Developmental functions of mammalian Hox genes

The structure of the four murine Hox complexes and the co-ordinate expression patterns of Hox genes have been elucidated for almost a decade. However, clues about their developmental functions have been recently uncovered from the analysis of loss-of-function mutants generated by the gene targeting technique, as well as from transgenic mice with altered Hox gene expression domains. The 'anterior' Hox genes control the morphogenetic programme of specific hindbrain segments (rhombomeres) or pharyngeal arch neural crest derivatives. Various studies indicate that Hox gene products act in a region-specific, combinatorial and partly redundant manner to specify the identities of developing vertebrae. In addition, 'posterior' HoxA and HoxD genes act coordinately to control the growth and morphogenesis of skeletal structures along the proximodistal axis of developing limbs. Studies in other vertebrate model systems suggest that the evolution of Hox gene functions has allowed for the acquisition of specific morphological features along both the vertebral column and limbs of tetrapods. Gene targeting studies have also revealed region-specific functions of Hox genes along the developing digestive and genito-urinary tracts.      

Characterization of BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance in unilateral and bilateral breast cancer: the WECARE study

BRCA1 and BRCA2 screening in women at high‐risk of breast cancer results in the identification of both unambiguously defined deleterious mutations and sequence variants of unknown clinical significance (VUS). We examined a population‐based sample of young women with contralateral breast cancer (CBC, n=705) or unilateral breast cancer (UBC, n=1398). We identified 470 unique sequence variants, of which 113 were deleterious mutations. The remaining 357 VUS comprised 185 unique missense changes, 60% were observed only once, while 3% occurred with a frequency of >10%. Deleterious mutations occurred three times more often in women with CBC (15.3%) than in women with UBC (5.2%), whereas combined, VUS were observed in similar frequencies in women with CBC and UBC. A protein alignment algorithm defined 16 rare VUS, occurring at highly conserved residues and/or conferring a considerable biochemical difference, the majority located in the BRCA2 DNA‐binding domain. We confirm a multiplicity of BRCA1 and BRCA2 VUS that occur at a wide range of allele frequencies. Although some VUS inflict chemical differences at conserved residues, suggesting a deleterious effect, the majority are not associated with an increased risk of CBC. © 2010 Wiley‐Liss, Inc.     

Pharmacological evaluation of a Br-76 analog of epibatidine: a potent ligand for studying brain nicotinic acetylcholine receptors

[(76)Br]-Norchlorobromoepibatidine ([(76)Br]BrPH) is a specific and high affinity radioligand for the nicotinic acetylcholine receptors (nAChRs). In vitro, on rat thalamus membranes [(76)Br]BrPH bound to two sites with apparent affinities of 8 pM and 3 nM. The density of binding sites were 1.9 and 70 fmol/mg protein, respectively. In vivo, in biodistribution and autoradiographic studies in rats the regional distribution of [(76)Br]BrPH paralleled the neuroanatomical localization of nAChRs. Two hours postinjection, the highest concentration in the brain was found in thalamus and colliculi (4% ID/g). Competition experiments with specific nicotinic, muscarinic, dopaminergic, and serotoninergic drugs confirmed that the in vivo binding of [(76)Br]BrPH was consistent with neuronal nicotinic receptors. PET imaging of [(76)Br]BrPH in baboon demonstrated a rapid and high uptake in the brain. Peak uptake occurred at 30-40 min for the thalamus. Due to the constant washout in the cerebellum, the thalamus to cerebellum ratio was 5 at 2 h postinjection. Subcutaneous injection of cytisine (1 mg/kg), 3 h postinjection of [(76)Br]BrPH reduced the radioactivity concentration in thalamus and cortex by 58 and 50%, respectively, as observed 1 h later. Cytisine pretreatment (5 mg/kg s.c.) inhibited completely the radioligand accumulation in the thalamus. Chronic MPTP pretreatment resulted in reduction of [(76)Br]BrPH uptake in all brain regions except in cerebellum. These preliminary results suggest that [(76)Br]BrPH has the potential to be a useful radioligand for studying the pharmacology of nicotinic acetylcholine receptors in preclinical experiments.     

In vivo detection of striatal dopamine release during reward: a PET study with [(11)C]raclopride and a single dynamic scan approach

A new simple method is proposed to detect, using PET and [(11)C]raclopride, changes in striatal extracellular dopamine concentration during a rewarded effortful task. This approach aimed to increase the sensitivity in detection of these effects. It requires a single-dynamic PET study and combines the classic kinetic compartmental model with the general linear model of SPM to provide statistical inference on changes in [(11)C]raclopride time-activity curve due to endogenous dopamine release during two short periods of activation. Kinetic simulations predicted that 100% dopamine increase during two 5-min periods starting at 30 and 60 min after the injection can be detected. Moreover the effects of dopamine release on the [(11)C]raclopride time-activity-curve are different from those induced by CBF increase. These simulated curves were used to construct the statistical linear model and to test voxel-by-voxel in healthy subjects the hypothesis that dopamine is released in the ventral striatum during periods of unexpected monetary gains, but not during periods of unexpected monetary loss. The experimental results are in line with the expected results although the amplitude of the effects due to dopamine release is moderate. The advantages and the limits of this method as well as the relevance of the results for dopamine involvement in reward processing are discussed.     

Discrepancies in the P-glycoprotein-Mediated Transport of 18F-MPPF: A Pharmacokinetic Study in Mice and Non-human Primates

Purpose

Several in vivo studies have found that the 5-HT_1A PET radioligand ¹⁸F-MPPF is a substrate of rodent P-glycoprotein (P-gp). However, in vitro assays suggest that MPPF is not a substrate of human P-gp. We have now tested the influence of inhibiting P-gp on the brain kinetics of ¹⁸F-MPPF in mice and non-human primates.

Methods

We measured the peripheral kinetics (arterial input function, metabolism, free fraction in plasma (f_P)) during ¹⁸F-MPPF brain PET scanning in baboons with or without cyclosporine A (CsA) infusion. We measured ³H-MPPF transport at the mouse BBB using in situ brain perfusion in P-gp/Bcrp deficient mice and after inhibiting P-gp with PSC833.

Results

There was an unexpected 1.9-fold increase in brain area under the curve in CsA-treated baboons (n?=?4), with no change in radiometabolite-corrected arterial input. However, total volume of distribution corrected for f_P (V_T/f_P) remained unchanged. In situ brain perfusion showed that P-gp restricted the permeability of the mouse BBB to ³H-MPPF while Bcrp did not.

Conclusion

These and previous in vitro results suggest that P-gp may not influence the permeability of human BBB to ¹⁸F-MPPF. However, CsA treatment increased ¹⁸F-MPPF free fraction, which is responsible for a misleading, P-gp unrelated enhanced brain uptake.     

Multiinjection approach for D2 receptor binding quantification in living rats using [11C]raclopride and the beta-microprobe: crossvalidation with in vitro binding data.

The purpose of this study was to quantify D2 receptors density and affinity in living rats using [11C]raclopride and to validate the multiinjection modelling approach. To this aim, we used an intracerebral beta+-sensitive probe as a highly sensitive system to quantify the radioligand activity using a single three-injection experimental paradigm. The study was divided into three main parts: (i) [11C]raclopride catabolism evaluation without and with cimetidine pretreatment (cytochrome P450 inhibitor); (ii) quantification of kinetics parameters in the striatum, enthorinal cortex, and cerebellum of living rats using a three-compartment model with an arterial input function; (iii) correlation study of in vivo and in vitro binding density and affinity values in the same striatal tissues. (i) raclopride catabolism was very reproducible between individuals; cimetidine pre-treatment resulted in a 30% reduction of raclopride metabolites. (ii) D2 striatal B'max and KdVr estimates obtained by compartmental modelling were 19.87+/-6.45 and 6.2+/-3.3 nmol/L, respectively. Cerebellum is the best candidate as a reference region with no specific binding detectable in vivo. (iii) When comparing density (Bmax/B'max) and affinity (Kd/KdVr) values in vivo and in vitro for each striatum, a high strict correlation was found (r2=0.90 and 0.72, for density and affinity, respectively). These results validate the multi-injection modelling approach coupled to beta-microprobe acquisitions as a mean to provide accurate and separate estimates of dopamine D2-receptor density and affinity, in the living rodent striatum.     

Secretion of human monocyte mature IL-1β: Optimization of culture conditions and inhibition by ICE inhibitors

Correspondence to: J. Uhl     

Construction of a high resolution linkage disequilibrium map to evaluate common genetic variation in TP53 and neural tube defect risk in an Irish population

Genetic and environmental factors contribute to the etiology of neural tube defects (NTDs). While periconceptional folic acid supplementation is known to significantly reduce the risk of NTDs, folate metabolic pathway related factors do not account for all NTDs. Evidence from mouse models indicates that the tumor protein p53 (TP53) is involved in implantation and normal neural tube development. To determine whether genetic variation in TP53 might contribute to NTD risk in humans, we constructed a high resolution linkage disequilibrium (LD) map of the TP53 genomic region based on genotyping 21 markers in an Irish population. We found that nine of these variants can be used to capture the majority of common variation in the TP53 genomic region. In contrast, the 3-marker haplotype commonly reported in the TP53 literature offers limited coverage of the variation in the gene. We used the expanded set of polymorphisms to measure the influence of TP53 on NTDs using both case-control and family based tests of association. We also assayed a functional variant in the p53 regulator MDM2 (rs2279744). Alleles of three noncoding TP53 markers were associated with NTD risk. A case effect was seen with the GG genotype of rs1625895 in intron 6 (OR = 1.37 [1.04-1.79], P = 0.02). A maternal effect was seen with the 135/135 genotype of the intron 1 VNTR (OR = 1.86 [1.16-2.96], P = 0.01) and the TT genotype of rs1614984 (RR = 0.58 [0.37-0.91], P = 0.02). As multiple comparisons were made, these cannot be considered definitive positive findings and additional investigation is required.     

δ-Opioid Mechanisms for ADL5747 and ADL5859 Effects in Mice: Analgesia, Locomotion, and Receptor Internalization

N,N-diethyl-4-(5-hydroxyspiro[chromene-2,4'-piperidine]-4-yl) benzamide (ADL5859) and N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4'-piperidine]-4-yl)benzamide (ADL5747) are novel δ-opioid agonists that show good oral bioavailability and analgesic and antidepressive effects in the rat and represent potential drugs for chronic pain treatment. Here, we used genetic approaches to investigate molecular mechanisms underlying their analgesic effects in the mouse. We tested analgesic effects of ADL5859 and ADL5747 in mice by using mechanical sensitivity measures in both complete Freund's adjuvant and sciatic nerve ligation pain models. We examined their analgesic effects in δ-opioid receptor constitutive knockout (KO) mice and mice with a conditional deletion of δ-receptor in peripheral voltage-gated sodium channel (Nav)1.8-expressing neurons (cKO mice). Both ADL5859 and ADL5747, and the prototypical δ agonist 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethyl-piperazin-1-yl]-(3-methoxyphenyl)methyl]-N,N-diethyl-benzamide (SNC80) as a control, significantly reduced inflammatory and neuropathic pain. The antiallodynic effects of all three δ-opioid agonists were abolished in constitutive δ-receptor KO mice and strongly diminished in δ-receptor cKO mice. We also measured two other well described effects of δ agonists, increase in locomotor activity and agonist-induced receptor internalization by using knock-in mice expressing enhanced green fluorescence protein-tagged δ receptors. In contrast to SNC80, ADL5859 and ADL5747 did not induce either hyperlocomotion or receptor internalization in vivo. In conclusion, both ADL5859 and ADL5747 showed efficient pain-reducing properties in the two models of chronic pain. Their effects were mediated by δ-opioid receptors, with a main contribution of receptors expressed on peripheral Nav1.8-positive neurons. The lack of in vivo receptor internalization and locomotor activation, typically induced by SNC80, suggests agonist-biased activity at the receptor for the two drugs.