Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.     

APOA5 and triglyceride metabolism, lesson from human APOA5 deficiency

PURPOSE OF REVIEW: In this review we compare the phenotype and lipoprotein abnormalities of some patients who were found to carry mutations in the APOA5 gene predicted to result in apolipoprotein A-V deficiency. RECENT FINDINGS: The sequencing of the APOA5 gene in patients with primary hypertriglyceridemia, in whom mutations of the LPL and APOC2 genes had been excluded, led to the identification of four families with two different mutations in this gene predicted to result in truncated apolipoprotein A-V. The first mutation (Q148X) was found in a homozygous state in a child with severe type V hyperlipidemia, some clinical manifestations of chylomicronemia syndrome and a slight reduction in plasma postheparin lipoprotein lipase activity. Carriers of a different mutation (Q139X) were recently reported. Four Q139X heterozygotes had type V hyperlipidemia and markedly reduced plasma postheparin lipoprotein lipase activity. The hypertriglyceridemic Q139X heterozygote had other factors that could have contributed to hypertriglyceridemia. ApoB-100 kinetic studies in hypertriglyceridemic Q139X heterozygotes revealed an impairment of very low-density lipoprotein catabolism. SUMMARY: Mutations in the APOA5 gene, leading to truncated apolipoprotein A-V devoid of lipid-binding domains located in the carboxy-terminal end of the protein, if present in the homozygous state, are expected to cause severe type V hyperlipidemia in patients with no mutations in LPL or APOC2 genes. If present in the heterozygous state, these mutations predispose to hypertriglyceridemia in combination with other genetic factors or pathological conditions.     

Indications for a dietary change in the extinct Bovid genus Myotragus (Plio-Holocene,Mallorca, Spain)

Evolution in isolated island has shaped a variety of endemic taxa with outstanding characteristics. Amongst them is the extinct bovid genus Myotragus, endemic to Mallorca and Menorca Island, for which six succeeding species have been described: M. palomboi, M. pepgonellae, M. antiquus, M. kopperi, M. batei and M. balearicus. Myotragus has developed special cranial and post-cranial adaptations to meet the specific ecological demands of its insular habitat, like progressive dwarfing and fused limb elements. During its evolution, the dentition of Myotragus underwent subsequent changes: firstly a reduction in the number of teeth, and secondly an increase in hypsodonty. The ecological conditions inducing this dental evolution, especially Myotragus’ diet, remain unknown. In this study, methods of 3D-dental topometry, enamel surface texture analysis according to ISO/FDIS 25178-2, and Scale-Sensitive Fractal Analysis (SSFA) are applied in order to infer palaeodiets of M. pepgonellae, M. kopperi, M. batei and M. balearicus, and to test the hypothesis that a dietary change may have occurred in the Myotragus lineage which relates to gradual morphological changes on upper second molars. We detect changes in the enamel/dentin ratio, enamel ridge length and enamel surface area within the lineage. Furthermore, Myotragus balearicus has enamel surface texture characteristics also present in extant browsing ungulates, while the three antecedent Myotragus species show an enamel surface texture signal similar to extant grazers. These results suggest a dietary change and are interpreted as a successive adaptation to limited resources in an isolated, insular environment. They can either be a consequence of a change in plant community structure or a successive expansion of Myotragus’ dietary range due to increased intraspecific competition.     

Rapid and transient activation of the ERK MAPK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on JAB1/CSN5 and Src kinase activity

Macrophage migration inhibitory factor (MIF) is a 12.5 kD polypeptide that serves as a critical regulator of cell functions such as gene expression, proliferation or apoptosis. However, the signal transduction pathways through which MIF takes part in cellular regulation are only incompletely understood. MIF leads to CD74-dependent "sustained" activation of ERK1/2 MAPK, but MIF's role in "transient" ERK activation and the involved upstream pathways are unknown. Here we report that the transient ERK pathway was markedly activated by MIF. This effect involved the phosphorylation and activation of Raf-1, MEK, ERK, and Elk-1. Of note, rapid and transient ERK phosphorylation by MIF was measurable in MIF-deficient cells, suggesting that MIF acted in a non-autocrine fashion. Applying the inhibitor genistein, a tyrosine kinase (TPK) activity was identified as a critical upstream signalling event in MIF-induced transient ERK signalling. Experiments using the Src kinase inhibitor PP2 indicated that the involved TPK was a Src-type tyrosine kinase. A role for an upstream Src kinase was proven by applying Src-deficient cells which did not exhibit transient ERK activation upon treatment with MIF, but in which MIF-induced ERK signalling could be restored by re-expressing Src. Intriguingly, JAB1/CSN5, a signalosome component, cellular binding protein of MIF and regulator of cell proliferation and survival, had a marked, yet dual, effect on MIF-induced ERK signalling. JAB1 overexpression inhibited sustained, but not transient, ERK phosphorylation. By contrast, JAB1-knock-down by siRNA revealed that minimum JAB1 levels were necessary for transient activation of ERK by MIF. In conclusion, MIF rapidly and transiently activates the ERK pathway, an effect that has not been recognized previously. This signalling pathway involves the upstream activation of a Src-type kinase and is co-regulated by the cellular MIF binding protein JAB1/CSN5. Our study thus has unravelled a novel MIF-driven signalling pathway and an intricate regulatory system involving extra- and possibly intracellular MIF, and which likely critically participates in controlling cell proliferation and survival.     

Teasing apart the contributions of hard dietary items on 3D dental microtextures in primates

3D dental microtexture analysis is a powerful tool for reconstructing the diets of extinct primates. This method is based on the comparison of fossils with extant species of known diet. The diets of primates are highly diversified and include fruits, seeds, grass, tree leaves, bark, roots, tubers, and animal resources. Fruits remain the main component in the diets of most primates. We tested whether the proportion of fruit consumed is correlated with dental microtexture. Two methods of microtexture analysis, the scale-sensitive fractal analysis (SSFA) and the Dental Areal Surface Texture Analysis (DASTA; after ISO/FDIS 25178-2), were applied to specimens of eight primate species (Alouatta seniculus, Gorilla gorilla, Lophocebus albigena, Macaca fascicularis, Pan troglodytes, Papio cynocephalus, Pongo abelii, Theropithecus gelada). These species largely differ in the mean annual proportion of fruit (from 0 to 90%) in their diet, as well as in their consumption of other hard items (seeds, bark, and insect cuticles) and of abrasive plants. We find the complexity and heterogeneity of textures (SSFA) to correlate with the proportion of fruits consumed. Textural fill volume (SSFA) indicates the proportion of both fruits and other hard items processed. Furthermore, anisotropy (SSFA) relates to the consumption of abrasive plants like grass and other monocots. ISO parameters valley height, root mean square height, material volume, density of peaks, and closed hill and dale areas (DASTA) describe the functional interaction between food items and enamel facets during mastication. The shallow, plastic deformation of enamel surfaces induced by small hard particles, such as phytoliths or dust, results in flat microtexture relief, whereas the brittle, deep fracture caused by large hard items such as hard seeds creates larger relief.     

Dominant TNF-α+ Mycobacterium tuberculosis-specific CD4+ T cell responses discriminate between latent infection and active disease

Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4+ T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-α Mtb-specific CD4+ T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry-based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion of single-positive TNF-α Mtb-specific CD4+ T cells is a new tool for the rapid diagnosis of active tuberculosis disease.