RESUMO
We studied the expression and function of the granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in male germ cells. RT-PCR showed expression of mRNAs encoding the alpha- and beta-subunits of the GM-CSF receptor in human testis, and the presence of the alpha- and beta-proteins was confirmed by immunoblotting with anti-alpha and anti-beta-antibodies. Immunolocalization studies showed the level of expression of GM-CSF alpha- and beta-subunits in the germ line in the testis and in ejaculated spermatozoa. Receptor binding studies using radiolabeled GM-CSF revealed that bull spermatozoa have about 105 high-affinity sites with a K(d) of 222 pM and approximately 1100 low-affinity sites with a K(d) of 10 nM. GM-CSF signaled, in a time- and dose-dependent manner, for an increased uptake of glucose and vitamin C.
Assuntos
Ácido Ascórbico/metabolismo , Glucose/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/biossíntese , Transdução de Sinais , Testículo/metabolismo , Animais , Sítios de Ligação , Bovinos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Cinética , Masculino , Ligação Proteica , Transporte Proteico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sêmen/metabolismo , Espermatozoides/metabolismo , Fatores de TempoRESUMO
Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.
Assuntos
Peroxidase do Rábano Silvestre/metabolismo , Mucosa Intestinal/metabolismo , Microvilosidades/metabolismo , Animais , Western Blotting , Carpas , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Ligação ProteicaRESUMO
The selective interaction of serum proteins with immobilized Cibacron Blue and the binding properties of the dye anilinonaphthalenesulphonate has been used to separate albumin and lipoproteins by affinity chromatography. The novel binding of anilinonaphthalenesulphonate to lipoproteins from the sera of lamprey, fish and mammals provides a simple procedure for the isolation of these plasma proteins, and permit preparation of specific antisera, tools particularly relevant for evolutionary and clinical studies.
Assuntos
Naftalenossulfonato de Anilina/farmacologia , Lipoproteínas/isolamento & purificação , Triazinas/farmacologia , Animais , Galinhas , Peixes , Humanos , Lampreias , Métodos , CoelhosRESUMO
A computer program written in BASIC language is described. The program allows processing and analysis of DNA data and has been designed to be used by persons with little or no computer experience. The operator using different options can search for direct homologies with varying degrees of matching, generate complementary strands, find restriction sites, invert the polarity of the sequence and edit a print-out.