RESUMEN
Cofilin, an actin-binding protein, plays an important role in the migration, phagocytosis, and superoxide production of activated phagocytes through cytoskeletal reorganization. In unstimulated phagocytes, cofilin is a major phosphoprotein. However, upon activation, the phosphoprotein is dephosphorylated and translocated from cytosol to plasma membranes. Only the unphosphorylated form of cofilin is an active form that binds actin, whereas the regulatory mechanisms of cofilin have not been elucidated. We found that 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phospholipase C (PLC), suppressed both opsonized zymosan (OZ)-induced dephosphorylation and translocation of cofilin in macrophage-like U937 cells at 4 microM concentration. OZ triggered an increase in inositol 1,4,5-trisphosphate (IP3), and U73122 inhibited it. 1-[6-[[17beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-pyrrodione-dione (U73343), which was employed as an inactive analogue, had no such inhibitory activities as did U73122. Furthermore, herbimycin A, an inhibitor of src-type tyrosine kinase, also inhibited OZ-triggered IP3 formation. These results suggest that the activity and localization of cofilin are regulated by PLC at the downstream of src-family tyrosine kinase.
Asunto(s)
Estrenos/metabolismo , Proteínas de Microfilamentos/metabolismo , Transporte de Proteínas/fisiología , Pirrolidinonas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Factores Despolimerizantes de la Actina , Membrana Celular/metabolismo , Estrenos/farmacología , Células HL-60 , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Activación de Macrófagos , Proteínas Opsoninas , Fosforilación , Pirrolidinonas/farmacología , Superóxidos/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Células U937 , Zimosan , Familia-src Quinasas/metabolismoRESUMEN
We previously noted that some aged human cortical specimens containing very low or negligible levels of amyloid beta-protein (As) by enzyme immunoassay (EIA) provided prominent signals at 6 approximately 8 kd on the Western blot, probably representing sodium dodecyl sulfate (SDS)-stable Abeta dimer. Re-examination of the specificity of the EIA revealed that BAN50- and BNT77-based EIA, most commonly used for the quantitation of Abeta, capture SDS-dissociable Abeta but not SDS-stable Abeta dimer. Thus, all cortical specimens in which the levels of Abeta were below the detection limits of EIA were subjected to Western blot analysis. A fraction of such specimens contained SDS-stable dimer at 6 approximately 8 kd, but not SDS-dissociable A(beta) monomer at approximately 4 kd, as judged from the blot. This A(beta) dimer is unlikely to be generated after death, because (i) specimens with very short postmortem delay contained the A(beta) dimer, and (ii) until 12 hours postmortem, such SDS-stable A(beta) dimer is detected only faintly in PDAPP transgenic mice. The presence of A(beta) dimer in the cortex may characterize the accumulation of A(beta) in the human brain, which takes much longer than that in PDAPP transgenic mice.
Asunto(s)
Envejecimiento/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Corteza Cerebral/metabolismo , Dodecil Sulfato de Sodio/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Anticuerpos Monoclonales , Dimerización , Estabilidad de Medicamentos , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Transgénicos/genética , Persona de Mediana Edad , Conformación MolecularRESUMEN
In this study we sought to learn about when and how amyloid beta-protein (A beta) accumulates in the cortex of normal individuals and about the difference in the A beta accumulation between normal aged and Alzheimer's disease (AD) brains. From consecutive autopsy cases and AD cases, hippocampus CA1 and occipitotemporal cortex T4 were sampled for A beta quantitation by the well characterized two-site enzyme immunoassays (EIAs). There was a strong tendency toward A beta 42 accumulation between the ages of 50 and 70 years in T4 and a little later in CA1. The A beta 42 levels were consistently higher in T4 than those in CA1 in any given case. The levels of A beta 42 in AD brains were significantly higher than those in control brains, and the extent of A beta 42 amino-terminal modification was also much greater in AD brains than that in control brains. Even in cases in which no senile plaques were immunocytochemically detected, EIAs clearly showed that significant amounts of A beta 42 already had accumulated. In contrast to A beta 42, A beta 40 showed no apparent age-dependent accumulation, and its high levels were found to be associated with AD.
Asunto(s)
Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Corteza Cerebral/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/química , Apolipoproteínas E/genética , Femenino , Genotipo , Hipocampo/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Procesamiento Proteico-Postraduccional , Transferrina/genéticaRESUMEN
Our previous study showed that non-reducing terminal galactose residues of N-linked sugar chains present in sheep erythrocyte membrane glycoproteins are important for rosette formation with T lymphoblastic cells [Ogasawara et al. (1995) Immunol Lett 48: 35-38]. As a first step to elucidate the significant structures of sugar chains involved in rosette formation, we analysed N-linked sugar chains released from the membrane glycoproteins by hydrazinolysis. The oligosaccharides were labeled with NaB3H4 and fractionated using columns of Aleuria aurantia lectin-Sepharose, MonoQ and Bio-Gel P-4. Structural analyses of oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the membrane glycoproteins contain bi- (19%), tri- (33%), and tetraantennary (44%) complex-type oligosaccharides and that the oligosaccharides having exposed galactose residues amount to 40% of the total.
Asunto(s)
Membrana Eritrocítica/química , Glicoproteínas de Membrana/química , Animales , Secuencia de Carbohidratos , Humanos , Metilación , Datos de Secuencia Molecular , Oligosacáridos/química , Formación de Roseta , Ovinos , Sialoglicoproteínas/química , Linfocitos T/citologíaRESUMEN
In this study, we found that rosette formation of T lymphoblastic Molt-3 cells with sheep erythrocytes is inhibited by addition of membrane glycoproteins which were solubilized from sheep erythrocyte ghosts by the lithium diiodosalicylate extraction methods. Their rosetting inhibitory activity was markedly reduced by digestion with N-glycanase, but not with O-glycanase. The inhibitory activity was also reduced by beta-galactosidase digestion, while it was enhanced by desialylation. These observations indicate that nonreducing terminal galactose residues of N-linked sugar chains included in membrane glycoproteins on sheep erythrocytes are important for rosette formation with T lymphocytes.
Asunto(s)
Membrana Eritrocítica/química , Glicoproteínas de Membrana/fisiología , Formación de Roseta , Animales , Línea Celular , Galactosa/química , Glicósido Hidrolasas/metabolismo , Ovinos/sangre , Linfocitos T/inmunologíaRESUMEN
Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.
Asunto(s)
ADN/metabolismo , Lectinas/biosíntesis , Plantas/química , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Elementos Transponibles de ADN , Electroforesis en Gel de Poliacrilamida , Galactosa/química , Expresión Génica , Biblioteca de Genes , Hidrólisis , Lectinas/química , Lectinas/genética , Datos de Secuencia Molecular , Lectinas de Plantas , Plásmidos , Albúmina Sérica Bovina/químicaRESUMEN
In order to examine the correlation between the amino acid sequence and sugar binding specificity of Bauhinia purpurea lectin (BPA), a galactose and lactose binding lectin, a peptide which interacts with lactose was purified from an Asp-N endoproteinase digest of BPA by means of affinity chromatography on a column of lactose-Sepharose. The amino acid sequence of this peptide is Asp-Thr-Trp-Pro-Asn-Thr-Glu-Trp-Ser. A tryptic fragment having the ability to interact with lactose was also purified and found to contain the above sequence, consisting of 9 amino acids. The chemical synthesis of this peptide was carried out by the solid-phase method and the synthetic peptide was found to exhibit lactose binding activity in the presence of calcium.