Functional calcitonin gene-related peptide receptors are formed by the asymmetric assembly of a calcitonin receptor-like receptor homo-oligomer and a monomer of receptor activity-modifying protein-1

In addition to their interactions with hetero-trimeric G proteins, seven-transmembrane domain receptors are now known to form multimeric complexes that can include receptor homo- or hetero-oligomers and/or accessory proteins that modulate their activity. The calcitonin gene-related peptide (CGRP) receptor requires the assembly of the seven-transmembrane domain calcitonin receptor-like receptor with the single-transmembrane domain receptor activity-modifying protein-1 to reach the cell surface and be active. However, the relative stoichiometric arrangement of these two proteins within a receptor complex remains unknown. Despite recent advances in the development of protein-protein interactions assays, determining the composition and stoichiometric arrangements of such signaling complexes in living cells remains a challenging task. In the present study, we combined bimolecular fluorescence complementation (BiFC) with bioluminescence resonance energy transfer (BRET) to probe the stoichiometric arrangement of the CGRP receptor complex. Together with BRET competition assays, co-immunoprecipitation experiments, and BiFC imaging, dual BRET/BiFC revealed that functional CGRP receptors result from the association of a homo-oligomer of the calcitonin receptor-like receptor with a monomer of the accessory protein receptor activity-modifying protein-1. In addition to revealing the existence of an unexpected asymmetric oligomeric organization for a G protein-coupled receptor, our study illustrates the usefulness of dual BRET/BiFC as a powerful tool for analyzing constitutive and dynamically regulated multiprotein complexes.     

Heterogeneous mechanomyographic absolute activation of paraspinal muscles assessed by a two-dimensional array during short and sustained contractions

Spatial dependency of paraspinal muscle activity was assessed using a new two-dimensional MMG recording system. MMG signals were detected over the left and right paraspinal muscles of 10 volunteers using a grid of 12 accelerometers. During two separate trials subjects maintained a 20 degrees flexed position and held loads that ranged from 0 to 15 kg (in 2.5 kg increments) for 20s; and 7.5 kg for 6 min. Maps of absolute and normalised (with respect to initial values) average rectified value, mean power frequency, variance and skewness of the power spectral density were obtained from the two-dimensional MMG recordings. For both the short duration and sustained contractions, the MMG absolute average rectified value, mean power frequency, variance and skewness depended on accelerometer location (P<0.05), while, with the exception of the skewness (P<0.05), normalised values did not. These results demonstrate both inhomogeneous MMG absolute activity and homogeneous MMG normalised activity in paraspinal muscles for short duration and sustained contractions. Moreover, the effect of accelerometer location on spectral variables confirmed the limited validity of general relationships between MMG spectral changes and motor unit recruitment strategies. This study underlines the importance of using multiple recording sites when assessing back muscle activity.     

Genomewide scan for linkage reveals evidence of several susceptibility loci for alopecia areata

Alopecia areata (AA) is a genetically determined, immune-mediated disorder of the hair follicle that affects 1%-2% of the U.S. population. It is defined by a spectrum of severity that ranges from patchy localized hair loss on the scalp to the complete absence of hair everywhere on the body. In an effort to define the genetic basis of AA, we performed a genomewide search for linkage in 20 families with AA consisting of 102 affected and 118 unaffected individuals from the United States and Israel. Our analysis revealed evidence of at least four susceptibility loci on chromosomes 6, 10, 16 and 18, by use of several different statistical approaches. Fine-mapping analysis with additional families yielded a maximum multipoint LOD score of 3.93 on chromosome 18, a two-point affected sib pair (ASP) LOD score of 3.11 on chromosome 16, several ASP LOD scores >2.00 on chromosome 6q, and a haplotype-based relative risk LOD of 2.00 on chromosome 6p (in the major histocompatibility complex locus). Our findings confirm previous studies of association of the human leukocyte antigen locus with human AA, as well as the C3H-HeJ mouse model for AA. Interestingly, the major loci on chromosomes 16 and 18 coincide with loci for psoriasis reported elsewhere. These results suggest that these regions may harbor gene(s) involved in a number of different skin and hair disorders.     

Na+ -D-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.     

Safeguarding marine life: conservation of biodiversity and ecosystems

Reviews in Fish Biology and Fisheries - Marine ecosystems and their associated biodiversity sustain life on Earth and hold intrinsic value. Critical marine ecosystem services include maintenance of...     

MMP-13 selective α-sulfone hydroxamates: a survey of P1' heterocyclic amide isosteres

Seeking compounds preferentially potent and selective for MMP-13, we reported in the preceding Letter on a series of hydroxamic acids with a flexible benzamide tail groups.^1a Here, we replace the amide moiety with non-hydrolyzable heterocycles in an effort to improve half-life. We identify a hydroxamate tetrazole 4e that spares MMP-1 and -14, shows >400-fold selectivity versus MMP-8 and >600-fold selectivity versus MMP-2, and has a 4.8 h half-life in rats. X-ray data (1.9 Å) for tetrazole 4c is presented.     

Historical and contemporary factors shape the population genetic structure of the broadcast spawning coral, Acropora millepora, on the Great Barrier Reef

Effective management of reef corals requires knowledge of the extent to which populations are open or closed and the scales over which genetic exchange occurs, information which is commonly derived from population genetic data. Such data are sparse for Great Barrier Reef (GBR) corals and other organisms, with the studies that are available being mostly based on a small number of sampling locations spanning only part of the GBR. Using 11 microsatellite loci, we genotyped 947 colonies of the reef-building coral Acropora millepora from 20 sites spanning almost the full length of the GBR (～12° of latitude and ～1550 km). The results show a major divide between the southernmost central to southern offshore populations and all other sampled populations. We interpret this divide as a signature of allopatric divergence in northern and southern refugia during the Pleistocene glaciations, from which the GBR was subsequently recolonized. Superimposed on this pattern is a cross-shelf genetic division, as well as a separation of inshore populations south of the Cape Clifton Front at ～21.5-22°S. Most inshore populations north of this, as well as mid-shelf populations in the northern and far northern GBR, are open, exchanging recruits frequently. In contrast, inshore populations south of the Cape Clifton Front and offshore populations in the central and southern GBR are largely self-seeding, at least within the spatial resolution that was achieved given our sampling intensity. Populations that have been impacted by recent disturbance events causing extensive coral mortality show no evidence of reduced genetic diversity.