RESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Peptídeo Hidrolases , Neoplasias da Mama/ultraestrutura , Linhagem Celular , Feminino , Humanos , Cinética , Manosefosfatos/metabolismo , Microscopia Eletrônica , Peso Molecular , Proteínas de Neoplasias/genética , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
PURPOSE: The purpose of this paper was to describe local control, overall survival, progression-free survival and toxicity of CyberKnife®-based stereotactic body radiation therapy of hepatocellular carcinoma. MATERIAL AND METHODS: Records of all the patients treated for hepatocellular carcinoma at the Eugene-Marquis cancer centre, Rennes and the Bretonneau hospital, Tours (France), between November 2010 and December 2016, were reviewed. Radiation therapy was performed as a salvage treatment, while awaiting liver transplantation or if no other treatment was possible. RESULTS: One hundred and thirty-six patients were consecutively included in the study. The median follow-up was 13months. Median total dose prescribed, fractionation and overall treatment time were respectively 45Gy, three fractions and 5 days. Overall survival, progression-free survival and local control rates at 1year and 2years were 79.8 % and 63.5 %, 61.3 % and 39.4 %; 94.5 % and 91 %. Two grade 3 acute toxicity events and two grade 4 late toxicity events corresponding to a duodenal ulcer have been reported. Seven patients underwent classic radiation-induced hepatitis and 13 patients showed non-classical radiation-induced hepatitis. Barcelona Clinic Liver Cancer stage, World Health Organisation grade and planning target volume were correlated with overall survival in univariate Cox analysis. CONCLUSION: Stereotactic body radiation therapy is effective and well-tolerated for inoperable hepatocellular carcinoma or as a bridge to liver transplantation. Toxicity is mainly related to cirrhotic background and requires a selection of patients and strict dose constraints.
Assuntos
Carcinoma Hepatocelular/radioterapia , Neoplasias Hepáticas/radioterapia , Radiocirurgia , Dor Abdominal/etiologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/mortalidade , Úlcera Duodenal/etiologia , Feminino , Seguimentos , França/epidemiologia , Hepatite/etiologia , Humanos , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Radiocirurgia/efeitos adversos , Dosagem Radioterapêutica , Estudos Retrospectivos , Terapia de SalvaçãoRESUMO
The management of the documentation is one of the key points regarding the efficacy and the performance of the quality management of health centres. It offers to all professionals the possibility to be informed on the procedures in use, leading to a pool of documents for improvement of organisations and for securing the critical steps of the patient management. In this paper, we will describe the optimal organisation of the documentation according to Haute autorité de santé (HAS) and ISO recommendations, then we will discuss in concrete terms the potential methods usable for the production of a tool well adapted to our routine practice, in order to achieve the objectives for security.
Assuntos
Documentação/normas , Radioterapia/normas , Idioma , Garantia da Qualidade dos Cuidados de Saúde , SegurançaRESUMO
PURPOSE: To analyse a new technique for prostate brachytherapy with permanent Iodine implants characterized by the use of a seed projector after a 3D dosimetric peroperative treatment planning (FIRST technique). PATIENTS AND METHOD: 395 patients have been treated in France with this technique in six radiotherapy centres between November 2002 and December 2005 for a localized prostate cancer. RESULTS: Thirteen patients (3.3%) developped a urinary retention, and respectively 7.8 and 26.5% an acute RTOG grade 3 and 2 toxicity. The 6-weeks IPSS score was equal or lower to 15 in 73% with a 11 median IPSS value. A failure of the loading with the seed-projector, leading to a manual loading of the seeds, occurred in 9 patients (2.3%) in two centres, directly related to the loading procedure with the seed-projector in 5 cases. The median duration of the procedure was reduced by 30 minutes for the patients treated in 2005. CONCLUSIONS: This multicenter study establishes the feasibility of the routine use of a seed projector for permanent iodine 125 prostate implants with an initial tolerance similar to the best results published for other implants techniques.
Assuntos
Braquiterapia/efeitos adversos , Braquiterapia/métodos , Radioisótopos do Iodo/administração & dosagem , Neoplasias da Próstata/radioterapia , Estudos de Viabilidade , Seguimentos , França , Humanos , Imageamento Tridimensional , Masculino , Estadiamento de Neoplasias , Próstata/patologia , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Planejamento da Radioterapia Assistida por Computador , Fatores de Tempo , Retenção Urinária/etiologiaRESUMO
Inner mitochondrial membranes from liver contain a dolichol kinase which required CTP as a phosphoryl donor. Kinase activity was linear with protein concentration and unlike other reported kinases, activated almost equally well by Mg2+, Mn2+ or Ca2+. Thin-layer chromatography showed that the reaction product co-migrated with authentic dolichyl monophosphate. The phosphorylation of dolichol did not occur in presence of ATP, GTP or UTP but required exogenous dolichol for maximal activity. Newly synthesized [3H]dolichyl monophosphate has been shown to be glycosylated in the presence of GDP[14C]mannose or UDP[14C]glucose. The double labeled lipids formed by the sugar nucleotide-dependent reactions were identified respectively as [14C]mannosylphosphoryl[3H]dolichol and [14C]glucosylphosphoryl [3H]dolichol. These results are discussed in terms of regulation of N-glycosylation processes in inner mitochondrial membranes from liver.
Assuntos
Dolicóis/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/metabolismo , Animais , Cátions Bivalentes , Fracionamento Celular , Detergentes/farmacologia , Glicosilação , Técnicas In Vitro , Camundongos , Mitocôndrias Hepáticas/ultraestrutura , Nucleotídeos/metabolismo , Fosfolipídeos/farmacologiaRESUMO
Mitochondrial outer membranes were prepared from mouse liver homogenates by swelling purified mitochondria in phosphate buffer and were purified on a discontinuous sucrose gradient. Assays for marker enzymes and controls in electron microscopy confirmed the purity and homogeneity of this subfraction. Mitochondrial outer membranes had significant galactosyltransferase activity when incubated with UDP-[14C]galactose: 14C-labelling was found in products extractable with organic solvents and in a residual precipitate. Addition of exogenous dolichylmonophosphate loaded into phosphatidylcholine liposomes strongly enhanced the incorporation of [14C]galactose into chloroform/methanol (2:1, v/v) -extractable products. Thin-layer chromatography of these 2:1 extracts showed that the increase of [14C]galactose incorporation was attributable to the synthesis of a new galactosylated lipid, 'lipid L'. This 'lipid L' has been purified on silicic acid columns by elution with chloroform/methanol (1:1, v/v). The purified 'lipid L' was labile in acid and released [14C]galactose. It had the same chromatographic behaviour as dolichylmonophosphate-mannose in neutral, acid and alkaline solvent systems. Upon incubation in presence of [3H]dolichylmonophosphate and UDP-[14C]galactose, purified 'lipid L' contained both 3H- and 14C-labelling. 'Lipid L', synthesized by mitochondrial outer membranes, was therefore characterized as dolichylmonophosphate-galactose.
Assuntos
Fosfatos de Dolicol/biossíntese , Galactose/biossíntese , Galactosiltransferases/metabolismo , Mitocôndrias/enzimologia , Fosfatos de Poli-Isoprenil/biossíntese , Animais , Camundongos , Uridina Difosfato Galactose/metabolismoRESUMO
In the rat small intestine, the glycosylation changes which normally take place at the weaning period are characterized by a shift from sialylation to fucosylation. The introduction of dietary fibers at weaning is one of the more striking nutritional modification so that some authors have suggested that the presence of fibers and the development of colonic fermentation might be important for the development of the small intestine, as for the colon. In order to define the respective contribution of ontogenic and nutritional factors to the intestinal glycosylation changes at this period, some aspects of the intestinal glycosylation were studied in five groups of rats (16-day-old suckling rats, prolonged nursing 23-day-old rats, 23-day-old rats weaned at day 19 with either a fiber-free, a cellulose or a pectin diet). Intestinal glycoproteins of suckling rats are characterized by a low fucose content and a high proportion of mannose. The amounts of the neutral sugars (fucose, mannose and galactose), expressed either per gram of intestine or for one intestine, are always higher in the fiber-fed groups than in the prolonged-nursing group or the group fed the fiber-free diet. Activities which promote fucosylation process (GDP-fucose production and fucosyltransferase activities) and those which are opposed to fucosylation (endogenous inhibitor of fucosyltransferase and GDP-fucose pyrophosphatase) are strongly modified in opposite ways at day 23 as compared to day 16. These modifications depend on the age of the animal (ontogenic factors) with additional modifications induced by the dietary factors. In particular, similar sugar contents and patterns are obtained with cellulose and pectin diets though the enzymatic activities of the fucosylation pathway are very different. No correlation was found between the caecal content of short chain fatty acids and any of the parameters under study. Thus, dietary fibers induce metabolic changes in the small intestine glycosylation in short-term experiments independently of colonic fermentation. Besides, these results point out that the consideration of fucosyl-transferase activities alone are not sufficient to predict glycoprotein fucose content and that other regulatory sites are involved. Dietary manipulations at the weaning period could represent a good model for the study of glycosylation regulation.
Assuntos
Ceco/metabolismo , Fibras na Dieta/administração & dosagem , Fucose/análise , Intestino Delgado/metabolismo , Desmame , Animais , Animais Lactentes , Peso Corporal , Glicoproteínas/isolamento & purificação , Hexosiltransferases/metabolismo , Masculino , Estado Nutricional , Tamanho do Órgão , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Membranas Intracelulares/enzimologia , Microssomos Hepáticos/enzimologia , Sialiltransferases/metabolismo , Transferases/metabolismo , Animais , Cinética , Lipídeos de Membrana/análise , Camundongos , Camundongos Endogâmicos , Fosfolipídeos/análise , Termodinâmica , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Asialomucin-sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity was solubilized from mouse liver microsomes by sonication. The catalytic activity was markedly inhibited by a series of lysophosphatidylcholines, particularly 1-palmitoyl-sn-glycero-3-phosphorylcholine. This lysophospholipid did not alter optimal conditions for enzyme activity. In contrast, it was found that affinities for binding of Mn2+, desialylated mucin and CMP-sialic acid were decreased by adding the lipid. A reasonable interpretation of these data is that the presence of phospholipid modifies the enzyme conformation.
Assuntos
Assialoglicoproteínas , Lisofosfatidilcolinas/farmacologia , Microssomos Hepáticos/enzimologia , Sialiltransferases/antagonistas & inibidores , Transferases/antagonistas & inibidores , Animais , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Manganês/metabolismo , Camundongos , Mucinas/metabolismo , Sialiltransferases/metabolismo , Solubilidade , Sonicação , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
A galactosyltransferase activity is located in the cell-sap of aortic intima-media cells. This enzymatic system calatyzes [14C]galactose transfer from UDP-[14C]galactose into endogenous and exogenous proteinic acceptors. Labelled products are isolated from the proteinic fraction obtained in 20% trichloroacetic acid pellet or from organic solvent extractions. Maximal [14C]galactose incorporation occurs at pH 7.8 in Tris-HCl buffer in the presence of 0.1 mM MnCl2 at 30 degrees C. The enzymatic activity is modified by phospholipids, particularly by phosphatidic acid and lysophosphatidylcholine, which behave as mixed inhibitors, while L-alpha-phosphatidylserine interacts as a competitive inhibitor. The effect of phospholipids is not stereospecific but appeared to be closely related to their polar headgroups, especially the acidic headgroups of phosphatidylcholine and phosphatidic acid. The chain length and the unsaturation degree of fatty acids involved in phospholipid structures are not a main factor of regulation. The lysophosphatidylcholine effect could be explained by its solubilization properties, as non-ionic detergents interact in the same way with galactosyltransferase activity. Exogenous phospholipids probably interact with the enzymatic environment by their own molecular arrangement and so could exert a control on galactosyltransferase activity or lead to a conformation change of this enzyme.
Assuntos
Aorta/enzimologia , Galactosiltransferases/metabolismo , Fosfolipídeos/farmacologia , Fenômenos Químicos , Química , Detergentes , Técnicas In Vitro , SolubilidadeRESUMO
Previous studies have shown the existence of an autonomous mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase, located in mitochondrial outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose:dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.
Assuntos
Guanosina Difosfato Manose/análise , Hexosiltransferases/análise , Membranas Intracelulares/enzimologia , Manosiltransferases/análise , Mitocôndrias Hepáticas/enzimologia , Açúcares de Nucleosídeo Difosfato/análise , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Ativação Enzimática/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Manosiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos , Mitocôndrias Hepáticas/ultraestrutura , Concentração Osmolar , Tripsina/farmacologiaRESUMO
The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated by choline-containing lipids such as phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl phosphate-mannose. The partially purified mannosyltransferase exhibits a Km of 1.33 microM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient centrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.
Assuntos
Hexosiltransferases/isolamento & purificação , Manosiltransferases/isolamento & purificação , Mitocôndrias Hepáticas/enzimologia , Animais , Centrifugação com Gradiente de Concentração , Cromatografia DEAE-Celulose , Cromatografia por Troca Iônica , Detergentes , Fosfatos de Dolicol/farmacologia , Lipossomos/isolamento & purificação , Camundongos , Fosfolipídeos/farmacologia , SolubilidadeRESUMO
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.
Assuntos
Encéfalo/enzimologia , Fucosiltransferases/metabolismo , Hexosiltransferases/metabolismo , Fosfolipídeos/farmacologia , Animais , Radioisótopos de Carbono , Detergentes , Guanosina Difosfato Fucose , Cinética , Octoxinol , Polietilenoglicóis , Ovinos , SolubilidadeRESUMO
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.
Assuntos
Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Assialoglicoproteínas , Encéfalo/enzimologia , Retículo Endoplasmático/enzimologia , Lisofosfatidilcolinas/farmacologia , Sialiltransferases/metabolismo , Estilbenos/farmacologia , Tripsina/farmacologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Animais , Fetuínas , Cinética , Masculino , Fosfolipídeos/farmacologia , Conformação Proteica , Ratos , Ratos Endogâmicos , Especificidade por Substrato , alfa-FetoproteínasRESUMO
Developmental changes in the fucoglycoproteins of the intestinal brush-border membranes were determined by lectin affinoblotting after electrophoresis. Whereas only two alpha(1-6)-fucoglycoproteins were detected in brush-border membranes from suckling rats, a large number of N-fucoglycoproteins with alpha(1-2)- and/or alpha(1-6)-linked fucose residues were detected in rat membranes after weaning. Dietary manipulations at weaning time were used to investigate the effect of nutritional factors in the development of fucosylation in the small intestine of prolonged-nursed rats fed with milk (a high-fat, low-carbohydrate diet) compared to rats weaned normally with a standard high-carbohydrate diet. The fucose content of the mucosa glycoproteins was lower in 22-day-old prolonged-nursed rats than in 22-day-old rats weaned normally with the standard diet. The appearance of fucoglycoproteins in the brush-border membranes, which was delayed by prolonged nursing, was accompanied by a concomitant delay in the increase of intestinal fucosyl-transferase activity and in the decrease of GDP-fucose substrate breakdown. The developmental decrease in the activity of the inhibitory protein which regulates the fucosyl-transferase activity was also delayed by prolonged nursing. The intestinal fucosylation of brush-border membrane glycoproteins (which include many digestive enzymes) displayed ontogenic changes on which were superimposed dietary influences at the time of weaning. The complete maturation of the brush-border membrane glycoproteins, and particularly their terminal fucosylation, is a developmental event which thus seems to be strongly influenced by the manipulation of nutritional factors during the weaning period.
Assuntos
Fucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Microvilosidades/metabolismo , Desmame , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta , Feminino , Fucose/metabolismo , Glicosilação , Guanosina Difosfato Fucose/metabolismo , Guanosina Difosfato Manose/metabolismo , Masculino , Peso Molecular , Ratos , Ratos Sprague-DawleyRESUMO
Human placental sphingomyelinase was highly purified through an original six-step scheme in order to raise a specific rabbit anti-sphingomyelinase antibody. Pure enzyme preparations showed specific activities ranging between 100 and 150 mumol/h per mg protein and gave two constant silver-stained bands (Mr 70,000 and 57,000) on acrylamide after electrophoresis under denaturing conditions. These two bands were the sole areas detected by the described antibody on Western blots from normal placental preparations at various stages of purification. A similar procedure was applied to the separate study of placental sphingomyelinase from two prenatally diagnosed foetuses with confirmed Niemann-Pick disease type A. During purification, the mutant enzyme could be followed owing to its minute but measurable level of catalytic activity, and behaved normally at the various chromatographic steps. In the purified preparations, specific activities of 0.18 and 0.49 mumol/h per mg protein, respectively, were reached. No alteration of the Km value (19 mumol/l) was observed, while the Vmax was 0.5-1% of normal. With immunostaining of Western blots obtained as above, results similar to those described for normal tissue were found, leading to the conclusion that immunoreactive sphingomyelinase is present in Niemann-Pick disease type A.
Assuntos
Doenças de Niemann-Pick/enzimologia , Diester Fosfórico Hidrolases/isolamento & purificação , Placenta/enzimologia , Esfingomielina Fosfodiesterase/isolamento & purificação , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoquímica , Gravidez , Coelhos , gama-GlobulinasRESUMO
The aim of this study was to examine the possible role of certain hormones (especially hydrocortisone) in the developmental variations of intestinal fucosyl-transferase activity in rats. Thyroxine and insulin, injected into suckling rats, did not induce significant modifications of the fucosyl-transferase activity, under the conditions used, whereas this enzyme activity was highly enhanced after administration of glucocorticoids (cortisone and hydrocortisone). Hydrocortisone administration to suckling rats induced a precocious and progressive activation of the fucosyl-transferase activity up to adult level as a function of the duration of treatment. The responsiveness of suckling rats to hydrocortisone, as shown by increased fucosyl-transferase activity, disappeared at the end of the third week (corresponding to the weaning time). These physiological periods of responsiveness and unresponsiveness to hydrocortisone could be related to the binding of the hormones to receptors since the antiglucocorticoid RU 38486 counteracted the effect of hydrocortisone in suckling rats but did not prevent the developmental rise of the fucosyl-transferase activity, when administered in the third week of life. These results suggest that the normal developmental rise of the fucosyl-transferase activity is independent of glucocorticoids.
Assuntos
Animais Lactentes/metabolismo , Fucosiltransferases/metabolismo , Hidrocortisona/farmacologia , Intestino Delgado/enzimologia , Desmame , Animais , Ativação Enzimática/efeitos dos fármacos , Fucosiltransferases/antagonistas & inibidores , Fucosiltransferases/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Hidrocortisona/antagonistas & inibidores , Insulina/farmacologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/crescimento & desenvolvimento , Mifepristona/farmacologia , Ratos , Ratos Endogâmicos , Tiroxina/farmacologiaRESUMO
Ceramides (Cer) are key intermediates in the metabolism of sphingomyelin and are also important second messengers. We report that natural long-chain ceramides added to the incubation medium in microgram amounts are internalized in HL-60 cells as well as the short-chain analogue C2-Cer and targeted to various subcellular compartments. No significant difference was detected in the ability of HL-60 cells to metabolize exogenous Cer containing a short (acetyl) versus long (palmitoyl or oleoyl) acyl chain. After a 2-h incubation time with [14C]-C16 ceramides, most of the cell-bound radioactivity was found in free ceramides. Sphingomyelin was the major metabolized sphingolipid containing labeled ceramides and only a small proportion of exogenous ceramides were converted to neutral glycolipids and gangliosides. Up to 20% of the exogenous ceramides taken up by the cells were recovered in mitochondria, mostly as authentic C16 ceramides and C16 sphingomyelin, along with a trace amount of labeled GM3 ganglioside. These results are consistent with the notion that exogenous natural ceramides enter cells, can be further metabolized in situ and partly targeted to mitochondria, which are known to be involved in the control of programmed cell death.
Assuntos
Ceramidas/metabolismo , Mitocôndrias/metabolismo , Radioisótopos de Carbono , Ceramidas/química , Ceramidas/farmacocinética , Gangliosídeo G(M3)/metabolismo , Glicolipídeos/metabolismo , Células HL-60 , Humanos , Esfingomielinas/metabolismo , Frações SubcelularesRESUMO
A sialyl transferase activity is found in purified mitochondria. It is not due to residual contamination and this enzymatic system is located in the outer mitochondrial membrane. This proves mitochondrial autonomy in regard to glyconconjugate sialylation.
Assuntos
Mitocôndrias/enzimologia , Sialiltransferases/metabolismo , Transferases/metabolismo , Membranas Intracelulares/enzimologiaRESUMO
Addition of low-density lipoprotein (LDL) to cholesterol-deprived human skin fibroblast cultures treated by imipramine at a 20 microM concentration induced a significant intracellular accumulation of unesterified cholesterol. Intracytoplasmic inclusions were already visible by histochemical filipin staining after 2 h of LDL uptake and were progressively mobilized towards the perinuclear region within 24 h. At this concentration of the drug, the rate of proteolytic 125I-LDL hydrolysis was similar in treated and untreated cells. Treated cells maintained in lipoprotein-deficient medium showed no abnormality, indicating the exogenous origin of the accumulated sterol. Further, the drug induced a drastic dose-dependent impairment of LDL-stimulated cholesterol esterification, not related to an inhibition of acyl CoA:cholesterol acyltransferase, and a significant delay in down-regulation of de novo cholesterol synthesis. However, imipramine did not affect 25-hydroxycholesterol-mediated regulation of the two latter processes. These results resemble those observed in Niemann-Pick type C disease and suggest an impaired mobilization of LDL-derived cholesterol in imipramine-treated cells.