Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Disposition kinetics of [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [H-3] palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [H-3] palmitate and metabolites were measured in four experimentalgroups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [H-3] palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [H-3] palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.

Reduced hepatic extraction of palmitate in steatosis correlated to lower level of liver fatty acid binding protein

Nonalcoholic fatty liver disease is the most common of all liver diseases. The hepatic disposition [H-3]palmitate and its low-molecular-weight metabolites in perfused normal and steatotic rat liver were studied using the multiple indicator dilution technique and a physiologically based slow diffusion/bound pharmacokinetic model. The steatotic rat model was established by administration of 17alpha-ethynylestradiol to female Wistar rats. Serum biochemistry markers and histology of treated and normal animals were assessed and indicated the presence of steatosis in the treatment group. The steatotic group showed a significantly higher alanine aminotransferase-to-aspartate aminotransferase ratio, lower levels of liver fatty acid binding protein and cytochrome P-450, as well as microvesicular steatosis with an enlargement of sinusoidal space. Hepatic extraction for unchanged [H-3]palmitate and production of low-molecular-weight metabolites were found to be significantly decreased in steatotic animals. Pharmacokinetic analysis suggested that the reduced extraction and sequestration for palmitate and its metabolites was mainly attributed to a reduction in liver fatty acid binding protein in steatosis.

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion. (c) 2006 Australian Society for Parasitology Inc Published by Elsevier Ltd. All fights reserved.

Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and other malignancies. WT1 is one of the earliest genes expressed during kidney development. WT1 proteins can activate and repress putative target genes in vitro, although the in vivo relevance of such target genes often remains unverified. To better understand the role of WT1 in tumorigenesis and kidney development, we need to identify downstream target genes. In this study, we have expression pro. led human embryonic kidney 293 cells stably transfected to allow inducible WT1 expression and mouse mesonephric M15 cells transfected with a WT1 antisense construct to abolish endogenous expression of all WT1 isoforms to identify WT1-responsive genes. The complementary overlap between the two cell lines revealed a pronounced repression of genes involved in cholesterol biosynthesis by WT1. This pathway is transcriptionally regulated by the sterol responsive element-binding proteins (SREBPs). Here, we provide evidence that the C-terminal end of the WT1 protein can directly interact with SREBP, suggesting that WT1 may modify the transcriptional function of SREBPs via a direct protein-protein interaction. Therefore, the tumour suppressor activities of WT1 may be achieved by repressing the mevalonate pathway, thereby controlling cellular proliferation and promoting terminal differentiation.

Sequence and tissue distribution of chicken cellular nucleic acid binding protein cDNA

We sequenced cDNAs coding for chicken cellular nucleic acid binding protein (CNBP). Two slightly different variations of the open reading frame were found, each of which translates into a protein with seven zinc finger domains. The longest transcript contains an in-frame insert of 3 bp. The sequence conservation between chick CNBP cDNAs with human, rat and mouse CNBP cDNAs is extreme, especially in the coding region, where the deduced amino acid sequence identity with human, rat and mouse CNBP is 99%. CNBP-like transcripts were also found in various tissues from insect, shrimp, fish and lizard. Regions with remarkable nucleotide conservation were also found in the 3' untranslated region, indicating important functions for these regions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that in the chick, CNBP is present in all tissues examined in approximately equal ratios to total RNA. RT-PCR of total RNA isolated from different phyla indicate CNBP-like proteins art widespread throughout the animal kingdom. The extraordinary level of conservation suggests an important physiological role for CNBP. (C) 1997 Elsevier Science Inc.

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

Lipid content and fatty acid composition of 11 species of Queensland (Australia) fish

The fatty acid composition of 11 species of fish caught off the northeast coast of Australia was determined. No fatty acid profiles have been previously published for fish from this area nor for nine of these species. Although the percentage of polyunsaturated fatty acid (PU FA) was the same as the calculated average for Australian fish (42.3%), the percentage of n-3 fatty acids was lower (24.4 +/- 5.4% vs. 30.7 +/- 10.1%) and the n-6 fatty acids higher (16.5 +/- 4.5% vs. 11.2 +/- 5.9%), P < 0.001 in each case. The major n-3 PUFA were docosahexaenoic (15.6 +/- 6.3%) and eicosapentaenoic acid (4.3 +/- 1.1%) while the major n-6 PUFA were arachidonic (8.3 +/- 3.2%) and n-6 docosatetraenoic acid (3.1 +/- 1.3%). The second-most abundant class of fatty acid was the saturates (31.6 +/- 3.5%) while the monounsaturates accounted for 17.4 +/- 4.3% of the total fatty acids. The monounsaturate with the highest concentration was octadecenoic acid (11.8 +/- 2.6%). There was a positive correlation between the total lipid content and saturated and monounsaturated fatty acids (r = 0.675 and 0.567, respectively) and a negative correlation between the total lipid content and PUFA(r = 0.774).

The human temporal cortex: Characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles

Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CH), in the inferotemporal gyros (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, GB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal calls, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.