Structural and molecular interactions of CCR5 inhibitors with CCR5

We have characterized the structural and molecular interactions of CC-chemokine receptor 5 (CCR5) with three CCR5 inhibitors active against R5 human immunodeficiency virus type 1 (HIV-1) including the potent in vitro and in vivo CCR5 inhibitor aplaviroc (AVC). The data obtained with saturation binding assays and structural analyses delineated the key interactions responsible for the binding of CCR5 inhibitors with CCR5 and illustrated that their binding site is located in a predominantly lipophilic pocket in the interface of extracellular loops and within the upper transmembrane (TM) domain of CCR5. Mutations in the CCR5 binding sites of AVC decreased gp120 binding to CCR5 and the susceptibility to HIV-1 infection, although mutations in TM4 and TM5 that also decreased gp120 binding and HIV-1 infectivity had less effects on the binding of CC-chemokines, suggesting that CCR5 inhibition targeting appropriate regions might render the inhibition highly HIV-1-specific while preserving the CC chemokine-CCR5 interactions. The present data delineating residue by residue interactions of CCR5 with CCR5 inhibitors should not only help design more potent and more HIV-1-specific CCR5 inhibitors, but also give new insights into the dynamics of CC-chemokine-CCR5 interactions and the mechanisms of CCR5 involvement in the process of cellular entry of HIV-1.     

Heme-copper/dioxygen adduct formation relevant to cytochrome c oxidase: spectroscopic characterization of [(6L)FeIII-(O2 2−)-CuII]+

In the further development and understanding of heme-copper dioxygen reactivity relevant to cytochrome c oxidase O(2)-reduction chemistry, we describe a high-spin, five-coordinate dioxygen (peroxo) adduct of an iron(II)-copper(I) complex, [((6)L)Fe(II)Cu(I)](BArF(20)) (1), where (6)L is a tetraarylporphyrinate with a tethered tris(2-pyridylmethyl)amine chelate for copper. Reaction of 1 with O(2) in MeCN affords a remarkably stable [t(1/2) (rt; MeCN) approximately 60 min] adduct, [((6)L)Fe(III)-(O(2) (2-))-Cu(II)](+) (2) [EPR silent; lambda(max)=418 (Soret), 561 nm], formulated as a peroxo complex based on manometry (1:O(2)=1:1; spectrophotometric titration, -40 degrees C, MeCN), mass spectrometry {MALDI-TOF-MS: (16)O(2), m/z 1191 ([((6)L)Fe(III)-((16)O(2) (2-))-Cu(II)](+)); (18)O(2), m/z 1195}, and resonance Raman spectroscopy (nu((O-O))=788 cm(-1); Delta(16)O(2)/(18)O(2)=44 cm(-1); Delta(16)O(2)/(16/18)O(2)=22 cm(-1)). (1)H and (2)H NMR spectroscopy (-40 degrees C, MeCN) reveals that 2 is the first heme-copper peroxo complex which is high-spin, with downfield-shifted pyrrole resonances (delta(pyrrole)=75 ppm, s, br) and upfield shifted peaks at delta= -22, -35, and -40 ppm, similar to the pattern observed for the mu-oxo complex [((6)L)Fe(III)-O-Cu(II)](BAr(F)) (3) (known S=2 system, antiferromagnetically coupled high-spin Fe(III) and Cu(II)). The corresponding magnetic moment measurement (Evans method, CD(3)CN, -40 degrees C) also confirms the S=2 spin state, with mu(B)=4.9. Structural insights were obtained from X-ray absorption spectroscopy, showing Fe-O (1.83 A) and Cu-O (1.882 A) bonds, and an Fe...Cu distance of 3.35(2) A, suggestive of a mu-1,2-peroxo ligand present in 2. The reaction of 2 with cobaltocene gives 3, differing from the observed full reduction seen with other heme-Cu peroxo complexes. Finally, thermal decomposition of 2 yields 3, with concomitant release of 0.5 mol O(2) per mol 2, as confirmed quantitatively by an alkaline pyrogallol dioxygen scavenging solution.     

New Insight on FGFR3-Related Chondrodysplasias Molecular Physiopathology Revealed by Human Chondrocyte Gene Expression Profiling

Endochondral ossification is the process by which the appendicular skeleton, facial bones, vertebrae and medial clavicles are formed and relies on the tight control of chondrocyte maturation. Fibroblast growth factor receptor (FGFR)3 plays a role in bone development and maintenance and belongs to a family of proteins which differ in their ligand affinities and tissue distribution. Activating mutations of the FGFR3 gene lead to craniosynostosis and multiple types of skeletal dysplasia with varying degrees of severity: thanatophoric dysplasia (TD), achondroplasia and hypochondroplasia. Despite progress in the characterization of FGFR3-mediated regulation of cartilage development, many aspects remain unclear. The aim and the novelty of our study was to examine whole gene expression differences occurring in primary human chondrocytes isolated from normal cartilage or pathological cartilage from TD-affected fetuses, using Affymetrix technology. The phenotype of the primary cells was confirmed by the high expression of chondrocytic markers. Altered expression of genes associated with many cellular processes was observed, including cell growth and proliferation, cell cycle, cell adhesion, cell motility, metabolic pathways, signal transduction, cell cycle process and cell signaling. Most of the cell cycle process genes were down-regulated and consisted of genes involved in cell cycle progression, DNA biosynthesis, spindle dynamics and cytokinesis. About eight percent of all modulated genes were found to impact extracellular matrix (ECM) structure and turnover, especially glycosaminoglycan (GAG) and proteoglycan biosynthesis and sulfation. Altogether, the gene expression analyses provide new insight into the consequences of FGFR3 mutations in cell cycle regulation, onset of pre-hypertrophic differentiation and concomitant metabolism changes. Moreover, impaired motility and ECM properties may also provide clues about growth plate disorganization. These results also suggest that many signaling pathways may be directly or indirectly altered by FGFR3 and confirm the crucial role of FGFR3 in the control of growth plate development.     

Comparative chewing efficiency in mammalian herbivores

Although the relevance of particle size reduction in herbivore digestion is widely appreciated, few studies have investigated digesta particle size across species in relation to body mass or digestive strategy. We investigated faecal particle size, which reflects the size of ingesta particles after both mastication and specialized processes such as rumination. Particle size was measured by wet sieving samples from more than 700 captive individuals representing 193 mammalian species. Using phylogenetic generalized least squares, faecal particle size scaled to body mass with an exponent of 0.22 (95% confidence interval: 0.16–0.28). In comparisons among different digestive strategies, we found that (1) equids had smaller faecal particles than other hindgut fermenters, (2) non-ruminant foregut fermenters and hindgut fermenters had similar-sized faecal particles (not significantly different), and (3) ruminants had finer faecal particles than non-ruminants. These results confirm that the relationship between chewing efficiency and body mass is modified by morphological adaptations in dental design and physiological adaptations to chewing, such as rumination. This allometric relationship should be considered when investigating the effect of body size on digestive physiology, and digestion studies should include a measure of faecal particle size.     

A Statistical Test of a Neutral Model Using the Dynamics of Cytonuclear Disequilibria

In this paper we use cytonuclear disequilibria to test the neutrality of mtDNA markers. The data considered here involve sample frequencies of cytonuclear genotypes subject to both statistical sampling variation as well as genetic sampling variation. First, we obtain the dynamics of the sample cytonuclear disequilibria assuming random drift alone as the source of genetic sampling variation. Next, we develop a test statistic using cytonuclear disequilibria via the theory of generalized least squares to test the random drift model. The null distribution of the test statistic is shown to be approximately chi-squared using an asymptotic argument as well as computer simulation. Power of the test statistic is investigated under an alternative model with drift and selection. The method is illustrated using data from cage experiments utilizing different cytonuclear genotypes of Drosophila melanogaster. A program for implementing the neutrality test is available upon request.     

Bellwether of the Canaries: anthropogenic effects on the land snail fauna of the Canary Islands

Natural areas near human-modified landscapes experience factors that may affect local biodiversity at levels commensurate with natural environmental factors. The land snails of the Canary Islands provide excellent opportunities to evaluate the importance of anthropogenic agents in mediating the diversity and distribution of species. Land snails are particularly sensitive to disturbance and are an integral part of terrestrial ecosystems. This study analyzed the distributions and abundances of terrestrial macrosnail shell assemblages at 60 localities throughout the coastal scrub biome of the Canary Islands. This was accomplished using data on natural and anthropogenic variables to assess their relative importance in governing land snail diversity. A total of 34,801 dead shells represented a diverse malacofauna with highly localized endemism. Due to uncertain species identifications, samples from the 18 sites from the two easternmost islands are described, but excluded from statistical analyses. Regression tree analysis indicated that proximity to agricultural sites was the most important predictor of species diversity. Sites with no or very little agricultural area (≤ 0.167 km²) within a 1 km radius had significantly higher richness and diversity. These results have implications for Canary Islands conservation. Protected areas that are patchworks of natural and agricultural landscapes are still subject to native biodiversity loss because of anthropogenic impacts even when the footprint of agriculture is small.