Intracellular lipid binding proteins including fatty acid solution binding proteins (FABPs)

Intracellular lipid binding proteins including fatty acid solution binding proteins (FABPs) 1 and 2 are highly expressed in tissues involved in the active lipid metabolism. displaced by oleic acid. In vivo experiments demonstrated for the first time that intestinal absorption of dietary BODIPY-FLC12 was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. and are the most strongly expressed FABP family members in the human small intestine (13 14 and these proteins are found in abundance in absorptive cells (39 40 In zebrafish the anterior intestine is the major site of fat absorption (41-43) and different genes were found expressed in the intestine including (41 44 The teleost ancestor experienced a whole-genome duplication event at the base of the teleost radiation (48) leading in some cases to the retention of pairs of duplicate genes (e.g. and found adjacent in the zebrafish genome (44). Zebrafish were used to study the expression pattern and dietary regulation of homologs to human and 7225625) corresponding to GenBank database dbEST zebrafish clone gb “type”:”entrez-nucleotide” attrs :”text”:”BC095259.1″ term_id :”66267583″ term_text :”BC095259.1″BC095259.1 was used to generate the RNA probe. The sense (ZF-fabp1b F1) 5-CAAGACTATTGTGAACAGAGA-3 and Quizartinib antisense (ZF-fabp1b R1) 5-TGAGATTGAGAACACTTTAATG-3 primers were designed from this clone and used for probe synthesis as previously described (43) except that the primer annealing temperature (Tm) in the thermal profile was 55°C for in the first PCR amplification. For the RNA probe the ZF-FB clone (41) was used corresponding to a 203 bp Quizartinib PCR product of GenBank database dbEST zebrafish clone gbAJ132590 after amplification with the sense (oligo ZFA1) 5-CTGTCATCATCATGACCTTCAACGG-3 and antisense (oligo ZF A3) 5-CCGCACACTGGAAATTAACTTTAC-3 primers subcloned using the pGEM-T Easy vector kit (Promega France). The second PCR was performed with the two pGEM-T Easy vector cDNAs using the T7 and SP6 universal primers as previously described (43). As the fragments were sense oriented in the vector the PCR template for the sense probe was produced using T7 universal primer while the PCR template for the antisense probe was produced using SP6 universal Quizartinib primer and the inverse for the probes. Both antisense and sense digoxigenin (DIG)-labeled RNA probes CCR5 were synthesized using the DIG RNA labeling kit (SP6/T7) (Roche Diagnostics Meylan France) following the manufacturer’s instructions. The 211 bp probe was able to hybridize to and transcript variants. The probe was 203 bp long. The protocol for in situ hybridization was as previously described (51 52 except that Quizartinib prehybridization and hybridization were conducted at 60°C. The Quizartinib posthybridization stringent baths were at hybridization temperature except for the last two baths in 0.2× SSC Tween at 57°C (30 min each). In contrast the hybridization buffer contained 50% formamide and the animals were incubated in preabsorbed sheep anti-DIG-AP Fab (Roche Diagnostics) fragments at 1:5 0 dilution at 4°C overnight. The antibody was rinsed in six PBS-Tween baths for 30 min each time. Amino acid sequence analyses Deduced protein sequences were extracted from the UniProt (53) database. Sequences were aligned (supplementary Fig. 1) using the ClustalW2 program (54). Recombinant Fabp1b.1 and Fabp2 production and purification Zebrafish and full coding cDNA sequences (supplementary Table 1) were cloned in the pET5a vector (Promega). pET5a-Fabp1b.1 and pET5a-Fabp2 constructs were transformed into BL21 (DE3) Star strain (Novagen). Bacterial cells containing the relevant expression plasmid were cultured in 2× yeast extract and tryptone media at 30°C for Fabp1b.1 and 37°C for Fabp2 for ～4 h before Fabp synthesis was induced by adding 0.4 mM isopropyl β-d-1-thiogalactopyranoside and incubating for a further 4 h. After centrifugation at 5 0 for 10 min the cells were collected and resuspended in cell lysis buffer (30 mM Tris-HCl 1 mM EDTA 1 mM DTT pH 8.3) and ruptured by sonication. Cell debris was removed by centrifugation at 15 0 at 4°C for 30 min and the supernatant was processed in two ammonium sulfate precipitation steps. (NH4)2SO4 was Quizartinib first added to a final concentration of 30% with stirring at room temperature for 2 h followed by centrifugation at 15 0 for 15 min. The supernatant fraction was treated with (NH4)2SO4 to a final concentration of 50% and centrifuged at 15.