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1.
Biochim Biophys Acta ; 451(1): 184-200, 1976 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-1009107

RESUMEN

Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [14C]glucosamine and [3H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as "intracellular", gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and "intracellular" material; it labelled extensively with both glucosamine and fucose and had a molecular fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.


Asunto(s)
Glicoproteínas/biosíntesis , Tráquea/metabolismo , Aminoácidos/análisis , Animales , Carbohidratos/análisis , Cricetinae , Fucosa/metabolismo , Glucosamina/metabolismo , Glicoproteínas/aislamiento & purificación , Técnicas de Cultivo de Órganos , Factores de Tiempo , Ácidos Urónicos/análisis
2.
FEBS Lett ; 163(1): 104-9, 1983 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-6628681

RESUMEN

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed-fed sheep. Analysis by gas-liquid chromatography and mass-spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with alpha-D-mannosidase and endo-beta-N-acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo-beta-N-acetylglucosaminidase was without effect.


Asunto(s)
Oligosacáridos/orina , Intoxicación por Plantas/veterinaria , Enfermedades de las Ovejas/orina , Acetilglucosamina/análisis , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Femenino , Cromatografía de Gases y Espectrometría de Masas , Manosa/análisis , Intoxicación por Plantas/orina , Ovinos
3.
FEBS Lett ; 163(1): 99-103, 1983 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-6628696

RESUMEN

Daily urine samples were collected from a locoweed-fed sheep, and the oligosaccharide content examined by thin-layer and liquid chromatography. An unusual pattern of urine oligosaccharides was observed, which appears to be characteristic of loco intoxication. Changes in the pattern could be correlated with the onset of visible disease, which occurred approximately 5 weeks after the typical urine sugars were first detected. HPLC showed that these sugars consisted of two homologous series of oligosaccharides containing one and two residues of 2-acetamido-2-deoxy-D-glucose, respectively.


Asunto(s)
Glicósidos/metabolismo , Manósidos/metabolismo , Oligosacáridos/orina , Intoxicación por Plantas/veterinaria , Enfermedades de las Ovejas/orina , Animales , Femenino , Intoxicación por Plantas/orina , Ovinos , Factores de Tiempo
4.
FEBS Lett ; 195(1-2): 247-52, 1986 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-3943609

RESUMEN

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.


Asunto(s)
Enfermedades de los Gatos/diagnóstico , Manosidasas/deficiencia , alfa-Manosidosis/veterinaria , Animales , Química Encefálica , Secuencia de Carbohidratos , Enfermedades de los Gatos/genética , Gatos , Cromatografía Líquida de Alta Presión , Ojo/análisis , Tamización de Portadores Genéticos , Hígado/análisis , Manosidasas/genética , Oligosacáridos/análisis , Placenta/análisis , alfa-Manosidosis/diagnóstico , alfa-Manosidosis/genética
5.
Neurology ; 31(9): 1159-62, 1981 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-7196541

RESUMEN

A trisaccharide, Man2glcNAc, was the most abundant urinary oligosaccharide (161-558 mumol per liter) in a patient with mannosidosis. By means of a newly developed high-performance liquid chromatography (HPLC) procedure, we were able to measure low levels of serum trisaccharide (0.1--0.4 nmol per milliliter). This is the first report of the measurements of serum oligosaccharides in mannosidosis. Our observations on the disparate relationship between serum and urinary concentrations of this trisaccharide suggest that a facilitated renal clearance of oligosaccharides may be related to the nonprogressive aspect of this disorder.


Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Manosa , Oligosacáridos/metabolismo , Trisacáridos/metabolismo , Adulto , Cromatografía Líquida de Alta Presión , Humanos , Masculino
6.
Placenta ; 8(5): 545-53, 1987.
Artículo en Inglés | MEDLINE | ID: mdl-3422924

RESUMEN

Lysosomal alpha-mannosidase activity, oligosaccharide profiles, light and electron microscopy and lectin histochemistry studies were performed on full-term placentae obtained from five litters of cats. They resulted from breeding related cats who are obligate heterozygotes for lysosomal alpha-mannosidase deficiency. alpha-Mannosidase activity in placentae from affected kittens was less than 10 per cent of control, while in placentae from presumptive heterozygotes the activity was less than 50 per cent of control. High-pressure liquid chromatographic analysis of oligosaccharides revealed massive accumulation of undegraded oligosaccharides in placentae of affected kittens. A small elevation was found in placentae from presumptive heterozygous kittens, and none was detected in placentae of normal kittens. Light and electron microscopic examinations revealed vacuolization of fetal endothelial and mesenchymal cells only in placentae of affected kittens. Succinylated wheat germ agglutinin and concanavalin A stained the fetal fibroblasts only in placentae of affected kittens.


Asunto(s)
Manosidasas/deficiencia , Placenta/enzimología , Animales , Gatos , Femenino , Heterocigoto , Histocitoquímica , Homocigoto , Lisosomas/enzimología , Manosidasas/genética , Placenta/ultraestructura , Embarazo , alfa-Manosidasa
7.
Am J Med Genet ; 42(4): 586-92, 1992 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-1609840

RESUMEN

In agreement with reports from other laboratories, we have shown that patients with the juvenile or late infantile forms of neuronal ceroid-lipofuscinosis (NCL) have greatly increased levels (5-fold to 20-fold) of dolichyl pyrophosphoryl oligosaccharides in their cerebral gray matter. Oligosaccharides containing 2 GlcNAc residues and 3 to 9 mannose residues were liberated by mild acid hydrolysis. The oligosaccharide profile given by brain tissue from 2 patients with infantile NCL was markedly different from that of late infantile and juvenile NCL brain, with Man9GlcNAc2 as the most abundant component and decreasing amounts of Man8- Man7- and Man6GlcNAc2. By contrast, Man5GlcNAc2 was the most abundant oligosaccharide present in all juvenile NCL brain samples analyzed. Both the susceptibility of the isolated Man5GlcNAc2 to endoglucosaminidase H digestion and permethylation analysis clearly indicated that it is not an intermediate in the biosynthesis of Glc3Man9GlcNAc2-PP-dolichol but has undergone catabolism, probably either in the endoplasmic reticulum or in the Golgi apparatus. Treatment of cultured skin fibroblasts for 7 days with N-methyldeoxynojirimycin, a potent inhibitor of the endoplasmic reticulum processing enzymes glucosidase I and II, resulted in an accumulation of the same Man5GlcNAc2-PP-dolichol species that was elevated in juvenile NCL brain. The level in untreated fibroblasts was undetectable, suggesting that inhibition of processing glucosidases has interfered with the regulation and compartmentalization of lipid-linked oligosaccharides.


Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Fosfatos de Dolicol/metabolismo , Lipofuscinosis Ceroideas Neuronales/metabolismo , Oligosacáridos/metabolismo , Oligosacáridos de Poliisoprenil Fosfato/metabolismo , alfa-Glucosidasas/metabolismo , Secuencia de Carbohidratos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Glucosamina/análogos & derivados , Glucosamina/farmacología , Humanos , Datos de Secuencia Molecular , alfa-Glucosidasas/efectos de los fármacos
8.
Am J Med Genet ; 31(1): 39-56, 1988 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-3146925

RESUMEN

Histopathologic, ultrastructural and Golgi impregnation studies disclosed lesions characteristic of a neuronal lysosomal storage disease in related sheep with onset of neurologic signs at 4-6 months. Biochemical and enzymatic evaluation disclosed storage of GM1 ganglioside, asialo-GM1, and neutral long chain oligosaccharides in brain, urinary excretion of neutral long chain oligosaccharides, and deficiencies of lysosomal beta-galactosidase and alpha-neuraminidase. Retrospective and limited prospective genetic studies suggested autosomal recessive inheritance. A gene-dosage effect on beta-galactosidase levels was documented in fibroblasts from putative heterozygous sheep. Fibroblasts from affected sheep did not have increased beta-galactosidase activity after incubation with the protease inhibitor, leupeptin. In some aspects this disease is similar to GM1 gangliosidosis, but is unique in that a genetic defect in lysosomal beta-galactosidase may cause the deficiency of lysosomal alpha-neuraminidase.


Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/veterinaria , Galactosidasas/deficiencia , Neuraminidasa/deficiencia , Enfermedades de las Ovejas/genética , beta-Galactosidasa/deficiencia , Animales , Encéfalo/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/enzimología , Errores Innatos del Metabolismo de los Carbohidratos/genética , Línea Celular , Femenino , Fibroblastos/enzimología , Lípidos/aislamiento & purificación , Masculino , Microscopía Electrónica , Neuronas/citología , Neuronas/ultraestructura , Oligosacáridos/análisis , Oligosacáridos/orina , Linaje , Ovinos , Enfermedades de las Ovejas/enzimología , Piel/enzimología , Médula Espinal/patología
9.
Pediatr Neurol ; 4(4): 207-12, 1988.
Artículo en Inglés | MEDLINE | ID: mdl-3072006

RESUMEN

A child with infantile sialic acid storage disease is reported. Ultrasonography demonstrated fetal ascites. At birth, the infant appeared hydropic and presented with numerous dysmorphic features, including sparse white hair, coarse facies, hypertelorism, epicanthal folds, anteverted nostrils, and a long philtrum. In addition, he had visceromegaly, bilateral inguinal hernias, and a slight gibbus deformity. Lymphocytes were vacuolated and bone marrow contained large numbers of foam cells. There were generalized vacuolations of both reticuloendothelial and parenchymal cells in the examined tissues. Neuropathologic studies revealed wide-spread neuronal storage, myelin loss, axonal spheroids, and gliosis. Neurons, endothelial cells, and Kupffer cells stained with wheat germ agglutinin indicated an accumulation of sialic acid. Free sialic acid was significantly increased in urine and serum, as well as in liver, heart, and brain tissues. The alpha-neuraminidase activity was normal. It is assumed that the basic defect of infantile sialic acid storage disease lies in impaired transport of sialic acid across the lysosomal membrane.


Asunto(s)
Enfermedades Renales/complicaciones , Enfermedades Metabólicas/complicaciones , Ácidos Siálicos/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Lactante , Enfermedades Renales/patología , Hígado/metabolismo , Hígado/patología , Enfermedades Metabólicas/patología , Miocardio/metabolismo , Miocardio/patología
10.
Carbohydr Res ; 180(2): 325-38, 1988 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-3203344

RESUMEN

Oligosaccharides were extracted from tissues and body fluids of five kittens with alpha-mannosidosis, three being from the same litter. The kittens were all of different ages at death and were compared to normal and heterozygote cats. The oligosaccharides were analyzed by high-pressure liquid chromatography after perbenzoylation and were identified by comparison with compounds of known structure. This provided a detailed picture of the distribution of oligosaccharides in each tissue, and a method for quantitation of the total oligosaccharides. With the exception of the youngest animal (death at day 2), the oligosaccharide elution profiles were broadly similar for all tissues and fluids, and were typical of feline alpha-mannosidosis. In contrast, concentrations of total oligosaccharides diverged widely from one source to another, from a high of 17.3 mumol/g to a low of 0.04 mumol/g. The results are interpreted in the context of glycoprotein catabolism.


Asunto(s)
Enfermedades de los Gatos/metabolismo , Oligosacáridos/metabolismo , alfa-Manosidosis/veterinaria , Animales , Líquidos Corporales/metabolismo , Secuencia de Carbohidratos , Gatos , Femenino , Masculino , Datos de Secuencia Molecular , Distribución Tisular , alfa-Manosidosis/metabolismo
12.
In Vitro ; 11(6): 347-53, 1975.
Artículo en Inglés | MEDLINE | ID: mdl-1238360

RESUMEN

Hamster tracheas were cultured in serum-free CMRL 1066 medium buffered with either NaHCO3 alone or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) plus NaHCO3 in an atmosphere of 95% O2 and 5% CO2. Afther 2 days in culture, tracheas maintained in HEPES plus NaHCO3-buffered medium showed an altered surface morphology. Histiological examination after 6 days of culture with HEPES plus NaHCO3 showed the presence of only scattered clumps of cilia. Incorporation of [14C]-glucosamine into intracellular glycoproteins was reduced by 75% and into secreted glycoproteins by 54% in cultures buffered with HEPES plus NaHCO3, compared to NaHCO3-buffered cultures. Incorporation of [H]fucose into intracellular glycoproteins was also reduced, although no effect was observed on secreted glycoproteins.


Asunto(s)
Glucosamina/metabolismo , Glicoproteínas/biosíntesis , HEPES/farmacología , Piperazinas/farmacología , Tráquea/metabolismo , Animales , Bicarbonatos/farmacología , Tampones (Química) , Cilios/fisiología , Cricetinae , Medios de Cultivo , Células Epiteliales , Fucosa/metabolismo , Glicopéptidos/análisis , Glicoproteínas/análisis , Movimiento , Técnicas de Cultivo de Órganos , Tráquea/citología
13.
Biochem J ; 221(3): 601-7, 1984 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-6433887

RESUMEN

Urinary oligosaccharides isolated from locoweed-intoxicated sheep were separated and quantified by reversed-phase high pressure liquid chromatography of the perbenzoylated alditols. Mannose-containing oligosaccharides were elevated as early as day 3 of feeding, but maximum levels (approx. 1 mumol/ml) were not attained until after 6 weeks of feeding. The relative abundance of individual oligosaccharides changed over the course of the feeding period. Man3GlcNAc2 reached a peak on day 3 and then rapidly declined. Two isomers were shown to be present in this fraction and the relative proportions altered with the duration of locoweed treatment. The major isomer present at early time points (less than 8 days) co-eluted with synthetic Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, was digested by endo-beta-N-acetyl-glucosaminidase D, and is probably derived from the trimannosyl core of complex glycoproteins synthesized prior to locoweed treatment. Man3GlcNAc2 isolated from day 53 urine was resistant to endo-beta-N-acetylglucosaminidase D digestion but was cleaved by endo-beta-N-acetylglucosaminidase H. This isomer has the probable structure Man(alpha 1-3)Man(alpha 1-6)Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, indicative of its origin from hybrid or high-mannose glycoproteins. Man5GlcNAc2 reached a peak on day 13 and then slowly declined, whereas Man4GlcNAc2 increased concomitantly. The rapid increase in Man5GlcNAc2 can probably be attributed to the breakdown of hybrid glycans produced as a result of swainsonine inhibition of Golgi alpha-D-mannosidase II. The onset of observable clinical signs on day 38 closely correlated with the time point at which the level of Man4GlcNAc2 exceeded Man5GlcNAc2. After locoweed feeding was discontinued, the amount of urinary oligosaccharides declined rapidly and reached baseline levels within 12 days.


Asunto(s)
Alcaloides/farmacología , Oligosacáridos/orina , Plantas Tóxicas , Acetilglucosaminidasa , Animales , Fenómenos Químicos , Química , Cromatografía Líquida de Alta Presión , Fabaceae , Femenino , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Plantas Medicinales , Ovinos , Swainsonina , Factores de Tiempo
14.
Glycobiology ; 4(5): 551-66, 1994 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-7881169

RESUMEN

Previously, alpha-mannosidases were classified as enzymes that process newly formed N-glycans or degrade mature glycoproteins. In this review, we suggest that two endoplasmic reticulum (ER) alpha-mannosidases, previously assigned processing roles, have important catabolic activities. Based on new evidence, we propose that the ER/cytosolic mannosidase is involved in the degradation of dolichol intermediates that are not needed for protein glycosylation, whereas the soluble form of Man9-mannosidase is responsible for the degradation of glycans on defective or malfolded proteins that are specifically retained and broken down in the ER. The degradation of oligosaccharides derived from dolichol intermediates by ER/cytosolic mannosidase now explains why cats and cattle with alpha-mannosidosis store and excrete some unexpected oligosaccharides containing only one GlcNAc residue. Similarly, the action of ER/cytosolic mannosidase, followed by the action of the recently described human lysosomal alpha(1 --> 6)-mannosidase, together explain why alpha-mannosidosis patients store and excrete large amounts of oligosaccharides that resemble biosynthetic intermediates, rather than partially degraded glycans. The relative contributions of the lysosomal and extra-lysosomal catabolic pathways can be derived by comparing the ratio of trisaccharide Man beta (1 --> 4)GlcNAc beta (1 --> 4)GlcNAc to disaccharide Man beta (1 --> 4)GlcNAc accumulated in tissues from goats with beta-mannosidosis. A similar determination in human beta-mannosidosis patients is not possible because the same intermediate, Man beta (1 --> 4)-GlcNAc is a product of both pathways. Based on inhibitor studies with pyranose and furanose analogues, alpha-mannosidases may be divided into two groups. Those in Class 1 are (1 --> 2)-specific enzymes like Golgi mannosidase I, whereas those in Class 2, like lysosomal alpha-mannosidase, can hydrolyse (1 --> 2), (1 --> 3) and (1 --> 6) linkages. A similar classification has recently been derived by others from protein sequence homologies. Based on this new classification of the alpha-mannosidases, it is possible to speculate about their probable evolution from two primordial genes. The first would have been a Class 1 ER enzyme involved in the degradation of glycans on incompletely assembled or malfolded glycoproteins. The second would have been a Class 2 lysosomal enzyme responsible for turnover. Later, other alpha-mannosidases, with new processing or catabolic functions, would have developed from these, by loss or gain of critical insertion or retention sequences, to yield the full complement of alpha-mannosidases known today.


Asunto(s)
Isoenzimas/metabolismo , Manosidasas/metabolismo , Animales , Evolución Biológica , Secuencia de Carbohidratos , Glicoproteínas/biosíntesis , Glicoproteínas/metabolismo , Isoenzimas/clasificación , Lisosomas/enzimología , Mamíferos , Manosa/análogos & derivados , Manosidasas/clasificación , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , alfa-Manosidasa
15.
J Biol Chem ; 267(14): 9706-12, 1992 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-1577805

RESUMEN

A novel lysosomal alpha-mannosidase, with unique substrate specificity, has been partially purified from human spleen by chromatography through concanavalin A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. This enzyme can catalyze the hydrolysis of only 1 mannose residue, that which is alpha(1----6)-linked to the beta-linked mannose in the core of N-linked glycans, as found in the oligosaccharides Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc and Man alpha(1----6)Man beta(1----4) GlcNAc. The newly described alpha-mannosidase does not catalyze the hydrolysis of mannose residues outside of the core, even if they are alpha(1----6)-linked, and is not active on the other alpha-linked mannose in the core, which is (1----3)-linked. The narrow specificity of the novel mannosidase contrasts sharply with that of the major lysosomal alpha-mannosidase, which is able to catalyze the degradation of oligosaccharides containing diverse linkage and branching patterns of the mannose residues. Importantly, although the major mannosidase readily catalyzes the hydrolysis of the core alpha(1----3)-linked mannose, it is poorly active towards the alpha(1----6)-linked mannose, i.e. the very same mannose residue for which the newly characterized mannosidase is specific. The novel enzyme is further differentiated from the major lysosomal alpha-mannosidase by its inability to catalyze the efficient hydrolysis of the synthetic substrate p-nitrophenyl alpha-mannoside, and by the strong stimulation of its activity by Co2+ and Zn2+. Similarly to the major mannosidase, it is strongly inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, but not by deoxymannojirimycin. The presence of this novel alpha-mannosidase activity in human tissues provides the best explanation, to date, for the structures of the oligosaccharides stored in human alpha-mannosidosis. In this condition the major lysosomal alpha-mannosidase activity is severely deficient, but apparently the alpha(1----6)-mannosidase is unaffected, so that the oligosaccharide structures reflect the unique specificity of this enzyme.


Asunto(s)
Lisosomas/enzimología , Manosidasas/metabolismo , Oligosacáridos/metabolismo , Bazo/enzimología , Animales , Secuencia de Carbohidratos , Cationes Bivalentes , Gatos , Cromatografía Líquida de Alta Presión , Humanos , Hígado/enzimología , Manosidasas/orina , Datos de Secuencia Molecular , Oligosacáridos/aislamiento & purificación , Páncreas/enzimología , Ovinos , Especificidad por Sustrato , Swainsonina/farmacología
16.
Eur J Biochem ; 114(2): 235-7, 1981 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-7215354

RESUMEN

Excessive gingival hyperplasia with storage of mannose-rich oligosaccharides appears to be a unique feature present in a 31-year-old mannosidosis patient. Fractionation and analysis of the gingiva established the presence of (Man)2GlcNAc (2.2 mumol/g), (Man)3GlcNAc (3.5 mumol/g), (Man)4GlcNAc (2.8 mumol/g) and higher oligomers (Man)5GlcNAc--(Man)8GlcNAc (0.5 mumol/g); (Man, mannose; GlcNAc, N-acetylglucosamine). Eight characteristic oligosaccharides were isolated from the patient's urine by thin-layer chromatography. The most abundant was (Man)2GlcNAc (161--558 mumol/l); decreasing amounts of higher homologues up to a dekasaccharide, (Man)9GlcNAc (1--4 mumol/l) were also present. In contrast to urine, in which the trisaccharide was predominant, tetrasaccharides and pentasaccharides were more abundant in gingiva.


Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Encía/análisis , Manosa/análisis , Manosidasas/deficiencia , Oligosacáridos/aislamiento & purificación , Acetilglucosamina/análisis , Adulto , Cromatografía en Capa Delgada , Humanos , Masculino , Oligosacáridos/orina
17.
Glycobiology ; 2(4): 327-36, 1992 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-1421754

RESUMEN

Normal human fibroblasts and fibroblasts from a patient with alpha-mannosidosis were grown in the presence or absence of 100 microM swainsonine for 7 days. Accumulated oligosaccharides were isolated and analysed by high performance liquid chromatography (HPLC) and methylation analysis. Man alpha 1----3Man beta 1----4GlcNAc and Man alpha 1----2Man alpha 1----3-Man beta 1----4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised greater than 80% of the total oligosaccharides in untreated mannosidosis cells. However, Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc was the major Man3GlcNAc isomer present after 7 days of swainsonine treatment. No mannose-containing oligosaccharides were detected in control fibroblasts in the absence of swainsonine but, in its presence, oligosaccharides containing 2-9 mannose residues accumulated. Man alpha 1----6[Man alpha 1----3]-Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc and Man alpha 1----6-[Man alpha 1----3]Man beta 1----4GlcNAc were the major components (67%). Surprisingly, Man alpha 1----3Man beta 1----4GlcNAc was only observed in swainsonine-treated control cells during the recovery period after removal of swainsonine. These studies suggest the presence of a second lysosomal alpha-mannosidase activity which is unaffected in genetic alpha-mannosidosis, but is inhibited by swainsonine. This enzyme would cleave the alpha(1----6)-linked mannose residue from branched Man3GlcNAc to form Man alpha 1----3Man beta 1----4GlcNAc. To confirm this hypothesis, fractions from alpha-mannosidosis and control fibroblasts that bound to concanavalin A (ConA)-Sepharose and were eluted with 0.5 M alpha-methyl mannoside were incubated at pH 4.0 with Man alpha 1----6[Man alpha 1----3]Man beta 1----4-GlcNAc. As anticipated, Man alpha 1----3Man beta 1----4GlcNAc was the sole product using enzyme from mannosidosis fibroblasts, while the major product from control fibroblasts was Man alpha 1----6Man beta 1----4GlcNAc. This confirmed the presence of a swainsonine-inhibitable alpha(1----6)-mannosidase activity unaffected by the disease. The differing substrate specificities of the alpha(1----6)-mannosidase and the major lysosomal alpha-mannosidase indicate that the alpha(1----6)-mannosidase plays an important role in the generation of the oligosaccharides accumulated in alpha-mannosidosis patients.


Asunto(s)
Fibroblastos/enzimología , Lisosomas/enzimología , Manosidasas/metabolismo , Polisacáridos/metabolismo , alfa-Manosidosis/enzimología , Secuencia de Carbohidratos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Humanos , Metilación , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Especificidad por Sustrato , Swainsonina/farmacología
18.
J Lipid Res ; 32(1): 157-64, 1991 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-2010687

RESUMEN

Previous studies from our laboratory have shown that male C57BL/6J mice excrete into the urine multilamellar lysosomal bodies that contain specific neutral glycosphingolipids. These mice excrete approximately 20-30% of their kidney glycolipids each day. The significance and function of this secretion of multilamellar lysosomal organelles is unknown. To characterize these excreted bodies further, we report here their neutral lipid and phospholipid composition. The bodies were collected by differential centrifugation, extracted with chloroform-methanol, and lipids were fractionated into neutral lipids, glycolipids, and phospholipids. The neutral lipids consisted primarily of cholesterol, dolichol, and ubiquinone. The phospholipid fraction consisted primarily of a single molecular species of phosphatidylcholine. This lipid which comprises more than 90% of the total phospholipids was found to contain 16:0 ether and C22:6 n-3 fatty acid as determined by gas-liquid chromatography-mass spectrometry. The glycosphingolipids as reported previously consisted primarily of galabiosylceramides and globotriaosylceramides. This membrane lipid composition is different from any previously reported cellular organelle.


Asunto(s)
Lípidos/orina , Lisosomas/química , Animales , Cromatografía Líquida de Alta Presión , Glucolípidos/orina , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfolípidos/orina , Orina/citología
19.
Ann Vasc Surg ; 9(1): 102-8, 1995 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-7703053

RESUMEN

We conducted this study to investigate the physiologic variations in venous valvular function and calf muscle pump function that occur in normal limbs after prolonged stationary standing. Twenty-two limbs from 11 healthy volunteers were studied after a brief period of activity and after 4 to 6 hours of stationary standing. Vein diameter, peak reflux flow velocity (PRFV), and valve closure time (VCT) were measured with duplex scanning in the standing position in the common femoral vein (CFV), superficial femoral vein (SFV), popliteal vein (POP), proximal greater saphenous vein (GSV), and greater saphenous vein at the knee (kGSV). Pneumatic rapid inflation-deflation cuffs were used to elicit reflux. Vein cross-sectional area (VA) and peak reflux volume (PRVol) were calculated. Venous volume (VV), venous filling index (VFI), ejection fraction (EF), residual volume fraction (RVF), and outflow fraction (OF) were measured with air plethysmography in all limbs. After stationary standing, there was no significant change or trend toward an increase in diameter or VA in any of the deep veins and there was no change in the PRFV or VCT. In the proximal GSV there was a significant increase in diameter (p = 0.0001) and VCT (p = 0.048) without a change in PRFV. No significant changes were noted in the kGSV. In the GSV the PRFV was significantly lower (p < 0.05) and the VCT significantly shorter (p < 0.05) compared with the SFV and POP but values were no different from those in the CFV. The PRFV was significantly higher in the SFV (p < 0.0001) and the POP (p < 0.002) compared with that in the CFV.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Pierna/irrigación sanguínea , Venas/fisiología , Adulto , Vena Femoral/fisiología , Humanos , Individualidad , Masculino , Pletismografía , Vena Poplítea/fisiología , Postura , Vena Safena/fisiología , Túnica Íntima/fisiología
20.
Biochem Biophys Res Commun ; 138(2): 902-9, 1986 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-3741440

RESUMEN

Ependymins, a family of extracellular glycoproteins of goldfish and mammalian brain, were shown to contain N-linked complex glycan chains. These glycoproteins reacted with a monoclonal antibody, HNK-1 which recognizes a membrane antigen on a subset of human lymphocytes, myelin-associated glycoprotein glycoprotein epitope reacting with HNK-1 antibody was previously shown to include a terminal 3-sulfoglucuronosyl residue present in certain glycolipids of the nervous tissue (Chou et al., Biochem. Biophys. Res. Commun. 1985, 128, 383-388). In this report, the presence of glucuronic acid in ependymins was demonstrated by gas-liquid chromatography and mass spectrometry. We suggest that a 3-sulfoglucuronosyl residue may be the common epitope on HNK-1-reactive glycoproteins.


Asunto(s)
Antígenos de Superficie/análisis , Química Encefálica , Glucuronatos/análisis , Glicoproteínas/análisis , Proteínas del Tejido Nervioso/análisis , Animales , Anticuerpos Monoclonales , Carbohidratos/análisis , Cromatografía de Gases y Espectrometría de Masas , Carpa Dorada
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