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First published online March 21, 2005
Journal of Experimental Biology 208, 1393-1399 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01512

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Undetectable apolipoprotein A-I gene expression suggests an unusual mechanism of dietary lipid mobilisation in the intestine of Cyprinus carpio

Margarita I. Concha, Rodrigo López, Julieta Villanueva, Nadya Báez and Rodolfo Amthauer^*

Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile

View larger version (120K): [in a new window]   Fig. 1. Immunohistochemical localisation of apoA-I in the carp intestinal epithelium. (A–G) Thin sections (5 µm) of each of carp intestinal segments 1–7, respectively. (H) A negative control incubated only with the conjugate secondary antibody. Scale bars, 50 µm.

View larger version (57K): [in a new window]   Fig. 2. Immunodetection of apoA-I in carp intestine. (A) SDS–PAGE separation of plasma proteins (lane 1) and proteins in the lumen of different intestinal samples (lanes 2, 3). (B) Western blot analysis of the gel in A. All lanes were loaded with 20 µg of proteins.

View larger version (40K): [in a new window]   Fig. 5. Interaction of apoA-I with brush border membrane vesicles (BBMV). (A) 125I-apoA-I bound to BBMV; S, washed peripheral membranes; P, integral membrane fractions. Fractions were separated on a 12% SDS polyacrylamide gel and the protein detected by autoradiography. (B) Endogenous content of apoA-I in BBMV and distribution in the peripheral membrane (S) and integral membrane (P) fractions analysed by western blot.

View larger version (41K): [in a new window]   Fig. 3. apoA-I gene expression in carp intestine. (A) Total RNA (30 µg) extracted from liver and each of the intestinal segments were analysed by northern blot. Lane 1, liver RNA; lanes 2–8, all segments of intestine, from proximal to distal. (B) Ethidium bromide-stained total RNA (30 µg) from liver (lane 1) and all intestinal segments (lanes 2–8). (C) RT–PCR products for apoA-I (top) and ß-actin (bottom) gene expression. Lanes 1, 2, liver RNA with and without reverse transcriptase, respectively; lanes 3–9, intestinal RNA from all segments.

View larger version (10K): [in a new window]   Fig. 4. Concentration and temperature effect on apoA-I binding to brush border membrane vesicles (BBMV). (A) BBMV were incubated with varying concentrations of 125I-apoA-I. The amount of bound apoA-I was determined as described in Materials and methods. Total apoA-I binding (circles) and non-specific binding (triangles) was determined by adding 100 times the molar ratio of unlabelled apoA-I to the binding assay. Values are means ± S.E.M. of triplicate determinations. (B) BBMV were incubated with 200 nmol l–1 125I-apoA-I at different temperatures. Values are means ± S.E.M. of triplicate determinations.

View larger version (15K): [in a new window]   Fig. 6. Dimyristoylphosphatidylcholine multilamellar vesicle (DMPC mLV) solubilisation by apoA-I. DMPC mLV were incubated in the absence (open circles) or presence (closed circles) of apoA-I under the conditions described in Materials and methods. The solubilisation kinetics of the multilamellar vesicles was followed by measuring the decrease in absorbance at 325 nm.