RESUMEN
OBJECTIVES: Systemic sclerosis (SSc) leads to pulmonary circulation dysfunctionand there are some indications of systemic circulation impairment. We evaluated the influence of SSc on the elastic properties of large systemic arterial walls and potential correlations between systemic and pulmonary circulation involvement. METHOD: We examined 75 consecutive women (mean age 53.13±10.1 years) with confirmed SSc [mean disease duration (DD) 7.1±9.1 years] and 21 age-matched female volunteers (mean age 52.6±8.3 years, ns). Pulse wave velocity (PWV) and transthoracic echocardiography were performed. SSc patients were divided into two groups according to the median of DD: ≤3 years (39 patients) and >3 years (36 patients). RESULTS: Patients with DD>3 years had higher PWV than those with DD≤3 years and controls (log PWV: 2.23±0.23 vs. 2.13±0.16 and vs. 2.11±0.16 m/s; p=0.028 and 0.029, respectively). In addition, echocardiographic indices showed impaired right ventricular (RV) function in the patients with DD>3 years. Also in these SSc patients, PWV correlated with clinical and echocardiographic parameters of pulmonary circulation: age (r=0.64, p<0.0001), acceleration time of pulmonary ejection (AcT; r=-0.38, p=0.021), and tricuspid regurgitation peak gradient (TRPG; r=0.34, p=0.04). Multiple linear regression analysis showed that PWV was independently associated with DD (ß=0.22, p==0.02), AcT (ß=-0.215, p=0.03), and age (ß=0.44, p<0.001). CONCLUSIONS: In patients with SSc lasting more than 3 years, the disease is characterized by increased stiffness of the large systemic arteries. Longer duration of SSc leads simultaneously to the increased stiffness of the large systemic arteries and to the progressive impairment of RV function and its coupling to the pulmonary arterial bed.
Asunto(s)
Arterias/fisiopatología , Arteria Pulmonar/fisiopatología , Esclerodermia Sistémica/fisiopatología , Remodelación Vascular/fisiología , Adulto , Arterias/diagnóstico por imagen , Estudios de Casos y Controles , Ecocardiografía , Elasticidad/fisiología , Femenino , Humanos , Hipertensión/epidemiología , Incidencia , Persona de Mediana Edad , Arteria Pulmonar/diagnóstico por imagen , Esclerodermia Sistémica/diagnóstico por imagen , Disfunción Ventricular Derecha/epidemiologíaRESUMEN
Human prolactin was expressed in insect culture cells by recombinant baculoviruses carrying prolactin gene cDNA placed under the transcriptional control of polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus. Preliminary results of recombinant human prolactin expression as extracellular as well as intracellular product of baculovirus expression system were presented at the FEBS Meeting in Nice, France, in 1999 (Abstracts, p. 288). In the present work prolactin was expressed as a hexahistidine-tagged fusion protein and recombinant protein was purified by metal affinity resin. Yields varied between approximately 20 and 35 mg/liter of medium. This recombinant prolactin was biologically active in Nb2 lymphoma cell proliferation assay and after simple purification could substitute for pituitary-derived prolactin.
Asunto(s)
Líquido Intracelular/metabolismo , Prolactina/biosíntesis , Prolactina/genética , Spodoptera/genética , Spodoptera/metabolismo , Animales , Baculoviridae/genética , Bioensayo , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Células Cultivadas , Clonación Molecular , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/aislamiento & purificación , Sustancias de Crecimiento/fisiología , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Peso Molecular , Nucleopoliedrovirus/genética , Prolactina/aislamiento & purificación , Prolactina/fisiología , Desnaturalización Proteica , Renaturación de Proteína , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Solubilidad , Spodoptera/citología , Spodoptera/virología , Células Tumorales CultivadasRESUMEN
Autoantibodies reacting with extracellular matrix proteins have been extensively studied in various autoimmune connective tissue diseases. Because of the possibility that such antibodies may play a role in orbital connective tissue inflammation in thyroid-associated ophthalmopathy (TAO), we studied the humoral immune response against specific extracellular matrix (ECM) proteins, namely: collagen types I, III, IV, V (CI, CIII, CIV, CV), fibronectin (FN), and laminin (LM). Anti-ECM antibodies of immunoglobulin G (IgG), IgA, and IgM classes were determined by enzyme linked immunosorbent assay (ELISA). Overall, sera from 50% of patients with TAO contained antibodies reactive against one or more ECM proteins, compared to 27% with Graves' disease (GD) without evident eye involvement, 28% with Hashimoto's thyroiditis (HT), and 9% of normal subjects. Serum anti-CI, anti-CIII, anti-CV and anti-LM levels were significantly (p<0.05) higher in patients with TAO than in normals. Anti-CI, anti-CV and anti-LM reactivity was antigen-specific in most TAO sera, while anti-CIII antibodies cross-reacted with other antigens. Anti-collagen antibodies were mainly of the IgG class. To determine the structural epitopes of these proteins, we performed immunoblotting studies on cyanogenbromide (CNBr)-derived peptides of CI and CV. While sera from 9 of 10 patients with TAO reacted with CI peptides, the response was polyclonal and uniform in all patients. However, only 2 of 10 TAO sera reacted with CV peptides. In conclusion, our study suggests that a variety of ECM proteins (CI, CV, LM) may be secondary autoantigens that are recognized by antibodies in TAO. While these antibodies appear to react with epitopes expressed on both native and denatured proteins, and may therefore have the potential to bind to ECM in vivo, their pathogenic role in TAO remains unknown.
Asunto(s)
Autoanticuerpos/sangre , Proteínas de la Matriz Extracelular/inmunología , Enfermedad de Graves/inmunología , Adolescente , Adulto , Anciano , Especificidad de Anticuerpos , Colágeno/inmunología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Enfermedad de Graves/sangre , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Laminina/inmunología , Masculino , Persona de Mediana Edad , Desnaturalización Proteica/inmunologíaRESUMEN
The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85-kDa band. GNA identifies mannose-rich oligosaccharides because it is highly specific for terminal alpha-mannose residues. After purification of this 85-kDa glycoprotein band by chromatography on GNA-agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N-Glycanase or endoglycosidase H produced a 65-kDa band, indicating that it consisted of about one-fourth N-linked oligomannosidic carbohydrate moieties. High-performance anion-exchange chromatography and fluorescence-assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85-kDa mannose-rich glycoprotein component contained the SHP substrate-1 for phosphotyrosine phosphatases and at least one other member of the signal-regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor-like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.
Asunto(s)
Química Encefálica/fisiología , Glicoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Galanthus , Glicoproteínas/genética , Hexosaminidasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Manósidos/metabolismo , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Ratas , Ratas Wistar , Proteínas Tirosina Fosfatasas con Dominio SH2 , Dominios Homologos src/fisiologíaRESUMEN
The myelin-associated glycoprotein (MAG) exhibits an abnormally high apparent molecular weight in sciatic nerve, but not in brain, of dysmyelinating trembler mutants (Inuzuka et al.: J Neurochem 44:793-797, 1985). Antibodies to the large and small isoforms of MAG (L- and S-MAG) and probes for oligosaccharide structure were used to determine if this was due to overexpression of L-MAG or increased glycosylation. Nerves from both control and trembler 36-day-old mice contained primarily S-MAG with only traces of L-MAG. The distribution of the two isoforms appeared normal in trembler mice, and both isoforms exhibited the higher apparent molecular weight. Lectin binding showed that, in contrast to brain in which most glycoproteins contain primarily alpha 2-3 linked sialic acid, most glycoproteins of both control and trembler nerve contained primarily alpha 2-6 linked sialic acid. Lectin binding and glycosidase treatments demonstrated that the higher molecular weight of MAG in trembler nerves was due to an increased content of alpha 2-3 linked sialic acid and galactose. The abnormal glycosylation of MAG in trembler mutants may contribute to the myelin pathology.
Asunto(s)
Galactosa/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Nervios Periféricos/metabolismo , Ácidos Siálicos/metabolismo , Animales , Glicoproteínas/metabolismo , Glicósido Hidrolasas/farmacología , Glicosilación , Isomerismo , Lectinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Peso Molecular , Glicoproteína Asociada a Mielina/química , Valores de ReferenciaRESUMEN
Human choriogonadotropin (hCG) is a glycoprotein hormone that activates adenylyl cyclase. The carbohydrate moieties of hCG are required for biological activity, but not for binding to the gonadotropin receptors. We modified N-acetylneuraminic acid (NeuAc) on the oligosaccharide moieties of hCG, and determined the effect on its biological activity by measuring hormone-stimulated adenylyl cyclase. Treating hCG with sodium periodate to remove two carbon atoms from NeuAc or quantitatively removing NeuAc from hCG reduced its biological activity by 36% and 50%, respectively. The galactose residues of asialo-hCG were reacted with NeuAc-hydrazone or a hydrazone of the oligosaccharide from the ganglioside GM1 (Gal(beta 1-3)GalNAc(beta 1-4) [NeuAc(alpha 2-3)]Gal(beta 1-4)Glc). The gonadotropin receptor had high affinity for both derivatives, but their biological activity was less than that of hCG. These results suggest that several structural aspects of NeuAc including carbon side chain, an intact ring structure, and the position of NeuAc relative to other carbohydrate residues are important for full biological activity of hCG.
Asunto(s)
Gonadotropina Coriónica/efectos de los fármacos , Ácidos Siálicos/fisiología , Transducción de Señal/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Asialoglicoproteínas/química , Asialoglicoproteínas/farmacología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Carbohidratos/análisis , Línea Celular , Toxina del Cólera/metabolismo , Gonadotropina Coriónica/química , Gonadotropina Coriónica/farmacología , Gonadotropina Coriónica/fisiología , Activación Enzimática/efectos de los fármacos , Gangliósido G(M1)/fisiología , Galactosa Oxidasa/farmacología , Glicosilación , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico , Oxidación-Reducción , Ácido Peryódico/farmacología , Procesamiento Proteico-Postraduccional , Transducción de Señal/fisiologíaRESUMEN
Quaking is an autosomal recessive hypo/dysmyelinating mutant mouse which has a 1-Mbp deletion on chromosome 17. The mutation exhibits pleiotrophy and does not include genes encoding characterized myelin proteins. The levels of the 67-kD isoform of the myelin-associated glycoprotein (S-MAG) relative to those of the 72-kD isoform (L-MAG) are increased in the quaking CNS, but not in other dysmyelinating mutants. Abnormal expression of MAG isoforms in quaking may result from altered transcription of the MAG gene or from abnormal sorting, transport, or targeting of L-MAG or S-MAG. To test these hypotheses, we have determined the distribution of L-MAG and S-MAG in cervical spinal cord of 7-, 14-, 21-, 28-, and 35-d-old quaking mice. In 7-d-old quaking and control spinal cord, L- and S-MAG was detectable in periaxonal regions of myelinated fibers and in the perinuclear cytoplasm of oligodendrocytes. Between 7 and 35 d, L-MAG was removed from the periaxonal membrane of quaking but not control mice. Compared to control mice, a significant increase in MAG labeling of endosomes occurred within oligodendrocyte cytoplasm of 35-d-old quaking mice. S-MAG remained in periaxonal membranes of both quaking and control mice. Analysis of the cytoplasmic domain of L-MAG identifies amino acid motifs at tyrosine 35 and tyrosine 65 which meet the criteria for "tyrosine internalization signals" that direct transmembrane glycoproteins into the endocytic pathway. These results establish that L-MAG is selectively removed from the periaxonal membrane of CNS-myelinated fibers by receptor-mediated endocytosis. The loss of L-MAG from quaking periaxonal membranes results from increased endocytosis of L-MAG and possibly a decrease in L-MAG production.
Asunto(s)
Endocitosis/fisiología , Ratones Quaking/fisiología , Vaina de Mielina/química , Glicoproteína Asociada a Mielina/análisis , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Axones/química , Axones/ultraestructura , Inmunohistoquímica , Isomerismo , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Proteína Proteolipídica de la Mielina/análisis , Proteína Proteolipídica de la Mielina/inmunología , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Asociada a Mielina/metabolismo , Médula Espinal/química , Fracciones Subcelulares/químicaRESUMEN
The relative expression of large (L) and small (S) isoforms of the myelin-associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using antisera specific for L- and S-MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetylneuraminic acid residues linked alpha 2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oligosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants.
Asunto(s)
Enfermedades Desmielinizantes/metabolismo , Modelos Animales de Enfermedad , Ratones Quaking/metabolismo , Proteínas de la Mielina/metabolismo , Amidohidrolasas , Animales , Metabolismo de los Hidratos de Carbono , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Glicósido Hidrolasas , Glicosilación , Immunoblotting , Lectinas , Masculino , Ratones , Peso Molecular , Proteínas de la Mielina/genética , Glicoproteína Asociada a Mielina , Neuraminidasa , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Nervio Ciático/metabolismo , Médula Espinal/metabolismoRESUMEN
A number of polyamine (PA) derivatives of thiosemicarbazone of 1,3-dichloroacetone (TDA) have been prepared and their effect on growth in vivo of tumorigenic but not metastatic cell strain (LY-R) of mouse lymphoma L5178Y has been investigated. Polyamine derivatives of TDA (PDT) were injected i.p. every third day (4 times, 10 or 25 mg/kg per injection) into DBA/2 mice inoculated i.p. or s.c. with LY-R cells. It has been found that disubstituted putrescine (Put), spermidine (Spd) and spermine (Spm) derivatives TDA exhibit a prometastatic activity as indicated by the appearance of solid tumor foci in subcutaneous tissues, liver and spleen. This activity depends mainly on the structure of the PA fragment and the presence of TDA. An increase in lipid bound sialic acid content after treating LY-R cells in vitro and in vivo with a Spm derivative has been found. These findings suggest that disubstituted PA derivatives of TDA and LY-R cells may be a useful model for investigation of the final steps in formation of metastases by lymphoma cells.
Asunto(s)
Acetona/análogos & derivados , Leucemia L5178/patología , Leucemia Experimental/patología , Poliaminas , Ácidos Siálicos/metabolismo , Tiosemicarbazonas/administración & dosificación , Animales , Gangliósidos/aislamiento & purificación , Leucemia L5178/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , Metástasis de la Neoplasia , Relación Estructura-ActividadAsunto(s)
Equipo Dental , Oclusión Dental , Humanos , Registro de la Relación Maxilomandibular , MandíbulaRESUMEN
A major mono- and a di-sialoganglioside were isolated and purified to homogeneity from a spontaneous thymoma that occurs in AKR mice. Compositional and methylation analyses and the use of exoglycosidases established the monosialoganglioside to be alpha Neu(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----4)Cer and the disialoganglioside to be alpha NeuAc(2----8)alpha NeuAc(2----3)beta Gal(1----3)beta GalNAc(1----4)beta Gal(1----4)Glc(1----1)Cer (GD1c). A possible pathway for the biosynthesis of this disialoganglioside is presented.
Asunto(s)
Gangliósidos/aislamiento & purificación , Timoma/análisis , Neoplasias del Timo/análisis , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía en Capa Delgada , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Glicósido Hidrolasas , Metilación , Ratones , Ratones Endogámicos AKRRESUMEN
Lymphocytes from spontaneous thymoma in AKR mice and from X-ray induced thymoma in C57B1/6 mice showed elevated levels (by 50% and 100%, respectively) of lipid-bound sialic acid as compared with lymphocytes from normal thymuses used as controls. Some ganglioside fractions in thymomas were elevated 4-6-fold over those in normal thymuses while other fractions decreased or disappeared. Neutral glycosphingolipid (NGSL) content in lymphocytes from thymomas was also changed. Thin-layer chromatography of NGSLs showed that the fractions migrating as ceramide monohexoside (CMH), dihexoside (CDH) and below globoside standards were increased, respectively, 2-3-fold, 3-6-fold and 2-fold in both types of thymomas. Methylation and gas-liquid chromatography analysis confirmed the presence of CMH, CDH and globoside in NGSLs isolated from X-ray induced thymoma.