RESUMEN
High density lipoprotein (HDL) has been shown to play an important role in the dietary lipid mobilisation in the carp. In spite of this, previous studies have failed to demonstrate the synthesis of the major protein component of HDL, apolipoprotein A-I (apoA-I), in the proximal intestine of the carp. Therefore, the aim of the present study was to evaluate the expression of apoA-I throughout the entire intestine. Curiously, no transcription of the apoA-I gene could be detected either by northern blot or RT-PCR assays in the intestinal mucosa, in clear contrast with the abundant cytosolic immunoreactive apoA-I detected in almost all intestinal segments, which suggests a different origin for this protein. In addition, the detection of specific, but low affinity, binding sites for apoA-I in the carp intestinal brush-border membranes (BBM), and the strong interaction with BBM, which is highly dependent on temperature, points to an important contribution of membrane lipids in apoA-I binding to the intestinal mucosa. This idea was reinforced by the ability of carp apoA-I to associate with multilamellar phospholipid vesicles.
Asunto(s)
Apolipoproteína A-I/metabolismo , Carpas/metabolismo , Grasas de la Dieta/metabolismo , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Animales , Northern Blotting , Cartilla de ADN , Dimiristoilfosfatidilcolina , Inmunohistoquímica , Microvellosidades/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , EspectrofotometríaRESUMEN
In a previous study we had demonstrated that a 15-kDa protein present in carp intestinal brush-border membrane vesicles (BBMV) was able to bind the endocytosis tracer horseradish peroxidase (HRP) with high specificity. Here we show that this protein corresponds to a peripheral membrane protein, identified by partial amino acid sequence analysis as the intestinal fatty acid-binding protein (I-FABP), a member of the small cytosolic fatty acid binding protein family (FABPs). The presence of I-FABP and its HRP-binding activity was demonstrated both in the cytosolic and membrane-associated fractions of intestinal mucosa by Western and ligand blot analyses, respectively. Also, both fractions displayed significant capacity to bind [(3)H]palmitic acid, a known ligand for I-FABP. Immunohistochemical analysis showed that I-FABP localizes both in the cytosol and in the brush-border membranes of epithelial cells. Taken together the unusual extra-cellular localization of I-FABP as well as its ability to interact with HRP suggests a novel function for this protein in the intestinal mucosa.
Asunto(s)
Carpas/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Endocitosis , Enterocitos/citología , Enterocitos/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Proteínas de Neoplasias , Secuencia de Aminoácidos , Animales , Western Blotting , Proteínas Portadoras/química , Polaridad Celular , Electroforesis en Gel de Poliacrilamida , Proteínas de Unión a Ácidos Grasos , Inmunohistoquímica , Microvellosidades/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Antimicrobial proteins and peptides play an important role in the primary defence of epithelial barriers in vertebrates and invertebrates. Here we report the detection of the apolipoproteins A-I and A-II in the epidermis and epidermal mucus of the carp (Cyprinus carpio L.) by immunohistochemistry and Western blot analysis. Both apolipoproteins are major constituents of high density lipoprotein and have been shown to display antiviral and antimicrobial activity in mammals. Therefore the aim of this study was to evaluate if they could be part of the innate immune system of teleost fish. A cDNA clone containing most of the coding region for carp apoA-I was isolated and used as a probe to demonstrate the expression of apoA-I gene in the skin. In addition, mucus apoA-I was shown to be associated to small particles that could correspond to nascent HDL. Finally, affinity purified plasma HDL displayed bactericidal activity in vitro against a non-pathogenic Escherichia coli strain, suggesting a defensive role for HDL and its associated proteins in the carp epidermis and mucus.
Asunto(s)
Apolipoproteína A-I/inmunología , Carpas/inmunología , Epidermis/inmunología , Secuencia de Aminoácidos , Animales , Apolipoproteína A-I/biosíntesis , Apolipoproteína A-I/genética , Apolipoproteína A-I/farmacología , Secuencia de Bases , Northern Blotting/veterinaria , Western Blotting/veterinaria , Carpas/genética , Carpas/metabolismo , ADN Complementario/química , ADN Complementario/genética , Epidermis/metabolismo , Regulación de la Expresión Génica/inmunología , Inmunohistoquímica/veterinaria , Lipoproteínas HDL/inmunología , Lipoproteínas HDL/farmacología , Datos de Secuencia Molecular , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Se sintetizó un análogo fotoactivable de biotina, el que se utilizó para marcar sondas de ácidos nucleicos. La marca se reveló con dos sistemas de detección avidina-peroxidasa y estreptavidina-fosfatasa alcalina, siendo ésta última la que demostró una mayor sensibilidad. Los plasmidos pSS1.8 y pSP64/U1 fueron fotobiotinilados y utilizados en ensayos de hibridación en gota con DNA extraido de leucocitos humanos. Despues de la incubacion con estreptavidina y fosfatasa alcalina biotinilada, la actividad de la enzima se reveló con un sustrato soluble. Los resultados obtenidos demuestran diferencias cuantitativas consistentes con el número de copias para globina y U1snRNA humano. El plasmido pSS1.8 fotobiotinilado se utilizó para identificar fragmentos de restricción de DNA genómico alterados en un paciente afectado de anemia de células falciformes. El gen de la globina mutado se detectó por digestión del DNA del paciente con la endonucleasa de restricción Dde I, seguido de una hibridación "Southern" con la sonda marcada