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Biomedical subjects

P Louisot

Publications and source records attributed to P Louisot.

At least 145 records · Page 8Linked to original sources
PubMed1987

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells.

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.

Breast Neoplasms↗
PubMed1987

Developmental changes in intestinal glycosyl-transferase activities.

The carbohydrate composition of some intestinal glycoproteins has been demonstrated previously to be modified during development. To evaluate the role of the enzymatic mechanisms of glycosylation, the activities of three soluble and microsomal glycosyl-transferases were studied during postnatal development in rat intestinal mucosa. Nonexistent as soluble forms in the cell sap from suckling rats, the membrane-bound N-acetylgalactosaminyl-, sialyl-, and fucosyl-transferases showed different activities in microsomal fractions before weaning. The N-acetylgalactosaminyl-transferase activity was constant whereas the sialyl-transferase activity, which was maintained at a rather high level until the age of 2 wk, decreased just before weaning, while the fucosyl-transferase activity declined progressively from birth to weaning. After weaning, the three enzymatic activities progressively enhanced until adult age. Their variable behaviors on several glycoprotein acceptors may suggest the presence of several sialyl--and fucosyl-transferases with differences in specificities, varying in different ways according to age. Such developmental modifications in intestinal glycosylation patterns may be linked with certain transformation observed in the carbohydrate level of mucus or membrane-bound glycoproteins and related to the profound modifications in nutritional status at the weaning period.

Animals↗
PubMed1986

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes.

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

A brain sialyltransferase having a narrow specificity for O-glycosyl-linked oligosaccharide chains.

The existence of a brain sialyltransferase catalyzing the specific transfer of NeuAc on native fetuin was demonstrated. This enzyme was not able to sialylate either asialofetuin or desialylated and nondesialylated orosomucoid, transferrin, and bovine submaxillary mucin. It required the presence of Mn2+ for optimal activity. Moreover, in fetuin, this activity was closely related to the proportion of NeuAc residues, but in liver tissue sialylation occurred only onto asialofetuin. In native fetuin, sialylation took place on O-glycan chains to give an O-disialyltetrasaccharidic structure. The Gal----GalNAc----protein was not an acceptor, but alpha-NeuAc-(2----3)-Gal----GalNAc----protein was, suggesting a specific transfer alpha-(2----6) to the GalNAc residue.

Animals↗
PubMed1985

Glycosyltransferase activities in liver mitochondria. Phospholipid-dependence of inner membrane mannosyltransferase.

The role of phospholipids in the activity of inner mitochondrial mannosyltransferase was investigated. This enzyme catalyzes the direct transfer from GDP-mannose to lipidic acceptor. Inner mitochondrial membranes from purified mice liver mitochondria are prepared by digitonin treatment. Swelling of mitoplasts leads to the formation of inner membrane vesicles, which are then purified on a discontinuous sucrose gradient. The validity of this fractionation procedure is controlled by measurements of specific enzymatic activities and by electron microscopy. Measurement of mannosyltransferase activity in native inner mitochondrial membranes is unsuccessful, even in the presence of exogenous dolichyl monophosphate. Treatment of inner membranes with specific phospholipid liposomes in the presence of exogenous dolichyl monophosphate is essential in order to measure this enzymatic activity. Addition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin in the presence of Mg2+ results in a high degree of activation of the mannosyltransferase system. Maximal enzymatic activity is obtained with an approximate 3:7 mass ratio of exogenous phospholipid to inner membrane proteins. These experiments establish that sensitivity to activation by phospholipids is an inherent property of inner membrane mannosyltransferase. Another approach to this problem was to reconstitute an in vivo lipidic environment of the inner membrane. The results of this procedure suggest that the activity of inner mitochondrial mannosyltransferase may be subject to modulation by outer membrane lipidic extract treatment.

Animals↗
PubMed1985

Biochemical studies in Niemann-Pick disease. III. In vitro and in vivo assays of sphingomyelin degradation in cultured skin fibroblasts and amniotic fluid cells for the diagnosis of the various forms of the disease.

Sphingomyelinase activities were assayed in vitro in cultured skin fibroblasts of 61 patients with Niemann-Pick disease (NPD). Residual activities found in type A and B were 1% and 4%, respectively, of the mean control values, i.e. significantly higher in type B. In 27 cases with NPD type C, the mean activity was 42% of that in controls, with residual activities ranging from 15% up to normal. Fifteen pregnancies at risk for NPD type A and B were monitored; 4 affected foetuses were found. The uptake of exogenously added radiolabelled sphingomyelin by cultured cells and metabolism of the choline moiety of this lipid were studied in 35 patients with NPD and 14 controls. No difference of uptake between normal and mutant cells was observed. Normally, 77 +/- 5% of the radioactivity taken up was converted to phosphatidylcholine after 18 h incubation, compared to 5 +/- 2% (n = 7) in NPD type A. A substantially greater hydrolysis (31 +/- 12%; n = 8) occurred in NPD type B, and the test allowed complete discrimination between these two types. In NPD type C, 16 patients showed an abnormally low rate of intracellular sphingomyelin degradation (48 +/- 5%) while 4 others were not distinguishable from controls. There was a correlation (r = 0.76) between the results of the in vitro and in vivo assays, but also between the severity of the clinical symptoms and the impairment in sphingomyelin degradation. For the diagnosis of NPD type C, the in vivo test gave more reproducible and more clearcut results than the in vitro assay.

Amniotic Fluid↗
PubMed1985

Involvement of phospholipids in the modulation of a membrane-bound brain fucosyltransferase.

Microsomal fucosyltransferase isolated from sheep brain is strongly enhanced by charged lysophospholipids such as lysophosphatidylinositol and lysophosphatidic acid, while the corresponding phospholipids are inhibitive. Lysophosphatidylcholine (lyso-PC) also greatly increases the enzymatic activity and leads to its solubilization. Its stimulatory effect is related to the length of the fatty acyl chain involved in the lyso-PC structure: fatty acids C18 and C20 are less activating than the fatty acids C14-C16. Stimulation is restored when C18 fatty acids are unsaturated (e.g., C18:1-C18:3). Enzymatic activity enhancement is decreased when phosphatidylcholine structures are reformed by the addition of lyso-PC and the corresponding fatty acid. The physical state of these structures has no influence. These data provide evidence that bilayer structures do not modify enzymatic activity, while micellar structures formed by detergents and lysophospholipids lead to a strong increase in fucosyltransferase activity. However, lyso-PC does not interact in exactly the same way as Triton X-100. Although they both enhance the maximal velocity of fucosyltransferase for its two substrates, GDP-fucose and asialofetuin, the effect with lyso-PC is greater, and it clearly enables a better affinity for GDP-fucose. Endogenous phospholipids are also able to modify enzymatic activity. Hydrolysis of phosphatidylcholine by phospholipase A2 leads to an enzymatic stimulation.

Animals↗
PubMed1984

Subcellular localisation of cerebral fucosyltransferase.

GDP-fucose: asialofetuin fucosyltransferase from sheep brain was fractionated on a sucrose gradient into two activity peaks. Using purification on Ficoll adapted from the proposed method [(1980) J. Neurochem. 35, 281-296], double localisation of cerebral fucosyltransferase was confirmed and the subcellular active fractions identified as light microsomes and mitochondria.

Acetylcholinesterase↗
PubMed1984

Lactic dehydrogenase isoenzyme electrophoretic patterns in the diagnosis of pleural effusion.

The relative value of each lactic dehydrogenase isoenzyme (iso LDH) was measured by electrophoretic separation in the serum and the pleural fluid of 100 patients. In each case, the cause of the pleural effusion was known. Two types of LDH isoenzyme pattern were found in the serum: a normal type with a low value of LDH 5 and an abnormal type with a high value of LDH 5. This high LDH-5 level is due to an impaired liver function. In the pleural fluid, the electrophoretic patterns of five LDH isoenzymes were found by computerized processing. During congestive heart failure (28 cases) the electrophoretic pattern of the LDH isoenzymes was always similar in the serum and in the pleural fluid (transudative pleural effusion). During thoracic empyema, the relative values of the isoenzymes in the pleural fluid were regularly increasing from LDH 1 to LDH 5. In this situation, the evaluation of LDH 5 appeared to emanate from the increased granulocytes in the pleural fluid. In 22 inflammatory pleural effusions, the relative values of the five isoenzymes were equal. During malignant effusions (35 cases) a high level of LDH 5 was found in 21 patients. LDH 5 is known to be secreted by malignant tissue, and the authors confirmed it by finding a high level of LDH 5 in biopsy specimens of patients with mesothelioma or epidermoid lung cancer (7 cases). Conversely, the level of LDH 5 was low in biopsy specimens from normal lung tissue or benign inflammatory pleuritis (6 cases). Among the 14 patients with low levels of LDH 5 in the pleural fluid during malignant pleural effusion, the authors found the malignant lymphomas (three cases) and the small cell lung carcinoma (five cases). In these cases, the low level of LDH 5 was in agreement with the result of a low level of LDH 5 found in the biopsy of a metastatic liver localization of a small cell lung carcinoma. So the electrophoretic determination of LDH isoenzymes pattern in pleural fluid is a sensitive tool for the management of pleural effusion.

Carcinoma, Squamous Cell↗
PubMed1984

Breaks in arrhenius plots of reactions involving membrane-bound and solubilized sialyltransferases, due to temperature dependence of kinetic parameters.

Temperature dependence of asialomucin-sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity is investigated. Discontinuities in Arrhenius plots are observed, whether the enzyme is membrane-associated or solubilized. These discontinuities cannot be firmly correlated with the phase-transition temperatures of either endogenous or exogenous phospholipids. Arrhenius plots of the kinetic parameters also exhibit sharp discontinuities, so that it is concluded that a significant change in Km and Vmax values occurs with varying temperature. Our results suggest that the biphasic behavior of Arrhenius plots may be attributed to the temperature dependence of the kinetic parameters for both membrane-associated and solubilized sialyltransferase activities.

Animals↗
PubMed1984

Modulation of solubilized brain fucosyltransferase activity by phospholipids.

Phospholipids interact on Triton X-100 solubilized GDP-fucose: asialofetuin fucosyltransferase (EC 2.4.1.68) isolated from sheep brain. This enzymatic activity is modulated by charged phospholipids. In particular, phosphatidic acid and analogues markedly inhibit the transfer of fucose from GDP-[14C]fucose. Kinetic studies show that phosphatidic acid interacts as a mixed inhibitor: the velocity and affinity of fucosyltransferase for the GDP-fucose and asialofetuin substrates are strongly decreased. However, this inhibitory effect is not related to stereospecificity, and the different parameters involved in the enzymatic reaction of glycosylation are not modified. The nature of fatty acids and chemical bond (ester or ether) occurring in the carbohydrate chain does not modify the behaviour of phosphatidic acid with respect to fucosyltransferase activity. Further, the physical state of phosphatidic acid (gel phase or liquid crystalline phase) has no influence. However, as the inhibition is closely pH-dependent, these data suggest that phosphatidic acid might directly interact with the active site of the enzyme and induce a conformational change.

Animals↗
PubMed1984

Involvement of some amino acid residues in the enzymatic activity of solubilized cerebral fucosyltransferase.

We have checked the effect of some chemical reagents specific for amino acid residues on the activity of a solubilized cerebral glycoprotein:fucosyltransferase. Diethylpyrocarbonate, 2,3-butanedione and tetranitromethane specific for histidyl, arginyl, and tyrosyl residues respectively, were strong inhibitors of the enzymatic activity This led us to conclude that these amino acid residues are "essential residues" in the cerebral fucosyltransferase activity.

Animals↗
PubMed1984

Temperature dependence of membranous and solubilized sialyltransferase activities in the presence of 1-palmitoyl-sn-glycero-3-phosphorylcholine and fatty acids.

The temperature dependence of sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetyl-neuraminyltrasferase, EC 2.4.99.1) inhibition is described when 1-palmitoyl-sn-glycero-3-phosphorylcholine, or a saturated fatty acid (lauric, myristic or palmitic acid) or an equimolar mixture of the two components are added. Lysophospholipid and fatty acids have no appreciable effect on the optimal temperature for sialyltransferase activity. In the presence of lysophospholipid, the membranous sialyltransferase activity is decreased for all the temperature range tested. In contrast, the solubilized sialyltransferase activity is decreased for temperatures exceeding 29 degrees C. In the presence of saturated fatty acids, the membranous activity is decreased above a chain-length dependent temperature: 22 degrees, 25 degrees and 30 degrees C for lauric, myristic and palmitic acids, respectively. In contrast, the solubilized activity remains unchanged. In the presence of equimolar mixtures of lysophospholipid and fatty acid, the membranous activity is decreased above the same critical temperature as that described for fatty acids added alone. In contrast, the solubilized activity is decreased above 29 degrees C. From these observations, it is suggested that lysophospholipid inhibits the solubilized enzyme when the temperature exceeds the critical micellar temperature of this lipid. The fatty acids inhibit the microsomal enzyme probably by incorporating into the membrane. It is also suggested that equimolar mixtures of lysophospholipid and fatty acid give rise to molecular analogs of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine.

Animals↗
PubMed1984

Glycosyltransferase activities in the rat intestinal mucosa: comparison between standard commercial and semi-synthetic diets.

Two groups of rats were fed either a commercial (C group) or a semi-synthetic (S group) diet of very similar quantitative composition, which induce similar growth in the animals. Glycosyltransferase activities are very different in the two groups: fucosyltransferase is lower in S group than in C group, regardless of the expression of the results (specific activity versus protein or DNA). The other activities (galactosyl-, N-acetylglucosaminyl-, N-acetylgalactosaminyltransferases) are modified when expressed versus DNA content; N-acetylneuraminyltransferase specific activity is not altered. Since the weight of mucosa is significantly lower in the S group than in the C group, all the total activities are decreased in the S group. The fucosylation process is further characterized by partial purification of the enzyme and study of the synthesis of GDP-fucose. In each case, interfering reactions (glycosyl-nucleotide pyrophosphatases and proteinases) are controlled. The results give evidence that glycosyltransferases are very sensitive to the qualitative composition of the diet.

Animals↗
PubMed1984

[Exclusive demonstration of creatine phosphokinase BB isoenzyme in serum, pleural fluid and neoplastic tissue in a case of small-cell pulmonary neoplasm].

The presence of circulating CPK BB isoenzyme is not as rare as was originally supposed. Theoretically damage of organes containing CPK activity should release soluble enzyme into the general circulation. So CPK BB has been reported in sera of patients with brain injury, prostate resection or with gastrointestinal malignant diseases. We present one case of small-cell lung carcinoma associated with 100% of CPK activity type BB in serum. This enzyme pattern was found also in malignant pleural effusion and in the biopsy of the malignant tumour tissue. In this tissue total CPK activity was found markedly more than in normal lung tissues or in epidermoid lung cancers. Brain type CPK is the fetal form of the enzyme and it is possible that our findings is yet another example of tumour expression of a fetal marker in the case of this very undifferentiated tumour.

Carcinoma, Small Cell↗
PubMed1983

Chromatofocusing of skin fibroblast sphingomyelinase: alterations in Niemann-Pick disease type C shared by GM1-gangliosidosis.

Sphingomyelinase activity of cultivated skin fibroblast extracts from normal individuals was resolved by chromatofocusing in the pH range 8-5 into three major components with pI's of 7.3, 6.3 and 5.9, respectively. Chromatofocusing proved a more efficient and reproducible separation technique than preparative flat-bed isoelectric focusing and it gave a constant profile even when detergent concentration varied. In skin fibroblasts from five patients with Niemann-Pick disease type C, a varying degree of reduction in the proportion of the 7.3 peak was observed. In a patient with clinical features of Niemann-Pick disease type C, the finding of such a profile would thus be a good argument for the diagnosis, but it is not pathognomonic as we found similar changes in two cases with GM1-gangliosidosis, while some cases of Niemann-Pick disease type C have borderline normal profiles. These results challenge the concept of a specific sphingomyelinase isoenzyme deficiency as the basic defect in Niemann-Pick disease type C.

Fibroblasts↗