Dalcetrapib: no off-target toxicity on blood pressure or on genes related to the renin-angiotensin-aldosterone system in rats.

BACKGROUND AND PURPOSE: The association between torcetrapib and its off-target effects on blood pressure suggested a possible class-specific effect. The effects of dalcetrapib (RO4607381/JTT-705) and torcetrapib on haemodynamics and the renin-angiotensin-aldosterone system (RAAS) were therefore assessed in a rat model. EXPERIMENTAL APPROACH: Arterial pressure (AP) and heart rate were measured by telemetry in normotensive and spontaneously hypertensive rats (SHR) receiving torcetrapib 10, 40 or 80 mg kg(-1) day(-1); dalcetrapib 100, 300 or 500 mg(-1) kg day(-1); or vehicle (placebo) for 5 days. Expression of RAAS genes in adrenal gland, kidney, aorta and lung from normotensive rats following 5 days' treatment with torcetrapib 40 mg kg(-1) day(-1), dalcetrapib 500 mg kg(-1) day(-1) or vehicle was measured by quantitative polymerase chain reaction. KEY RESULTS: Torcetrapib transiently increased mean AP in normotensive rats (+3.7 +/- 0.1 mmHg), whereas treatment in SHR resulted in a dose-dependent and sustained increase [+6.5 +/- 0.6 mmHg with 40 mg kg(-1) day(-1) at day 1 (P < 0.05 versus placebo)], which lasted over the treatment period. No changes in AP or heart rate were observed with dalcetrapib. Torcetrapib, but not dalcetrapib, increased RAAS-related mRNAs in adrenal glands and aortas. CONCLUSIONS AND IMPLICATIONS: In contrast to torcetrapib, dalcetrapib did not increase blood pressure or RAAS-related gene expression in rats, suggesting that the off-target effects of torcetrapib are not a common feature of all compounds acting on cholesteryl ester transfer protein.

Homeobox genes in the developing mouse brain.

Any list of past and recent findings on vertebrate brain prenatal development would have to include the fundamental roles of homeobox genes, the genes encoding the nuclear regulatory homeodomain proteins. The discovery of homeobox genes and their involvement as master regulatory elements in programming the development of an embryo into a complete adult organism has provided a key to our understanding of ontogenesis. Also, the correlation of mouse developmental mutants and their corresponding human syndromes with mutations in homeobox genes has provided further evidence for the fundamental role of homeobox genes during the vertebrate brain embryonic development. Here, we review the expression patterns and the phenotypes of gene mutations that implicate a large repertoire of mouse homeobox genes in the specification of neuronal functions during brain embryogenesis.

Pharmacological characterisation of the recombinant human CRF binding protein using a simple assay.

We present the pharmacological characterisation of the recombinant human corticotropin releasing factor binding protein (hCRF-BP) using a simple assay. In this assay we employed [3H]urocortin as the radioligand and, as a means to separate bound and free ligand, adsorption to activated charcoal. Using this method, approximately 60-70% of total binding was specific. Kinetic analysis revealed that association of specific [3H]urocortin binding was monophasic and slow and that the binding was irreversible. Saturation analysis showed a single saturable site of relatively high density (94 fmol per 10 microl of medium from cells transfected with the recombinant CRF binding protein). The apparent Kd for [3H]urocortin binding of 0.25 nM is similar to previously reported affinities of rat urocortin for hCRF-BP. A range of CRF-related peptides potently competed for specific [3H]urocortin binding. The rank order of potency of these agents was human/rat CRF = urotensin 1 > human urocortin > CRF6-33 > sauvagine > ovine CRF. The non-peptide CRF1 receptor antagonists CP 154,526 (N-butyl-N-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]p yri midin-4-yl]-N-ethylamine) and SC 241 ([3-(2-bromo-4-isopropyl-phenyl)-5-methyl-3H-[1,2,3]triazo lo[4,5-d]pyrimidin-7-yl]-bis-(2-methoxy-ethyl)-amine) were not active at the highest concentration tested (10(-6) M). We conclude that this is a simple and accurate assay for characterisation of the pharmacology of the recombinant CRF-BP. This assay should assist with further study of the pharmacology and function of the CRF-BP.

The high-mobility group protein T160 binds to both linear and cruciform DNA and mediates DNA bending as determined by ring closure.

The high-mobility group protein T160 was isolated by screening a phage library from a murine pre-B-cell line L1210. South-Western experiments have previously shown that this protein binds to V-(D)-J recombination signal sequences, suggesting that it may be a sequence-specific DNA-binding protein. However, neither gel-shift nor footprinting analyses have been successfully employed with the T160 protein, despite an extensive effort. In this study, the T160 protein or truncated forms made soluble through denaturing and renaturing cycles in urea were successfully used in gel-shift experiments showing that T160 binds to cruci-form or linear duplex DNA with no apparent sequence specificity. Furthermore, fragments longer than 100 bp efficiently formed covalently closed circular monomers in the presence of T160 and T4 DNA ligase, indicating that the protein is capable of introducing bends into the duplex. Last, tissue distribution by Western blotting analysis showed that the T160 protein is expressed in various murine tissues in addition to those of lymphoid origin. Considering its broad evolutionary conservation (from plants to mammals) also, these results suggest that the functional role of the T160 protein is not limited to V-(D)-J recombination, but might be involved in basic processes such as DNA replication and repairing, where irregular DNA structures are generated and very likely recognized by HMG domain proteins.

Labelling of CRF1 and CRF2 receptors using the novel radioligand, [3H]-urocortin.

The binding of the novel radioligand, [3H]-rat urocortin to homogenates of rat cerebellum and homogenates of cells stably transfected with the human CRF1, rat CRF2alpha and rat CRF2beta receptors was examined. In each case, specific reversible high affinity binding was observed (K[d]s between 0.18 and 0.31 nM). The density of sites was relatively low in the cerebellum (9 fmol/mg tissue) but high in the recombinant systems with expression levels of between 1.4 and 6.3 pmol/mg protein. Agents known to interact with CRF receptors potently competed for binding in each case. The pharmacological profile of binding to the recombinant receptors were consistent with data previously published using other radioligands. Thus, for the recombinant CRF1 receptor, binding was inhibited with similar affinity by Urocortin, sauvagine, Urotensin 1 and CRF. The non-peptidic CRF antagonists (e.g. CP 154,526 and SC 241) also potently inhibited binding. The CRF2alpha and CRF2beta receptor recombinant systems had a very similar pharmacological profile with a clear rank order of potency for the peptide ligands (Urocortin > Sauvagine > Urotensin 1 > CRF), whereas the non-peptide CRF receptor antagonists had no measurable affinity. The pharmacological profile of specific [3H]-urocortin binding to homogentates of rat cerebellum was consistent with specific labelling of a CRF1 receptor. We conclude that [3H]-urocortin is a useful tool for the study of CRF receptors with the advantages that a filtration assay can be used, all CRF receptors can be labelled with the same ligand and the benefits associated with the low energy emittor, 3H.

Expression of a novel murine homeobox gene in the developing cerebellar external granular layer during its proliferation.

The TGIF homeobox gene encodes a homeoprotein that represses the 9-cis retinoic acid receptor-dependent transcription activation. To investigate the potential role of this gene in vertebrate development, we have isolated cDNA clones of the murine TGIF (mTGIF) gene and analyzed its expression pattern during mouse embryogenesis and postnatal development by Northern analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization histochemistry. mTGIF transcripts were detected at day E16 in the emerging external granular layer (EGL), the cells of which arise from the proliferating cerebellar neuroepithelium. Expression of mTGIF transcripts was also detected at day E16 in the proliferating cells in the neuroepithelium of the hippocampal formation. Following gestation, mTGIF expression increases to a maximum between postnatal days 5 and 10 (PN5 and PN10) in the rapidly expanding cerebellar EGL. mTGIF transcripts are no longer detectable when EGL proliferation ceases on approximately day PN15. Throughout embryo development and in the adult mice, TGIF is detected in a restricted number of tissues, mostly in proliferating and differentiating cell lineages, such as tongue and testis. Our results suggest that the TGIF gene regulates target genes involved in the proliferation, migration, and/or differentiation of particular neuronal cell lineages in the developing brain.

A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoid-responsive motif.

We describe here a novel homeobox gene, denoted TGIF (5"TG3' interacting factor), which belongs to an expanding TALE (three amino acid loop extension) superclass of atypical homeodomains. The TGIF homeodomain binds to a previously characterized retinoid X receptor (RXR) responsive element from the cellular retinol-binding protein II promoter (CRBPII-RXRE), which contains an unusual DNA target for a homeobox. The interactions of both the homeprotein TGIF and receptor RXR alpha with the CREBPII-RXRE DNA motif occur on overlapping areas and generate a mutually exclusive binding in vitro. Transient cellular transfections demonstrate that TGIF inhibits the 9-cis-retinoic acid-dependent RXR alpha transcription activation of the retinoic acid responsive element. TGIF transcripts were detected in a restricted number of tissues. The canonical binding site of TGIF is conserved and is an integral part of several responsive elements which are organized like the CRBPII-RXRE. Hence, a novel auxiliary factor to the steroid receptor superfamily may participate in the transmission of nuclear signals during development and in the adult, as illustrated by the down-modulation of the RXR alpha activities.

The Oct-2 protein binds cooperatively to adjacent octamer sites.

Recombinant proteins derived from the cloned human oct-2 gene were used to investigate cooperative binding by Oct-2 to adjacent DNA-binding sites. Oct-2, a B-cell-specific transcription factor, binds tightly to the octamer sequence in immunoglobulin promoters. A second apparently unrelated consensus sequence in heavy chain promoters, the heptamer site, also is recognized by the Oct-2 protein but with 1000-fold lower affinity. Simultaneous occupancy of both the octamer and heptamer sites is favored by cooperative interactions. The heptamer site is probably recognized by the same binding surface in the Oct-2 protein as the octamer site and thus is conserved as a lower-affinity binding site. This permits the immunoglobulin promoter to respond to a much broader range of levels of Oct-2 protein. Substitution of prototype octamer sequences for heptamer sequences yields a probe with two octamer sites spaced by 2 nucleotides, which also binds Oct-2 protein cooperatively. Only the POU domain in the Oct-2 protein is required for this cooperative interaction. Similar protein-protein interactions between bound Oct-2 proteins may promote promoter-enhancer synergism in the heavy chain gene.

Molecular cloning of sequence-specific DNA binding proteins using recognition site probes.

Genes encoding sequence-specific DNA binding proteins can be isolated by screening lambda gt11 expression libraries with recognition site DNAs. This strategy is derived from that developed for the isolation of genes using antibody probes. Many different genes encoding transcriptional regulatory proteins have been cloned using this strategy. The DNA binding domains of these regulatory proteins contain different structural motifs including the helix-turn-helix, the "zinc finger" and the "leucine zipper". Various aspects of the screening strategy are evaluated and a detailed protocol is provided. In addition to binding site DNAs, protein and nucleotide probes have been successfully used to screen expression libraries. Therefore ligand based expression screening may be quite general in scope.

The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains.

Transcription of promoters of immunoglobulin genes is controlled by an octanucleotide sequence element. The sequence of a cDNA encoding a B-cell-specific protein, Oct-2, has been determined. This protein specifically recognizes the octanucleotide element and is part of the previously identified NF-A2 family of proteins. The DNA-binding domain of Oct-2 is structurally related to the homeo box consensus and thus contains a potential helix-turn-helix sequence. Oct-2 also possesses a potential 'leucine zipper' domain, where four leucines are each separated by exactly seven residues. Comparisons of Oct-2 with protein Oct-1, which also recognizes the octanucleotide element but is constitutively expressed in all cell types, show high sequence conservation through the 60-residue DNA-binding domain, as well as an adjacent tract of 75 residues. The latter conserved region is also found in regulatory genes expressed in pituitary cells and nematodes and has been termed a POU box. Because two different cDNAs were isolated, it is proposed that the oct-2 gene is expressed as multiple mRNAs that vary in splicing patterns. Most interestingly, the oct-2 cDNA contains a second overlapping open reading frame, 278 residues in length, which might also specify a protein important for B-cell development.

Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif.

An octamer DNA sequence plays a critical role in directing transcription of immunoglobulin genes in B lymphocytes. A new technique of direct binding of radioactive DNA was used to screen a complementary DNA expression library from the BJAB cell line in lambda gt11 phage to derive molecular cDNA clones representing a putative B lymphocyte-specific octamer binding protein. The plaques were screened with DNA containing four copies of the octamer sequence and positive phage recombinants were identified. The fusion protein produced on inducing a lysogen of one phage bound to a monomeric octamer probe. The cDNA insert from this phage hybridized to messenger RNA found in B lymphocytes, but not in most other cells. Thus, this cDNA derives from a gene (oct-2) that specifies an octamer binding protein expressed preferentially in B lymphocytes, proving that, for at least one gene, a cell-specific transcription factor exists and its amount is controlled through messenger RNA availability.

Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation.

The cellular mechanisms involved in chronic inflammatory processes are poorly understood. This is especially true for the role of macrophages, which figure prominently in the inflammatory response. Two proteins, MRP8 and MRP14, which are expressed in infiltrate macrophages during inflammatory reactions but not in normal tissue macrophages, have been characterized. Here we report that MRP8 and MRP14 mRNAs are specifically expressed in human cells of myeloid origin and that their expression is regulated during monocyte-macrophage and granulocyte differentiation. To initiate the analysis of cis-acting elements governing the tissue-specific expression of the MRP genes, we cloned the human genes encoding MRP8 and MRP14. Both genes contain three exons, are single copy, and have a strikingly similar organization. They belong to a novel subfamily of highly homologous calcium-binding proteins which includes S100 alpha, S100 beta, intestinal calcium-binding protein, P11, and calcyclin (2A9). A transient expression assay was devised to investigate the tissue-specific regulatory elements responsible for MRP gene expression after differentiation in leukemia HL60 cells. The results of this investigation demonstrated that the cis-acting elements responsible for MRP expression are present on the cloned DNA fragment containing the MRP gene loci.

Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis.

The aetiology and cellular mechanism of chronic inflammatory processes are poorly understood. Macrophages act prominently in the inflammatory response and we report here that they express two calcium-binding proteins. The expression of these proteins, referred to as MRP-8 and MRP-14, is specific for cells of myeloid origin, namely granulocytes, monocytes and macrophages, and is observed in blood granulocytes and monocytes but not in normal tissue macrophages. In acutely inflamed tissues, macrophages can express MRP-14 but not MRP-8, and in chronic inflammations, such as primary chronic polyarthritis, infiltrate macrophages express both MRP-8 and MRP-14. Characterization of MRP-8 and MRP-14 could therefore be useful to the understanding of cellular processes induced in chronic inflammation.

Transcription of a cloned Xenopus laevis H4 histone gene in the homologous frog oocyte system depends on an evolutionary conserved sequence motif in the -50 region.

A cloned Xenopus laevis H4 histone gene has been expressed in the X.laevis oocyte nucleus. The homologous histone H4 gene can be correctly and efficiently expressed in the frog oocyte even in presence of bacterial vector DNA. As revealed by both analytical gel electrophoresis and S1 mapping, two H4 mRNAs are specified with different transcriptional efficiencies from the tandemly repeated promoter. Results from deletion mapping of the sequences essential for promoting H4 transcription show that drastic reduction of transcription is obtained when the sequences lying between -64 and -35 bp from the mRNA cap site are removed. We demonstrate by DNA sequence comparison using a novel computer program that this important area of the H4 promoter contains two highly conserved DNA motifs near positions -51 to -46 upstream from the cap site in all H4 gene promoters analysed.