Structure of bovine rhodopsin in a trigonal crystal form

We have determined the structure of bovine rhodopsin at 2.65 A resolution using untwinned native crystals in the space group P3(1), by molecular replacement from the 2.8 A model (1F88) solved in space group P4(1). The new structure reveals mechanistically important details unresolved previously, which are considered in the membrane context by docking the structure into a cryo-electron microscopy map of 2D crystals. Kinks in the transmembrane helices facilitate inter-helical polar interactions. Ordered water molecules extend the hydrogen bonding networks, linking Trp265 in the retinal binding pocket to the NPxxY motif near the cytoplasmic boundary, and the Glu113 counterion for the protonated Schiff base to the extracellular surface. Glu113 forms a complex with a water molecule hydrogen bonded between its main chain and side-chain oxygen atoms. This can be expected to stabilise the salt-bridge with the protonated Schiff base linking the 11-cis-retinal to Lys296. The cytoplasmic ends of helices H5 and H6 have been extended by one turn. The G-protein interaction sites mapped to the cytoplasmic ends of H5 and H6 and a spiral extension of H5 are elevated above the bilayer. There is a surface cavity next to the conserved Glu134-Arg135 ion pair. The cytoplasmic loops have the highest temperature factors in the structure, indicative of their flexibility when not interacting with G protein or regulatory proteins. An ordered detergent molecule is seen wrapped around the kink in H6, stabilising the structure around the potential hinge in H6. These findings provide further explanation for the stability of the dark state structure. They support a mechanism for the activation, initiated by photo-isomerisation of the chromophore to its all-trans form, that involves pivoting movements of kinked helices, which, while maintaining hydrophobic contacts in the membrane interior, can be coupled to amplified translation of the helix ends near the membrane surfaces.     

Crystals of native and modified bovine rhodopsins and their heavy atom derivatives

Rhodopsin, the pigment protein responsible for dim-light vision, is a G protein-coupled receptor that converts light absorption into the activation of a G protein, transducin, to initiate the visual response. We have crystallised detergent-solubilised bovine rhodopsin in the native form and after chemical modifications as needles 10-40 microm in cross-section. The crystals belong to the trigonal space group P3(1), with two molecules of rhodopsin per asymmetric unit, related by a non-crystallographic 2-fold axis parallel with the crystallographic screw axis along c (needle axis). The unit cell dimensions are a=103.8 A, c=76.6 A for native rhodopsin, but vary over a wide range after heavy atom derivatisation, with a between 101.5 A and 113.9 A, and c between 76.6 A and 79.2 A. Rhodopsin molecules are packed with the bundle of transmembrane helices tilted from the c-axis by about 100 degrees . The two molecules in the asymmetric unit form contacts along the entire length of their transmembrane helices 5 in an antiparallel orientation, and they are stacked along the needle axis according to the 3-fold screw symmetry. Hence hydrophobic contacts are prominent at protein interfaces both along and normal to the needle axis. The best crystals of native rhodopsin in this crystal form diffracted X-rays from a microfocused synchrotron source to 2.55 A maximum resolution. We describe steps taken to extend the diffraction limit from about 10 A to 2.6 A.     

Dominantly inherited cutaneous small-vessel lymphocytic vasculitis maps to chromosome 6q26–q27

Outside the context of hereditary deficiencies of complement and IgA, Mendelian inherited predisposition to small vessel lymphocytic vasculitis (SVLV) has rarely been documented. Here we report a large, multigenerational family segregating symmetrical cutaneous SVLV affecting the cheeks, thighs and hands. In all affected family members the disease presented in early infancy and there was no evidence for an association with systemic disease. Skin biopsy of lesions showed a lymphocytic vasculitis with red blood cell extravasation. Complementary studies, with extensive investigation focused on dysfunction of the immunological system were negative. The pattern of inheritance of SVLV in the family was compatible with an autosomal dominantly acting disease gene with incomplete penetrance. To localize the disease causing gene in the family a genome-wide linkage search was conducted using a high-density SNP array. Haplotype construction and analysis of recombination events permitted the minimal interval defining the disease locus to be refined to a 4.7 Mb region on chromosome 6q26–q27. The genes CCR6 and GPR31, which map to the linked region represent plausible candidates for the disease on the basis of their biological function. Extensive screening of both genes by mutational analysis failed to identify a deleterious mutation in the family.     

Dual Action of Myricetin on Porphyromonas gingivalis and the Inflammatory Response of Host Cells: A Promising Therapeutic Molecule for Periodontal Diseases

Periodontitis that affects the underlying structures of the periodontium, including the alveolar bone, is a multifactorial disease, whose etiology involves interactions between specific bacterial species of the subgingival biofilm and the host immune components. In the present study, we investigated the effects of myricetin, a flavonol largely distributed in fruits and vegetables, on growth and virulence properties of Porphyromonas gingivalis as well as on the P. gingivalis-induced inflammatory response in host cells. Minimal inhibitory concentration values of myricetin against P. gingivalis were in the range of 62.5 to 125 μg/ml. The iron-chelating activity of myricetin may contribute to the antibacterial activity of this flavonol. Myricetin was found to attenuate the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including proteinases (rgpA, rgpB, and kgp) and adhesins (fimA, hagA, and hagB). Myricetin dose-dependently prevented NF-κB activation in a monocyte model. Moreover, it inhibited the secretion of IL-6, IL-8 and MMP-3 by P. gingivalis-stimulated gingival fibroblasts. In conclusion, our study brought clear evidence that the flavonol myricetin exhibits a dual action on the periodontopathogenic bacterium P. gingivalis and the inflammatory response of host cells. Therefore, myricetin holds promise as a therapeutic agent for the treatment/prevention of periodontitis.     

Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.     

Gap analyses of priority wild relatives of food crop in current ex situ and in situ conservation in Indonesia

Conservation programmes are always limited by available resources. Careful planning is therefore required to increase the efficiency of conservation and gap analysis can be used for this purpose. This method was used to assess the representativeness of current ex situ and in situ conservation actions of 234 priority crop wild relatives (CWR) in Indonesia. This analysis also included species distribution modelling, the creation of an ecogeographical land characterization map, and a complementarity analysis to identify priorities area for in situ conservation and for further collecting of ex situ conservation programmes. The results show that both current ex situ and in situ conservation actions are insufficient. Sixty-six percent of priority CWRs have no recorded ex situ collections. Eighty CWRs with ex situ collections are still under-represented in the national genebanks and 65 CWRs have no presence records within the existing protected area network although 60 are predicted to exist in several protected areas according to their potential distribution models. The complementarity analysis shows that a minimum of 61 complementary grid areas (complementary based on grid cells) are required to conserve all priority taxa and 40 complementary protected areas (complementary based on existing protected areas) are required to conserve those with known populations within the existing in situ protected area network. The top ten of complementary protected areas are proposed as the initial areas for the development of CWR genetic reserves network in Indonesia. It is recommended to enhanced coordination between ex situ and in situ conservation stakeholders for sustaining the long term conservation of CWR in Indonesia. Implementation of the research recommendations will provide for the first time an effective conservation planning of Indonesia’s CWR diversity and will significantly enhance the country’s food and nutritional security.

     

High genetic diversity despite drastic bottleneck in a critically endangered,long‐lived seabird,the Mascarene Petrel Pseudobulweria aterrima

The Mascarene Petrel Pseudobulweria aterrima is a critically endangered seabird endemic to Reunion Island, with an extremely small population suffering several threats. Fifteen polymorphic microsatellite loci were isolated from this species to analyse genetic diversity, estimate contemporary effective population size, search for evidence of a population bottleneck and see whether results support the hypothesis that life history traits could preserve allelic diversity in small populations. Results from 22 individuals found grounded as a consequence of light pollution highlight a surprisingly high genetic diversity, an absence of inbreeding, a contemporary effective population size estimated at approximately 1211 individuals and a probable bottleneck around 10 000 generations ago. Additional studies on genetic diversity and structure from a larger number of samples are thus required to evaluate the evolutionary potential of this critically endangered species.     

Beyond the senses: perception,the environment,and vision impairment

The ‘sensory turn’ in anthropology has generated a significant literature on sensory perception and experience. Whilst much of this literature is critical of the compartmentalization of particular ‘senses’, there has been limited exploration of how anthropologists might examine sensory perception beyond ‘the senses’. Based on ethnographic fieldwork with people who have impaired vision walking the South Downs landscape in England, this article develops such an approach. It suggests that the experiences of seeing in blindness challenge the conceptualization of ‘vision’ (and ‘non-vision’). In place of ‘vision’ (as a sense), the article explores ‘activities of seeing’ – an approach that contextualizes the visual to examine the biographically constituted and idiosyncratic nature of perception within an environment. Through an ethnography of seeing with anatomical eyes and ‘seeing in the mind's eye’, it articulates an approach that avoids associating perception with anatomy, or compartmentalizing experience into ‘senses’.     

Polymorphism in Gag gene cleavage sites of HIV-1 non-B subtype and virological outcome of a first-line lopinavir/ritonavir single drug regimen

Virological failure on a boosted-protease inhibitor (PI/r) first-line triple combination is usually not associated with the detection of resistance mutations in the protease gene. Thus, other resistance pathways are being investigated. First-line PI/r monotherapy is the best model to investigate in vivo if the presence of mutations in the cleavage sites (CS) of gag gene prior to any antiretroviral treatment might influence PI/r efficacy. 83 patients were assigned to initiate antiretroviral treatment with first-line lopinavir/r monotherapy in the randomised Monark trial. We compared baseline sequence of gag CS between patients harbouring B or non-B HIV-1 subtype, and between those who achieved viral suppression and those who experienced virological failure while on LPV/r monotherapy up to Week 96. Baseline sequence of gag CS was available for 82/83 isolates; 81/82 carried at least one substitution in gag CS compared to HXB2 sequence. At baseline, non-B subtype isolates were significantly more likely to harbour mutations in gag CS than B subtype isolates (p<0.0001). Twenty-three patients experienced virological failure while on lopinavir/r monotherapy. The presence of more than two substitutions in p2/NC site at baseline significantly predicted virological failure (p = 0.0479), non-B subtype isolates being more likely to harbour more than two substitutions in this specific site. In conclusion, gag cleavage site was highly polymorphic in antiretroviral-naive patients harbouring a non-B HIV-1 strain. We show that pre-therapy mutations in gag cleavage site sequence were significantly associated with the virological outcome of a first-line LPV/r single drug regimen in the Monark trial.