RESUMEN
A novel glycosphingolipid containing inositol phosphate as an acidic group has been demonstrated in whole tissues of the porcine roundworm, Ascaris suum. The thin layer chromatographic pattern of the total acidic glycolipid revealed the presence of several components, of which a major component (named AGL) with positive reactions toward both orcinol-sulfuric acid (sugar) and molybdate (phosphate) spray reagents was isolated and purified by the use of successive column chromatography on DEAE-Sephadex and silicic acid (latrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, methylation analysis, periodate oxidation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, the structure of AGL was deduced to be Gal alpha 1-2Ins(1-->)-P-Cer. Aliphatic constituents were lignoceric acid and its 2-hydroxy homologue as the principal fatty acids, and octadecasphinganine and branched heptadecasphinganine as the major sphingoids.
Asunto(s)
Ascaris suum/química , Glicoesfingolípidos/química , Fosfatos de Inositol/química , Animales , Borohidruros/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Carbohidratos/análisis , Cromatografía de Gases , Cromatografía en Capa Delgada , Galactosa/química , Cromatografía de Gases y Espectrometría de Masas , Glicoesfingolípidos/análisis , Hidrólisis , Fosfatos de Inositol/análisis , Espectroscopía de Resonancia Magnética , Metilación , Conformación Molecular , Datos de Secuencia Molecular , Oxidación-Reducción , Ácido Peryódico/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The novel amphoteric glycosphingolipids containing a choline phosphate were purified from whole tissues of the earthworm, Pheretima hilgendorfi. Their chemical structures were completely characterized as cholinephosphoryl-->6(Man alpha 1-4)Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylmannosylneogalabiaosylceramide, named PGL3a) and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylneogalatriaosylceramide, named PGL3b) by compositional sugar, fatty acid and sphingoid analyses, hydrogen fluoride degradation, partial acid hydrolysis, methylation analysis, exoglycosidase degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide moieties of these lipids consisted of 22:0, 23:0 and 24:0 acids as major fatty acids, and branched octadeca- and nonadeca-4-sphingenines and octadeca-4-sphingenine as main sphingoids. Since the oligosaccharides and the ceramide moieties of PGL3a and PGL3b were identical with those of neutral glycosphingolipids found in this organism, the biosynthesis of the amphoteric glycolipids may occur by the addition of a choline phosphate residue to the corresponding neutral glycolipids, Man alpha 1-4Gal beta 1-6Gal beta 1-1Cer or Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer.
Asunto(s)
Glicoesfingolípidos/química , Oligoquetos/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Ceramidas/química , Cromatografía de Gases , Cromatografía en Capa Delgada , Ácidos Grasos/análisis , Ácidos Grasos/química , Glucolípidos/análisis , Glucolípidos/química , Glicoesfingolípidos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Metilación , Datos de Secuencia Molecular , Oligosacáridos/química , Fosforilcolina/química , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría Infrarroja , Esfingosina/análogos & derivados , Esfingosina/análisis , Esfingosina/químicaRESUMEN
Amines, diamines and polyamines inhibit the erythropoietin-stimulated incorporation of 59Fe into newly-synthesized heme by fetal mouse liver cells in short-term culture. As assessed by cellular retention of lactic dehydrogenase, most of these compounds affect the viability of the cells very little at concentrations that substantially inhibit heme synthesis. In the families of amines tested, compounds of chain length greater than five carbons are more effective inhibitors the longer the chain, and monoamines are more inhibitory than diamines. The naturally-occurring polyamines spermine and spermidine are among the most potent amine inhibitors tested.
Asunto(s)
Aminas/farmacología , Eritropoyetina/antagonistas & inhibidores , Hemo/biosíntesis , Hígado/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Diaminas/farmacología , Feto , Técnicas In Vitro , Radioisótopos de Hierro , L-Lactato Deshidrogenasa/metabolismo , Ratones , Peso Molecular , Poliaminas/farmacología , Espermina/farmacología , Relación Estructura-ActividadAsunto(s)
Proteínas Sanguíneas/metabolismo , Diálisis Peritoneal Ambulatoria Continua/efectos adversos , Diálisis Peritoneal/efectos adversos , Proteínas/metabolismo , Animales , Permeabilidad Capilar , Humanos , Concentración de Iones de Hidrógeno , Peso Molecular , Cavidad Peritoneal/metabolismo , Albúmina Sérica/metabolismo , SolucionesRESUMEN
A monounsaturated and a triunsaturated form of phytanic acid (3,7,11,15-tetramethylhexacosanoate) were isolated from plasma lipids of a patient with Refsum disease. Both were converted to their methyl esters, oxidized to polyhydroxy acids by treatment with OsO4 and converted to their vicinal trimethylsilyl ethers. These derivatives were analyzed by gas chromatography-mass spectrometry using both electron impact ionization (at 21 and 70 eV) and chemical ionization conditions to obtain clear evidence to establish the structure of the monounsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-15-monoenoic acid and that of the triunsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-6,10,14-trienoic acid. The possible metabolic and dietary sources for these novel fatty acids are discussed.
Asunto(s)
Ácidos Eicosanoicos/aislamiento & purificación , Ácido Fitánico/aislamiento & purificación , Enfermedad de Refsum/metabolismo , Fenómenos Químicos , Química , Cromatografía de Gases y Espectrometría de Masas , Humanos , Espectrometría de Masas , Oxidación-ReducciónRESUMEN
The high-performance liquid chromatographic (HPLC) method for the determination of unsaturated sulfated disaccharides is a comprehensive and reliable method which expedites ensymatic studies of isomeric chondroitin sulfates. Responses for these unsaturated disaccharides derived from urinary chondroitin sulfates were linear from 100 ng to 10 micrograms injected and good quantitation was obtained for 25 microliters or less of samples placed on the column. This method which is more sensitive and accurate than methods now being used has considerable potential for the chemical diagnosis of patients with mucopolysaccharidoses and for the clarification of glycosaminoglycan structure. The isomeric chondroitin sulfates in urines from patients with mucopolysaccharidoses were studied by enzyme digestion with chondroitinases followed by HPLC determination of the sulfated unsaturated disaccharides produced. Evaluation by HPLC of the unsaturated 4-sulfated disaccharide produced by digestion of the urinary GAG with chondroitinases ABC and AC revealed rapidly and quantitatively the large amounts of dermatan sulfate present in Hurler, Hunter, and Maroteaux-Lamy urines. Chondroitin 4-sulfate predominated in Sanfilippo urinary isomeric chondroitin sulfates whereas chondroitin 6-sulfate and chondroitin 4-sulfate were shown to be present in nearly equal amounts in Morquio urine. An oversulfated chondroitin sulfate was detected in small amounts in some of these urines. This was demonstrated by the detection of an unsaturated disulfated disaccharide after digestion with chondroitinase ABC but not with chondroitinase AC.
Asunto(s)
Sulfatos de Condroitina/orina , Condroitín/análogos & derivados , Mucopolisacaridosis/orina , Condroitinasas y Condroitín Liasas , Cromatografía Líquida de Alta Presión , Glicosaminoglicanos/orina , Humanos , IsomerismoRESUMEN
Two pregnancies at risk for Farber's disease were monitored with amiocentesis at 15 and 16 weeks' gestation. In the first pregnancy tested, cultured amniotic-cell ceramidase activity was 7.8% of the control mean and an affected fetus was predicted. The pregnancy was terminated at 22 weeks' gestation and the diagnosis was confirmed by the demonstration of considerably elevated renal and hepatic ceramide concentrations and severe reduction of ceramidase activity in fetal brain and cultured fibroblasts. In the second pregnancy tested, cultured amniotic-cell ceramidase activity was within the control range, and the prediction of an unaffected fetus was confirmed in the newborn.
Asunto(s)
Ceramidas/deficiencia , Diagnóstico Prenatal , Esfingolipidosis/diagnóstico , Amidohidrolasas/deficiencia , Amniocentesis , Encéfalo/enzimología , Femenino , Fibroblastos/enzimología , Edad Gestacional , Heterocigoto , Homocigoto , Humanos , Embarazo , Esfingolipidosis/genéticaRESUMEN
Since plant lectins were used to help define differences between normal and transformed cell surfaces (reviewed in References 1-4), they have been employed in many other situations where their sugar-recognition specificities could be used to advantage. One of these applications has been the purification and characterization of enzymes and other proteins; this work is reviewed here in order to define some of the variables that affect binding of glycoproteins to lectins, as well as to demonstrate how this technique has been profitably exploited for isolation of purified glycoproteins, and for their better understanding.
Asunto(s)
Glicoproteínas/metabolismo , Lectinas/metabolismo , Unión Competitiva , Cromatografía en Gel , Concanavalina A , Enzimas/aislamiento & purificación , Glicósidos/metabolismo , Concentración de Iones de Hidrógeno , Concentración Osmolar , Especificidad por Sustrato , Temperatura , Factores de TiempoRESUMEN
Two novel branched-chain fatty acids, which appear to be unsaturated analogs of phytanic acid, have been observed in sera and urine of patients with Refsum's disease. They occur in both phospholipids and neutral lipids, and have been isolated and characterized.
Asunto(s)
Ácidos Eicosanoicos/metabolismo , Ácido Fitánico/metabolismo , Enfermedad de Refsum/metabolismo , Ácidos Grasos Insaturados/orina , Humanos , Lípidos/orina , Fosfatidilcolinas/orina , Fosfolípidos/orina , Ácido Fitánico/análogos & derivados , Ácido Fitánico/orina , Enfermedad de Refsum/orina , Triglicéridos/orinaRESUMEN
The enzyme defect in Farber disease, a deficiency of acid ceramidase, has been demonstrated in cultured skin fibroblasts, which provides a means of confirming the diagnosis during life. The assay can also be performed using cultured amniotic fluid cells and is a potential tool for detection of carriers of the disease.
Asunto(s)
Amidohidrolasas/deficiencia , Ceramidas/metabolismo , Fibroblastos , Granuloma/diagnóstico , Lipidosis/diagnóstico , Femenino , Fibroblastos/enzimología , Granuloma/enzimología , Humanos , Embarazo , Piel/patologíaRESUMEN
The mean specific activity of alph-L-iduronidase in leukocytes from six obligate heterozygotes for Hurler's syndrome was found to be slightly less than one-half of the mean in normal controls; no overlap of normal and known heterozygote values was encountered. The assay has been applied with success to six potential heterozygotes, siblings of a child with Hurler's syndrome. Thus heterozygote detection of Hurler's syndrome is clearly possible; this finding, as well as the ready availability of leukocytes for screening tests, recommends their use for examination of potential carrier status in this disorder.
Asunto(s)
Glicósido Hidrolasas/sangre , Heterocigoto , Iduronidasa/sangre , Leucocitos/enzimología , Mucopolisacaridosis I/enzimología , Adulto , Niño , Femenino , Hexosaminidasas/sangre , Humanos , Masculino , Mucopolisacaridosis I/diagnóstico , Mucopolisacaridosis I/genética , LinajeRESUMEN
It has been shown that tissues of patients with Farber's disease characteristically lack acid (pH 4.0) ceramidase. In normal cerebellum, however, ceramide cleavage and the reverse reaction, free fatty acid-dependent ceramide synthesis, both occur not only at pH 4.0 but also at pH 9.0, although normal kidney exhibits these activities only at pH 4.0. Both tissues are capable of snythesizing ceramide via an acyl-COA-dependent pathway at neutral pH. The synthetic analog of ceramide, N-oleoyl-ethanolamine, is a potent inhibitor of ceramidase.
Asunto(s)
Amidohidrolasas/metabolismo , Ceramidas/biosíntesis , Cerebelo/metabolismo , Riñón/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Recién Nacido , Artropatías/congénito , Artropatías/enzimología , Cinética , Lipidosis/congénito , Lipidosis/enzimología , Síndrome de Dificultad Respiratoria del Recién Nacido/enzimología , SíndromeAsunto(s)
Glicosaminoglicanos/orina , Antígenos HLA , Antígenos de Histocompatibilidad , Discapacidad Intelectual/terapia , Transfusión de Leucocitos , Mucopolisacaridosis/terapia , Supervivencia Celular , Fenómenos Químicos , Química , Niño , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Cromosomas Humanos 1-3 , Ciclofosfamida/uso terapéutico , Fucosa , Glicósido Hidrolasas/análisis , Hexosaminidasas/análisis , Prueba de Histocompatibilidad , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/inmunología , Discapacidad Intelectual/orina , Leucocitos/enzimología , Linfocitos/inmunología , Masculino , Mucopolisacaridosis/genética , Mucopolisacaridosis/inmunología , Mucopolisacaridosis/orina , Nitrofenoles , Síndrome , Trasplante HomólogoRESUMEN
Conditions have been determined for the benzoylation of ceramides containing nonhydroxy and hydroxy fatty acids, and a high performance liquid chromatography system for the separation and measurement of these derivatives has been devised that is capable of good resolution and high sensitivity. These methods have been used to determine quantitatively the levels of ceramides in human tissues, and in serum and urine, and to demonstrate elevated amounts of ceramide in Farber's disease urine and tissues.
Asunto(s)
Ceramidas/metabolismo , Lipidosis/metabolismo , Compuestos de Bencilo , Ceramidas/análisis , Cerebelo/metabolismo , Preescolar , Cromatografía , Creatinina/orina , Estudios de Evaluación como Asunto , Mucosa Gástrica/metabolismo , Humanos , Recién Nacido , Discapacidad Intelectual/metabolismo , Artropatías/congénito , Artropatías/metabolismo , Riñón/metabolismo , Lipidosis/congénito , Pulmón/metabolismo , Masculino , Métodos , Miocardio/metabolismo , Especificidad de Órganos , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , Dióxido de Silicio , SíndromeRESUMEN
The livers of four patients with metachromatic leukodystrophy contained galactosyl sulfatide and lactosyl sulfatide, whereas these substances were undetectable in normal human liver. On the basis of methanolysis and permethylation studies, both sulfatides were shown to be substituted with sulfate at the C-3 position of the galactose moiety. Examination of the fatty acid compositions of these sulfatides showed that C(22:0) and higher 2-hydroxy and nonhydroxy fatty acids predominated in both. Both sulfatides contained the same long-chain bases, predominantly sphingosine, dihydrosphingosine, and phytosphingosine. Using as criteria the proportion of lactosyl sulfatide to galactosyl sulfatide, and the fatty acid and long-chain base compositions, the liver sulfatides from subjects with metachromatic leukodystrophy closely resemble those in the kidney and differ from those in brain and peripheral nerve.