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1.
Arch Biochem Biophys ; 394(2): 201-8, 2001 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-11594734

RESUMEN

Cytosolic sulfotransferases play essential roles in regulating the activities and transfer of steroids. To evaluate their biological significance in the murine uterus and placenta during the course of gestation, we determined their activities with several steroids as substrates. Activated estrogen sulfotransferase (EST) was found in the placenta and uterus during the late gestational period. Reverse-transcribed cDNA of murine placental EST (mpEST) was isolated from mouse placenta at 18 days of gestation and its expression in the tissue coincided with a change in its enzyme activity. The open-reading frame of mpEST encodes a protein composed of 296 amino acids with a predicted molecular mass of 35.5 kDa and was revealed to be an isoform of the murine testis-specific EST gene (99.7%). Also, the amino acid sequence of mpEST showed 49.6 and 77.9% homology with human placental and endometrial EST, respectively, showing that it corresponds to human endometrial EST. COS-7 cells transfected with mpEST exhibited sulfotransferase activity with the phenolic hydroxy groups of steroids and artificial substrates. The best acceptor substrate was estrogen.


Asunto(s)
Placenta/enzimología , Sulfotransferasas/biosíntesis , Testículo/enzimología , Animales , Secuencia de Bases , Células COS , Clonación Molecular , ADN Complementario/aislamiento & purificación , Activación Enzimática , Femenino , Expresión Génica , Edad Gestacional , Isoenzimas/biosíntesis , Isoenzimas/genética , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Especificidad de Órganos , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Sulfotransferasas/genética , Transfección , Útero/enzimología
2.
Arch Biochem Biophys ; 388(2): 207-15, 2001 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-11368156

RESUMEN

We cloned three members of a GDP-fucose:beta-galactoside alpha1,2-fucosyltransferase (alpha1,2-fucosyltransferase) family, MFUT-I, -II, and -III, from a cDNA of murine small intestine, and determined their enzymatic properties after transfection of the genes into COS-7 cells, and their expression in murine tissues by Northern blotting. MFUT-I, -II, and -III exhibited sequence homology with the human H (78.4%), Se (79.0%), and Sec1 (74.9%) gene products, respectively. COS-7 cells transfected with MFUT-I and -II exhibited alpha1,2-fucosyltransferase activity and the best acceptor substrate for both gene products was GA1 to yield a fucosyl GA1 structure, but no activity was detected in COS-7 cells with MFUT-III. MFUT-II yielded a 3.5-kb mRNA transcript in several tissues, whereas MFUT-I and -III were predominantly expressed in epididymis and testis, respectively. The administration of microbes into germ-free mice resulted in a rapid increase of the MFUT-II gene (Se gene) for the synthesis of fucosyl GA1 in the intestine.


Asunto(s)
Fucosiltransferasas/genética , Intestinos/enzimología , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Helmínticos , Secuencia de Bases , Células COS , Clonación Molecular , ADN/análisis , Fucosiltransferasas/clasificación , Fucosiltransferasas/aislamiento & purificación , Proteínas del Helminto/química , Humanos , Intestinos/microbiología , Ratones , Datos de Secuencia Molecular , Proteínas Munc18 , Proteínas del Tejido Nervioso/química , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Transfección , Galactósido 2-alfa-L-Fucosiltransferasa
3.
Dig Dis Sci ; 46(1): 69-74, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11270796

RESUMEN

The Mongolian gerbil has been used as an excellent experimental animal model for studying Helicobacter pylori infection because it can stably colonize and induce severe chronic gastritis, ulceration, and cancer-simulating human diseases in this animal. In contrast, H. pylori can only induce mild inflammation in many mouse models. The aim in this study is to clarify the difference of induction of pathological lesions in the two animal models. SPF ICR mice and Mongolian gerbils were inoculated with a clinically isolated strain of H. pylori. Six weeks after inoculation, bacteria colonizing the stomach were counted. Immunohistochemical staining and biochemical analyses of three putative receptor glycolipids were performed with monoclonal antibodies to the respective glycolipids. Significantly higher numbers of H. pylori were recovered from the stomachs of Mongolian gerbils than mice (5.77 +/- 0.46 log CFU vs 4.17 +/- 0.55 log CFU, P < 0.01). Immunohistochemical studies showed that sulfatide expression in the gastric mucosa of Mongolian gerbils was much stronger than that in mice, whereas the expression of Lewis(b) glycolipid and GM3 were almost equal. Quantitative analysis of each glycolipid by thin-layer chromatography confirmed the results of immunohistochemical study, showing 4.1 times higher sulfatide content in the Mongolian gerbil stomach. The content of both Lewis(b) and GM3 was almost equivalent in these two animals. In conclusions, higher levels of sulfatide expression, a putative adhesion receptor, in the gastric mucosa of Mongolian gerbils may allow abundant colonization by H. pylori, resulting in the development of gastric lesions in this animal model.


Asunto(s)
Helicobacter pylori/crecimiento & desarrollo , Receptores de Superficie Celular/análisis , Animales , Cromatografía en Capa Delgada , Modelos Animales de Enfermedad , Gerbillinae , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Organismos Libres de Patógenos Específicos , Sulfoglicoesfingolípidos/análisis
4.
Biochim Biophys Acta ; 1535(3): 285-93, 2001 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-11278168

RESUMEN

Peroxisome biogenesis disorders (PBD) are classified into Zellweger syndrome (ZS), infantile Refsum disease (IRD) and neonatal adrenoleukodystrophy. Disturbances in the differentiation of neural cells such as migration arrest are characteristic of PBD. So far the pathogenesis of these disturbances is not clearly understood. We describe an altered metabolism of glycosphingolipids in PBD which has not yet been investigated. We observed an increased amount of a-series gangliosides, GM2, GM1 and GD1a, in the fibroblasts of patients with ZS and IRD. Gangliosides GM1 and GD1a were not present in detectable amounts in normal subjects. A key step in the synthesis of a-series gangliosides is a transfer of GalNAc to ganglioside GM3, so we determined the level of ganglioside GM3 by immunohistochemical methods. We found a granular structure, which was positive toward anti-ganglioside GM3 antibody in the cytoplasm of the patients' fibroblasts. In control cells, the cell membrane was slightly positive toward anti-GM3 antibody. These results may help to clarify the pathogenesis of PBD with respect to the functional roles of glycosphingolipids in cell differentiation, proliferation and apoptosis.


Asunto(s)
Fibroblastos/metabolismo , Gangliósidos/metabolismo , Trastorno Peroxisomal/metabolismo , Gangliósido G(M3)/análisis , Gangliósidos/análisis , Gangliósidos/biosíntesis , Humanos , Inmunohistoquímica , Lactante , Trastorno Peroxisomal/etiología , Enfermedad de Refsum/metabolismo , Síndrome de Zellweger/metabolismo
5.
Biochim Biophys Acta ; 1531(1-2): 90-8, 2001 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-11278175

RESUMEN

In order to clarify the pathological outcome of congenital diaphragmatic hernia (CDH), we devised an animal model of CDH by administration of 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) to pregnant rats, and determined the level and distribution of lung surfactant using the monoclonal antibody toward sphingomyelin and disaturated phosphatidylcholine (disat-PC). In control rats, the concentration of disat-PC was found to increase greatly from 16 to 18 days of gestation. Intragastric administration of nitrofen to pregnant rats at day 9 of gestation resulted in CDH in 42.7% of fetuses delivered after 20 days of gestation. In nitrofen-treated fetuses, the concentration of disat-PC in the lungs was lower than those in control fetuses, and surfactant apoprotein SP-A was similarly reduced in nitrofen-treated fetuses. However, the concentration of disat-PC in nitrofen-treated fetuses was higher than that in control fetuses at 18 days of gestation, indicating a synthetic potential of surfactant in nitrofen-treated fetuses comparable to that at the late stage of normal gestation. Immunohistochemical study with the antibody revealed that surfactant phospholipid was mainly in the form of intracellular granules in nitrofen-treated fetuses, probably causing the hypoplastic lungs and then CDH, in contrast to the uniform distribution on the pulmonary alveolar surface in control fetuses.


Asunto(s)
Hernias Diafragmáticas Congénitas , Pulmón/metabolismo , Surfactantes Pulmonares/metabolismo , Líquido Amniótico/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Peso Corporal , Cromatografía en Capa Delgada , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Herbicidas , Hernia Diafragmática/inducido químicamente , Inmunohistoquímica , Pulmón/anomalías , Pulmón/química , Tamaño de los Órganos , Éteres Fenílicos , Fosfatidilcolinas/análisis , Surfactantes Pulmonares/química , Ratas , Ratas Sprague-Dawley , Esfingomielinas/análisis
6.
Biochim Biophys Acta ; 1487(2-3): 268-74, 2000 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-11018478

RESUMEN

Cholesterol sulfate (CS) and sulfatides in the epithelium of the digestive tract were found in the 1000xg supernatants of digestive fluid, particularly in gastric juices containing the duodenal contents and bile acids, there being 14-131 microg of CS and 3-54 microg of sulfatides per mg of protein in the fluid, respectively. CS and sulfatides dissolved in detergents including bile acids inactivated pancreatic trypsin to the same level as by DMSO-solubilized sulfated lipids at 37 degrees C. Similarly, pancreatic DNase I was inhibited by CS solubilized with DMSO or bile acids, but not by sulfatides or other membrane lipids at 37 degrees C. Both the sulfate group and the hydrophobic side chain of CS were indispensable structures for the inhibition of DNase I. Also, the optimum molar ratio of bile acids to CS was important for expression of the inhibitory activity of CS toward DNase I, it being 0.18 of the optimum ratio for sodium taurocholate, and the molar ratio of CS to DNase I for complete inhibition was 342:1. Thus, CS was shown to play a role as an epithelial inhibitor of DNase I in concert with bile acids.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Ésteres del Colesterol/metabolismo , Desoxirribonucleasa I/antagonistas & inhibidores , Jugo Gástrico/metabolismo , Páncreas/enzimología , Adulto , Anciano , Ésteres del Colesterol/química , Ésteres del Colesterol/farmacología , Detergentes , Endopeptidasas/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Lípidos/análisis , Lípidos/farmacología , Masculino , Persona de Mediana Edad , Inhibidores de Serina Proteinasa/farmacología , Sulfatos/análisis , Sulfoglicoesfingolípidos/química , Tripsina/química
7.
Biochim Biophys Acta ; 1487(2-3): 275-85, 2000 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-11018479

RESUMEN

A shift from sialylation to fucosylation of mucosal glycoconjugates occurred in the mammalian digestive tract in the weaning period, but mice under germ-free conditions were found to express both fucosyl GM1 (FGM1) and fucosyl asialo GM1 (FGA1) in the stomach, cecum and colon, but not in the small intestine. By host-microbe interactions and administration of cycloheximide, FGA1 was quickly induced in the small intestine, but the concentrations of fucosylated glycolipids in the other regions were not altered significantly. Their expression coincided with the activity of GDP-fucose:GA1 alpha(1, 2)-fucosyltransferase (alpha1,2-FT), and we isolated a cDNA with an open reading frame encoding the murine alpha1,2-FT (MFUT-II) of 347 amino acids with a predicted molecular mass of 39.21 kDa. The intraperitoneal injection of cycloheximide induced the mRNA and activity of alpha1,2-FT (MFUT-II) in the small intestine of germ-free mice, whereas no change in the mRNA or activity was observed in the stomach, cecum and colon, indicating that expression of FGA1 in response to microbial colonization or cycloheximide is transcriptionally regulated in a restricted region of the murine digestive tract. At 24 h after the administration of cycloheximide, FGA1 was preferentially produced in the upper half of the duodenal microvilli.


Asunto(s)
Cicloheximida/farmacología , Sistema Digestivo/metabolismo , Fucosiltransferasas/biosíntesis , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/análisis , Autorradiografía , Secuencia de Bases , Células COS , Cromatografía en Capa Delgada , Clonación Molecular , Sistema Digestivo/enzimología , Epitelio/metabolismo , Fucosiltransferasas/genética , Vida Libre de Gérmenes , Glucolípidos/análisis , Inmunohistoquímica , Intestino Delgado/enzimología , Intestino Delgado/microbiología , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección
8.
Glycoconj J ; 17(11): 787-93, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11443280

RESUMEN

Amphipathic sulpholipids have been shown to inhibit pancreatic serine proteases due to their detergent-like properties. To evaluate the structural requirement for this inhibitory activity, we examined the effects of various derivatives of sulphoglycolipids, some of which were prepared by deacylation with sphingolipid ceramide N-deacylase, followed by acylation with acyl chloride, on the activity of pancreatic trypsin. Both deacylated sulphatides and seminolipids exhibited inhibitory activity towards trypsin without any requirement for solubilisation and preincubation. On the other hand, stronger inhibition was observed for acylated sulphatides than for deacylated ones, but increasing the chain length of the fatty acid moiety resulted in the need for a solubilisation agent and preincubation in order to achieve maximal inhibitory activity. The structural isomers of sulphoglycolipids, such as I(6)SO(3)-GalCer, and phytosphingosine- and diglyceride-containing sulphoglycolipids, showed similar inhibitory activity, indicating the involvement of sulphate and hydrophobic groups, irrespective of the fine structure, in the inhibition. Among the sulphoglycolipids examined, II(3)SO(3)-LacCer was found to exhibit the highest inhibitory activity.


Asunto(s)
Glucolípidos/farmacología , Páncreas/enzimología , Sulfoglicoesfingolípidos/farmacología , Inhibidores de Tripsina/farmacología , Tripsina/efectos de los fármacos , Animales , Detergentes/síntesis química , Detergentes/farmacología , Glucolípidos/síntesis química , Sulfoglicoesfingolípidos/síntesis química , Porcinos , Inhibidores de Tripsina/síntesis química
9.
Biochim Biophys Acta ; 1438(1): 55-62, 1999 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-10216280

RESUMEN

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.


Asunto(s)
Glucolípidos/metabolismo , Glicoesfingolípidos/metabolismo , Trastorno Peroxisomal/etiología , Animales , Células CHO , Cricetinae , Mutación
10.
J Biochem ; 125(3): 594-601, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10050049

RESUMEN

Thrombin and plasmin, both of which are serine proteases in the plasma of vertebrates, play essential roles in blood clotting and fibrinolysis, respectively, and regulation of their activities is important to suppress the excessive reactions within the vascular network and to prevent tissue injury. Along with the peptidic inhibitors belonging to the serpin family, we found that cholesterol sulfate (CS), which is present at the concentration of 2.0+/-1.2 nmol/ml in human plasma, was a potent inhibitor of both plasma thrombin and plasmin. Thrombin, as determined both using a chromogenic substrate and the natural substrate, fibrinogen, was inactivated upon reaction with CS in a dose-dependent manner, but not in the presence of the structurally related steroid sulfates, I3SO3-GalCer and II3NAalpha-LacCer, suggesting that both the sulfate group and the hydrophobic side chain of CS are necessary for the inhibitory activity of CS. Preincubation of thrombin with CS at 37 degrees C for 10 min was required to achieve maximum inhibition, and virtually complete inhibition was achieved at a molar ratio of CS to thrombin of 18:1. CS-treated thrombin had the same Km and a lower Vmax than the original enzyme, and a higher molecular weight. The molecular weight and activity of the original enzyme were not observed on the attempted separation of the CS-treated enzyme by gel permeation chromatography and native PAGE, indicating that the inactivation of thrombin by CS is irreversible. In contrast, CS was readily liberated from the enzyme by SDS-PAGE, suggesting that hydrophobic interactions are involved in the CS-mediated inactivation of thrombin. When acidic lipids were reacted with thrombin after dissolving them in DMSO, I3SO3-GalCer, steroid sulfates and II3NAalpha-LacCer, as well as CS, but not SDS and sodium taurocholate, exhibited inhibitory activity, probably due to micellar formation facilitating interaction between thrombin and negatively charged lipids. On the other hand, plasmin, as determined using a chromogenic substrate, was more susceptible to acidic lipids than thrombin. CS, I3SO3-GalCer and II3NAalpha-LacCer, all of which are present in serum, inhibited the activity of plasmin in aqueous media, as well as in DMSO-mediated lipid solutions. Thus, acidic lipids in plasma were demonstrated to possess regulatory activity as endogenous detergents toward both enzymes for blood clotting and fibrinolysis.


Asunto(s)
Ésteres del Colesterol/farmacología , Fibrinolisina/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/farmacología , Trombina/antagonistas & inhibidores , Ésteres del Colesterol/metabolismo , Fibrinolisina/metabolismo , Fibrinólisis/efectos de los fármacos , Humanos , Inhibidores de Serina Proteinasa/metabolismo , Trombina/metabolismo
11.
J Invest Dermatol ; 111(6): 973-81, 1998 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9856804

RESUMEN

Recent evidence suggests that cholesterol sulfate may be an important second messenger involved in signaling epidermal differentiation in skin. The activity of cholesterol sulfotransferase (Ch-ST) is increased during squamous differentiation of keratinocytes and is believed to be a marker enzyme for terminal differentiation. The primary objective of this study was to examine changes in levels of cholesterol sulfate (CS) and activity of its biosynthetic enzyme, Ch-ST, during multistage carcinogenesis in mouse skin. Using SENCAR mice, we determined the activity of Ch-ST in normal epidermis, in tumor promoter-treated epidermis, in epidermis during wound healing, and in mouse skin tumors generated by initiation-promotion regimens. A single topical application of tumor promoters led to significantly elevated levels of Ch-ST activity and of CS. Epidermal Ch-ST activity was also elevated during wound healing. Dramatic increases in CS levels and in the activity of Ch-ST were found in nearly all of the papillomas and squamous cell carcinomas examined. The increased levels of CS and activity of Ch-ST in tumor promoter-treated epidermis were accompanied by increased transglutaminase-I activity. In contrast, transglutaminase I activity was not elevated in primary papillomas or squamous cell carcinomas. Finally, Ch-ST activity was significantly elevated in the epidermis of newborn HK1.ras transgenic mice, whereas transglutaminase I activity did not correlate with Ch-ST activity in these mice. These results demonstrate that diverse tumor-promoting stimuli all produce elevated CS levels and Ch-ST activity and that CS levels and Ch-ST activity were constitutively elevated in both papillomas and squamous cell carcinomas. The data also suggest a mechanism for upregulation of Ch-ST in skin tumors involving activation/upregulation of Ha-ras.


Asunto(s)
Anticarcinógenos/metabolismo , Ésteres del Colesterol/metabolismo , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/metabolismo , Sulfotransferasas/metabolismo , Administración Tópica , Animales , Antracenos/farmacología , Carcinógenos/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Sulfato de Deshidroepiandrosterona/farmacología , Epidermis/efectos de los fármacos , Epidermis/enzimología , Femenino , Queratinocitos/citología , Ratones , Ratones Endogámicos SENCAR , Ratones Transgénicos , Ácido Ocadaico/farmacología , Neoplasias Cutáneas/etiología , Acetato de Tetradecanoilforbol/farmacología , Transglutaminasas/metabolismo , Cicatrización de Heridas/fisiología
12.
Clin Cancer Res ; 4(12): 2985-90, 1998 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9865910

RESUMEN

The expression of cholesterol sulfate (CS) is known to increase during squamous differentiation of keratinocytes and to activate the epsilon, eta, and zeta forms of protein kinase C as a signal transduction molecule for the subsequent expression of transglutaminase-1 (TG-1) and cytokeratins. To gain further insight into the regulation of cellular differentiation and tumorigenesis by CS, we examined the concentration and the potential for synthesis of CS in seven and four surgical specimens from human ovarian and uterine cervical cancer patients, respectively, and eight cell lines established from human uterine cervical cancer patients and compared them for the rate of expression of cytokeratin. CS was present in all of the uterine cervical cancer tissue specimens but only in the mucinous type of cystadenocarcinoma among ovarian cancer tissue specimens, and cytokeratin was highly expressed in the tissues with a high concentration of CS, which were classified as well-differentiated on the basis of morphological examination. Similarly, cells derived from a keratinizing type of well-differentiated cervical carcinoma demonstrated strong potential for synthesis of CS, stained positive with anti-cytokeratin antibody, and exhibited a higher specific activity of TG-1, whereas the cells without CS did not stain positive with anti-cytokeratin antibody and exhibited a lower specific activity of TG-1. These findings indicate that CS is coexpressed with TG-1 and cytokeratin in the well-differentiated types of squamous cell cancers as a tumor marker.


Asunto(s)
Biomarcadores de Tumor/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Ésteres del Colesterol/biosíntesis , Queratinas/biosíntesis , Neoplasias del Cuello Uterino/metabolismo , Neoplasias Uterinas/metabolismo , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Inmunohistoquímica , Metabolismo de los Lípidos , Lípidos , Neoplasias Ováricas/metabolismo , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/patología , Neoplasias Uterinas/patología
13.
Jpn J Cancer Res ; 89(9): 923-32, 1998 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9818028

RESUMEN

Cells of the human tumor cell line RMG-1, derived from a clear-cell adenocarcinoma of the ovary, were injected intraperitoneally into nude mice, and the cells obtained from the tumor nodules in the mesenterium were found to form a larger number of, and larger-sized, tumor nodules than the original RMG-1 cells. The RMG-1-h cells, transferred into culture from the tumor nodules after a 4th in vivo passage, showed a dissemination potential as high as that of cells disseminating directly from the tissues, and exceedingly higher than that of RMG-1 cells. To assess the molecular bases of the different biological properties of RMG-1 and RMG-1-h cells, we compared the content and expression of various carbohydrate antigens in both cells. The chromosomal profile of RMG-1-h cells revealed their human origin and was identical to that of the original RMG-1 cells. In contrast to the broad histogram for the Le(x)-bearing cells among RMG-1 cells in flow cytometry, the weakly and moderately positive cells toward anti-Le(x) antibody were found to be eliminated from the histogram for the RMG-1-h cells, resulting in the enrichment of cells strongly expressing Le(x), which may account for the high dissemination potential. In addition, the adhesion of RMG-1 cells to mesothelial cells was found to be significantly inhibited by pretreatment of the cells with anti-Le(x) antibody, indicating Le(x)-mediated cell-to-cell interaction between ovarian cancer cells and mesothelial cells. By TLC-immunostaining, two Le(x)-glycolipids, III3Fuc alpha-nLc4Cer and V3Fuc alpha-nLc6Cer were detected in both RMG-1 and RMG-1-h cells, and their total concentrations were not significantly different from each other. However, the hydrophobic moieties of Le(x)-glycolipids in RMG-1-h cells were different from those in RMG-1 cells, suggesting that a difference in the structure of the hydrophobic moieties of Le(x) is partly involved in the enhanced reactivity of RMG-1-h cells toward anti-Le(x) antibody. Thus, the high dissemination potential of ovarian cancer cells was shown to be mediated by the Le(x)-determinant and the Le(x)-bearing cells are enriched by repeated in vivo passage of the cells into nude mice.


Asunto(s)
Adenocarcinoma de Células Claras/patología , Antígeno Lewis X/fisiología , Metástasis de la Neoplasia , Neoplasias Ováricas/patología , Adenocarcinoma de Células Claras/inmunología , Animales , Adhesión Celular , Separación Celular , Cromatografía en Capa Delgada , Epitelio/patología , Femenino , Citometría de Flujo , Glucolípidos/análisis , Humanos , Cariotipificación , Antígeno Lewis X/análisis , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Ováricas/inmunología , Espectrometría de Masa Bombardeada por Átomos Veloces , Células Tumorales Cultivadas
14.
Biochim Biophys Acta ; 1406(3): 327-35, 1998 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-9630707

RESUMEN

Niemann-Pick disease type C (NP-C) is an autosomal recessive disorder characterized by the sequestration and trapping of endocytosed cholesterol in lysosomes. The NPC1 gene on chromosome 18 was recently identified but its physiological function remains unknown. We have studied the lipid compositions of cultured human NP-C fibroblasts and mouse SPM-3T3 cell line derived from the C57BL/KsJ NP-C model mouse, which belongs to the same complementation group. Fibroblasts derived from apparently normal age-matched individuals and a subline of SPM-3T3 cells which restores cholesterol metabolism by transfer of human chromosome 18 were used as controls. Levels of free cholesterol in whole cell homogenates increased about 1.5-fold in human NP-C fibroblasts and mouse SPM-3T3 cells, while in the plasma membrane, cholesterol content did not significantly change in NP-C fibroblasts but rather decreased in SPM-3T3 cells. The total phospholipid content did not significantly change; however, among phospholipid head groups, increases in sphingomyelin and decreases in other classes were observed in human NP-C fibroblasts and mouse SPM-3T3 cells. The ratios of saturated fatty acids to unsaturated fatty acids increased in both human and mouse cells. The increase was also confirmed in the plasma membrane fraction of SPM-3T3 cells. Membrane fluidity was examined using a 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probe. The DPH anisotropy values were markedly increased in NP-C fibroblasts and in SPM-3T3 cells. The results suggest that a NP-C mutation causes complex alterations in cellular lipid contents and biophysical properties of the membrane.


Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fluidez de la Membrana , Enfermedades de Niemann-Pick/metabolismo , Enfermedades de Niemann-Pick/patología , Células 3T3 , Adulto , Animales , Línea Celular , Células Cultivadas , Niño , Preescolar , Colesterol/metabolismo , Ácidos Grasos/análisis , Ácidos Grasos Insaturados/análisis , Polarización de Fluorescencia , Humanos , Interfase , Lisofosfatidilcolinas/metabolismo , Ratones , Persona de Mediana Edad , Fosfolípidos/metabolismo
15.
J Biochem ; 123(1): 107-14, 1998 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9504416

RESUMEN

Sulfated lipids, cholesterol sulfate (CS) and I3SO3-GalCer, are commonly present in the epithelia of the digestive tracts of pigs, humans, rabbits, and rats. CS was the only sulfated lipid in the esophageal epithelia of these mammals, and I3SO3-GalCer, together with CS, was detected in the epithelia of the gastrointestinal tracts, at a concentration higher than 0.05 micromol per gram of dry weight. Although no sulfated lipids were present in the pancreatic duct, they were found in relatively high concentrations in the duodenal, jejunal, and ileal epithelia. To elucidate the functional significance of sulfated lipids in the digestive tract, we determined the effect of CS and I3SO3-GalCer on the activities of pancreatic and Pseudomonas aeruginosa elastases and found that both characteristically inhibited the pancreatic elastase but not the P. aeruginosa elastase. Desulfation of CS and I3SO3-GalCer abolished their inhibitory activity, and other membrane constituents including free fatty acids, phospholipids, and gangliosides failed to inhibit pancreatic elastase. In addition, steroid sulfates, such as dehydroepiandrosterone sulfate and pregnenolone sulfate, did not exhibit any inhibitory activity toward pancreatic elastase, indicating that the sulfate group and a suitable hydrophobic side chain are required in the inhibition of elastase. Inhibition of elastase by sulfated lipids occurred in a dose-dependent manner, and the molar ratios of CS and I3SO3-GalCer to elastase at which the enzyme activity was inhibited to 50% of the maximum level were 6:1 and 9:1, respectively. CS-treated elastase had the same Km and a lower Vmax compared with the untreated enzyme, and sulfated lipids were observed to bind tightly to the enzyme, suggesting irreversible inhibition. Thus, CS and I3SO3-GalCer in the digestive tracts of mammals were shown to function as epithelial inhibitors of pancreatic elastase.


Asunto(s)
Proteínas Bacterianas , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos , Elastasa Pancreática/antagonistas & inhibidores , Sulfatos/metabolismo , Sulfoglicoesfingolípidos , Animales , Ésteres del Colesterol/metabolismo , Galactosilceramidas/metabolismo , Humanos , Mucosa Intestinal/enzimología , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Elastasa Pancreática/metabolismo , Conejos , Ratas , Porcinos
16.
Biochem Biophys Res Commun ; 237(2): 262-5, 1997 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-9268697

RESUMEN

Cholesterol sulfate and I3SO3-GalCer, which were commonly contained in the epithelia of mammalian digestive tracts, were found to inhibit the activities of typical digestive enzymes, pancreatic trypsin and chymotrypsin, but steroid sulfates, gangliosides and the other membrane constituents did not show any inhibitory activity. The preincubation of trypsin with I3SO3-GalCer at 37 degrees C for 10min was necessary to inhibit the activity of trypsin, but cholesterol sulfate showed its inhibitory activity without preincubation. Sulfated lipid-treated enzyme gave the same Km as and lower Vmax than those of the original enzymes. Also, both sulfated lipids inhibited pronase from Streptomyces griseus, but not lysyl endopeptidase from Achromobacter lyticus. These findings indicate that cholesterol sulfate and I3SO3-GalCer in the digestive tract function as epithelial inhibitors to prevent tissue injury by endogenous and exogenous proteases.


Asunto(s)
Quimotripsina/antagonistas & inhibidores , Mucosa Gástrica/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Lípidos/farmacología , Páncreas/efectos de los fármacos , Inhibidores de Tripsina/farmacología , Animales , Mucosa Gástrica/enzimología , Humanos , Mucosa Intestinal/enzimología , Lípidos/química , Páncreas/enzimología , Conejos , Ratas , Sulfatos/química
17.
Jpn J Cancer Res ; 88(7): 669-77, 1997 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9310140

RESUMEN

We have developed a new procedure for the selective determination of beta 1-3 and beta 1-4 galactosyltransferases with Lc3Cer as the substrate and the microsomes of fetal and adult porcine livers as the enzyme sources. This method was based on the detection of such products as Lc4Cer for beta 1-3 galactosyltransferase (beta 1-3GT) and nLc4Cer for beta 1-4 galactosyltransferase (beta 1-4GT), with monoclonal anti-Lc4Cer and anti-nLc4Cer antibodies, respectively. This method thus enabled us to differentiate the activity of beta 1-3GT from that of beta 1-4GT with a high degree of sensitivity. The method was then used to determine the activities of both enzymes in human gynecological carcinoma-derived cells. Four of the five cell lines derived from uterine endometrial cancer expressed significantly high levels of specific activity of beta 1-3GT among the cell lines examined, while their beta 1-4GT activities were less than 20% of that for beta 1-3GT in the endometrial carcinoma-derived cells. On the other hand, a higher specific activity of beta 1-4GT than that of beta 1-3GT was detected in the cell lines derived from uterine cervical and ovarian cancers. These findings were thus found to correlate closely with the rate of expression of Lc4Cer- and nLc4Cer-based carbohydrate chains in the cell lines based on the results of immunohistochemical staining.


Asunto(s)
Neoplasias Endometriales/enzimología , Galactosiltransferasas/metabolismo , Uridina Difosfato Galactosa/biosíntesis , Animales , Secuencia de Carbohidratos , Carbohidratos/biosíntesis , Cromatografía en Capa Delgada , Ensayo de Inmunoadsorción Enzimática , Femenino , Neoplasias de los Genitales Femeninos/enzimología , Neoplasias de los Genitales Femeninos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Porcinos , Células Tumorales Cultivadas
18.
Lipids ; 32(6): 599-604, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9208388

RESUMEN

Cholesterol sulfate (CS) recently has been shown to be involved in signal transduction pathway. To evaluate its functional significance, we determined the concentration of CS, and the specific activities of cholesterol sulfotransferase and CS sulfatase in various tissues of rabbit, and compared them with the concentration of sulfoglycolipids in rabbit tissues. CS was present in the epithelia and mucosa, but not in the tunica muscularis, of the digestive tract, trachea, uterine endometrium and uterine cervix. It was also present in lung, spleen, kidney, prostate, skin, hair, and nail at relatively high concentrations. Its concentration in the uterine endometrium was nine times higher in pseudopregnant rabbits than in nonpregnant rabbits because of activation of cholesterol sulfotransferase and inhibition of CS sulfatase in the pseudopregnant rabbits. Sulfoglycolipids were not detected in the uterine endometria of either non-pregnant- or pseudopregnant rabbits. However, sulfoglycolipids were detected at relatively high concentrations in the cerebrum, cerebellum, stomach, duodenum, jejunum, testis, and kidney of rabbits and thus the tissues in which both sulfolipids were detected were the gastrointestinal tract and kidney. In the digestive tract, the concentration of CS decreased in the order esophagus, stomach, duodenum, and jejunum, but that of sulfatide increased in the same order, indicating distribution of CS in the squamous epithelium. In addition, both CS and sulfatide were detected in the serum. On the other hand, CS sulfatase activity was detected in all tissues examined, even in hair, from which the enzyme was liberated by brief sonication, and its highest specific activity was detected in the liver. The specific activity of cholesterol sulfotransferase varied among the tissues examined and was found to be significantly high in the esophageal epithelium and the uterine endometrium of pseudopregnant rabbit, indicating involvement of cholesterol sulfation in the formation of epithelium.


Asunto(s)
Arilsulfatasas/metabolismo , Ésteres del Colesterol/metabolismo , Sulfotransferasas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Secuencia de Carbohidratos , Ésteres del Colesterol/análisis , Cromatografía en Capa Delgada/métodos , Densitometría , Epítopos , Femenino , Glucolípidos/química , Glucolípidos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Conejos , Esteril-Sulfatasa , Sulfoglicoesfingolípidos/inmunología , Distribución Tisular
19.
Am J Obstet Gynecol ; 176(1 Pt 1): 142-9, 1997 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9024105

RESUMEN

OBJECTIVE: Our purpose was to investigate the metabolic background of the expression of sulfoglycolipids in human endometrium during the luteal phase. STUDY DESIGN: We investigated the expression of sulfoglycolipids by thin-layer chromatography immunostaining and the activities of galactosylceramide sulfotransferase and arylsulfatase A, which regulate the synthesis and degradation of sulfoglycolipid. In addition, arylsulfatase A messenger ribonucleic acid was studied by reverse transcriptase polymerase chain reaction. RESULTS: Sulfoglycolipid expression showed a marked increase in the luteal phase but not in the follicular phase, whereas sialoglycolipids remained relatively constant. The increase of sulfoglycolipids was found to be due to 4.5-fold increased activation of sulfotransferase and a concurrent reduction of arylsulfatase A activity in the luteal phase. Arylsulfatase A messenger ribonucleic acid was detected in both phases and showed no significant changes. CONCLUSIONS: These findings suggest that increased sulfoglycolipid expression in the luteal phase is due to the simultaneous regulation of sulfotransferase and arylsulfatase A, probably by sex steroid hormones.


Asunto(s)
Endometrio/metabolismo , Glucolípidos/biosíntesis , Fase Luteínica/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Cerebrósido Sulfatasa/genética , Cerebrósido Sulfatasa/metabolismo , Endometrio/enzimología , Femenino , Humanos , ARN Mensajero/biosíntesis , Sulfotransferasas/metabolismo
20.
J Clin Gastroenterol ; 25 Suppl 1: S135-40, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9479640

RESUMEN

Immunohistochemical staining using a specific monoclonal antibody against sulfatide was performed to examine cellular localization of sulfatides in normal human gastric mucosa and in various types of gastric carcinoma. In normal gastric mucosa without Helicobacter pylori infection, both epithelial and glandular cells were densely stained with anti-sulfatide antibody. Sulfatide staining was more abundant in the apical area of normal epithelium compared with cytosol or basolateral areas. This tendency was stronger in the antrum than the gastric body and was more intensified in glandular cells of the pyloric glands. The levels of sulfatide expression were decreased in papillary and well-differentiated adenocarcinomas compared with normal mucosa, were markedly attenuated in moderately differentiated adenocarcinomas, and were very low in poorly differentiated adenocarcinomas. The polarity of the stains was preserved in gastric cancers that exhibited differentiated tissue organization. The levels of sulfatide expression were highly variable in signet ring carcinoma cells. The cancer cells that expressed sulfatides did not show any polarity of staining.


Asunto(s)
Adenocarcinoma Papilar/metabolismo , Adenocarcinoma/metabolismo , Carcinoma de Células en Anillo de Sello/metabolismo , Mucosa Gástrica/metabolismo , Neoplasias Gástricas/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad
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