohydrate pretreated controls. Alcohol pretreatment or addition of alcohol in vitro had no effect on acetoacetate utilization by rat diaphragms, and decreased only slightly the conversion of beta-hydroxybutyrate-(14)C to (14)CO(2). Thus, the hyperketonemia and ketonuria observed after alcohol consumption cannot be attributed to an immediate effect of alcohol, but is the consequence of a delayed change in intermediary metabolism characterized by increased hepatic ketone production from fatty acids, possibly linked to ethanol-induced glycogen depletion and depression of citric acid cycle activity.
Replacement of dietary triglycerides containing long-chain fatty acids (LCFA) by triglycerides containing medium-chain fatty acids (MCFA) markedly reduced the capacity of alcohol to produce fatty liver in rats. After 24 days of ethanol and MCFA, the increase in hepatic triglycerides was only 3 times that of controls, whereas an 8-fold rise was observed after ethanol and LCFA. The triglyceride fatty acids that accumulated in the liver after feeding of ethanol with MCFA contained only a small percentage of the MCFA; their composition also differed strikingly from that of adipose lipids. To study the mechanism of the reduction in steatosis, we compared oxidation to CO(2) and incorporation into esterified lipids of (14)C-labeled chylomicrons or palmitate-(14)C (representing LCFA), and of octanoate-(14)C (as MCFA) in liver slices and isolated perfused livers, in the presence or absence of ethanol. Ethanol depressed the oxidation of all substrates to CO(2); MCFA, however, was much more oxidized and reciprocally much less esterified than LCFA, with a 100-fold difference in the ratio of esterified lipid-(14)C to (14)CO(2). Furthermore, in hepatic microsomal fractions incubated with alpha-glycerophosphate, octanoate was much less esterified than palmitate. This propensity of MCFA to oxidation rather than esterification represents a likely explanation for the reduction in alcoholic steatosis upon replacement of dietary LCFA by MCFA.
In this review, we present some studies on genetic analysis of proteome and transcriptome variations, which exemplify new strategies for a better understanding of the molecular and genetic bases of complex traits. A large genetic variability was revealed at the proteome expression level, which raised the possibility to predict phenotypical performance on the basis of gene product variability. This approach yielded limited results, but could be re-newed by extensive identification of proteins now allowed by mass spectrometry. The dissection of the genetic basis of the variation of individual protein amounts proves very powerful to select 'candidate' proteins, physiologically relevant for a given phenotypical trait, as shown by a study on the effect of water stress in maize. In order to investigate factors of grain quality in maize, we selected a regulatory locus known to control the expression of several storage protein genes, Opaque-2, and investigated the relationships between variability in zein amount and composition and the molecular polymorphism at this locus. Moreover, a QTL analysis revealed that the variability in Opaque-2 transcript abundance was controlled by several polymorphic trans-acting regulators unlinked to the Opaque-2 structural gene. Such genetic approaches should represent additional tools for physiological analysis of the huge amounts of data generated by transcritome and proteome projects.
The role of retinoids in the regulation of epididymal fluid protein expression was investigated. We compared the patterns of two-dimensional electrophoretic gels of proteins from luminal fluids, cytosols and spermatozoa (from control rats only) of control, retinoid-depleted, retinoid-depleted retinoic acid-complemented and retinoid-depleted testosterone-supplemented rats. This study compared the luminal fluid patterns from the 4 diets and observed 13 proteins whose expression was dependent on nutritional status. Eight were either absent or very weakly expressed in retinoid-depleted animals only, while their presence was obvious in control rats and in the retinoid-deficient retinoic acid- and testosterone-complemented groups. The expression of 8 proteins was greatly enhanced in retinoid-depleted testosterone-supplemented fluids as compared to control fluids. Five of the regulated proteins seemed to be captured by spermatozoa as they were observed in sperm protein patterns of control rats. These results clearly show that the synthesis of several epididymal proteins is influenced by retinoids. Since testosterone-supplemented animals on retinoid-free diet elicited the same response as retinol and retinoic acid ones, testosterone is likely to be the mediator of retinoid action on epididymal protein synthesis. Nevertheless, the observation of one protein whose expression is stimulated by retinoic acid only and is totally independent of testosterone also favors the direct influence of this retinoid.
The adjuvant and immunostimulating activities of a glycoprotein preparation (GP) extracted from Klebsiella pneumoniae have been studied. When injeted with an ovalbumin in incomplet Freund adjuvant emulsion, the GP induces delayed hypersensitivity to the antigen and increases the level of antibody developed. When it is injected before the antigen to C57Bl/6 mice, the GP provokes an increase of plaque forming cells, rosette forming cells, and antibody responses. With the doses that are used, no mechanism can be detected which could be due to an antigenic activity of the GP. The immunostimulating properties decribed cannot be due to a lipopolysaccharide since the preparation contains less than 1 p. 100 of endotoxin.