Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase

To investigate the role of protein cavities in facilitating movement of the substrates, methane and dioxygen, in the soluble methane monooxygenase hydroxylase (MMOH), we determined the X-ray structures of MMOH from Methylococcus capsulatus (Bath) cocrystallized with dibromomethane or iodoethane, or by using crystals pressurized with xenon gas. The halogenated alkanes bind in two cavities within the alpha-subunit that extend from one surface of the protein to the buried dinuclear iron active site. Two additional binding sites were located in the beta-subunit. Pressurization of two crystal forms of MMOH with xenon resulted in the identification of six binding sites located exclusively in the alpha-subunit. These results indicate that hydrophobic species bind preferentially in preexisting cavities in MMOH and support the hypothesis that such cavities may play a functional role in sequestering and enhancing the availability of the physiological substrates for reaction at the active site.     

Phylogenetic analysis of the Monocotylidae (Monogenea) inferred from 28S rDNA sequences

The current classification of the Monocotylidae (Monogenea) is based on a phylogeny generated from morphological characters. The present study tests the morphological phylogenetic hypothesis using molecular methods. Sequences from domains C2 and D1 and the partial domains C1 and D2 from the 28S rDNA gene for 26 species of monocotylids from six of the seven subfamilies were used. Trees were generated using maximum parsimony, neighbour joining and maximum likelihood algorithms. The maximum parsimony tree, with branches showing less than 70% bootstrap support collapsed, had a topology identical to that obtained using the maximum likelihood analysis. The neighbour joining tree, with branches showing less than 70% support collapsed, differed only in its placement of Heterocotyle capricornensis as the sister group to the Decacotylinae clade. The molecular tree largely supports the subfamilies established using morphological characters. Differences are primarily how the subfamilies are related to each other. The monophyly of the Calicotylinae and Merizocotylinae and their sister group relationship is supported by high bootstrap values in all three methods, but relationships within the Merizocotylinae are unclear. Merizocotyle is paraphyletic and our data suggest that Mycteronastes and Thaumatocotyle, which were synonymized with Merizocotyle after the morphological cladistic analysis, should perhaps be resurrected as valid genera. The monophyly of the Monocotylinae and Decacotylinae is also supported by high bootstrap values. The Decacotylinae, which was considered previously to be the sister group to the Calicotylinae plus Merizocotylinae, is grouped in an unresolved polychotomy with the Monocotylinae and members of the Heterocotylinae. According to our molecular data, the Heterocotylinae is paraphyletic. Molecular data support a sister group relationship between Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis to the exclusion of the other species of Neoheterocotyle and recognition of Troglocephalus renders Neoheterocotyle paraphyletic. We propose Troglocephalus incertae sedis. An updated classification and full species list of the Monocotylidae is provided.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Effects of Nosema bombi and its treatment fumagillin on bumble bee (Bombus occidentalis) colonies

We examined the effects of Nosema bombi (Microsporidia: Nosematidae) on colonies of bumble bees, Bombus occidentalis Greene (Hymenoptera: Apidae), used to pollinate tomatoes in commercial greenhouses. We assessed methods of detecting N. bombi and tested the effectiveness of fumagillin to control this parasite. N. bombi did not affect adult population size or amount of brood in B. occidentalis colonies. Fumagillin was not effective against N. bombi at the doses we tested, and frass samples did not provide accurate estimates of the intensity of N. bombi infections. The number of N. bombi spores per bee was highly variable among bumble bees within colonies, and accurate estimates could only be obtained by sampling a large proportion of bees in each colony. Therefore, whole bee and frass sampling is useful for determining if N. bombi is present or absent, but not for obtaining accurate estimates of the intensity of N. bombi infections.     

Salt bridges do not stabilize polyproline II helices

Interactions between side chains, and in particular salt bridges, have been shown to be important in the stabilization of secondary structure. Here we investigate the contribution of a salt bridge formed between a lysine and a glutamate to the polyproline II (P(II)) helical content of proline-rich peptides. Since this structure has precisely three residues per turn, charged residues spaced three residues apart are on the same side of the helix and are best situated to interact. By contrast, computer simulations show that charged residues spaced four residues apart are both too far apart to interact strongly and are oriented such that interactions are unlikely. We have measured the P(II) content of peptides containing a lysine and glutamate pair spaced three or four residues apart using circular dichroism spectroscopy. Somewhat surprisingly we find that the P(II) content is insensitive to both the spacing and the pH. These findings indicate that i --> i + 3 salt bridges do not stabilize the P(II) helical conformation. The implications of these observations for both P(II) helix formation and denatured protein conformations are discussed.     

Creation of genome-wide protein expression libraries using random activation of gene expression

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.     

Euzetia occultum n. g., n. sp. (Euzetiinae n. subf.), a monocotylid monogenean from the gills of Rhinoptera neglecta (Rhinopteridae) from Moreton Bay,Queensland, Australia

Euzetia occultum n. g., n. sp. (Monogenea: Monocotylidae) is described from the gills of the Australian cownose ray Rhinoptera neglecta Ogilby collected in Moreton Bay, Queensland, Australia. Euzetia has one central and ten peripheral loculi, which is similar to species in Decacotyle Young, 1967. However Euzetia is distinguished from other genera in the family by the presence of an additional loculus on either side of the central loculus. Because Euzetia does not fit into any of the six existing subfamilies in the Monocotylidae Taschenberg, 1879, as currently recognised, we propose the Euzetiinae n. subf. to accommodate the new genus. Euzetia occultum is described and illustrated fully. This is the first published record of a monocotylid from a species of Rhinoptera Cuvier.     

Use of growth indices from radiometric culture for quantification of sheep strains of Mycobacterium avium subsp. paratuberculosis

A simple method for using growth indices from radiometric BACTEC cultures was evaluated for the enumeration of Australian sheep strains of Mycobacterium avium subsp. paratuberculosis. The numbers of viable organisms in inocula were determined by end-point titration in BACTEC cultures. Growth indices were measured by using a BACTEC 460 machine. There was a linear relationship between the number of days taken for the cumulative growth index to reach 1,000 (dCGI1000) and log(10) inoculum size. The use of dCGI1000 was shown to be as effective as the use of growth index data from the entire growth cycle for the estimation of inoculum size. For particular isolates characterized by end-point titration, the dCGI1000 of a single BACTEC vial provided estimates of viable numbers within narrow prediction limits. Predictive relationships were also established for the enumeration of M. avium subsp. paratuberculosis from field samples by using the dCGI1000 of a single BACTEC vial, with prediction limits of +/-1 to 2 log units. Organisms from feces or contaminated soil grew more slowly than those from cultures or tissues, and separate equations were developed for enumeration from these sources.     

Morphology and development of the haptors among the Monocotylidae (Monogenea)

Monocotylid monogeneans inhabit a wide diversity of sites on their chondrichthyan hosts including the skin, gills, nasal fossae, urogenital system and coelom. The large variation in the morphology of the haptor appears to reflect this diversity in attachment sites. We demonstrate that the complexity of the haptor can be related to the habitat of the parasite. Generally, those parasites which live in habitats subject to strong water currents such as the gills and dorsal skin surface have more complex haptors than those in environments exposed to weaker or no water currents including the nasal fossae, urogenital system and body cavity. However, there can be considerable variation in haptoral components, even among congeners, living on the ‘gills’ of their hosts. The microhabitat was determined for Monocotyle helicophallus and M. spiremae, both from the gills of the pink whipray, Himantura fai, and M. corali from the gills of the cowtail ray, Pastinachus sephen. We demonstrate that differences in the morphology of the hamuli and the number and morphology of septal sclerites and marginal papillae among these species of Monocotyle can be related directly to their microhabitat. It also appears that different haptoral structures are important for attachment to the host at different stages in the development of the parasite, based on studies on the development and distribution of Neoheterocotyle rhinobatidis from the gills of the common shovelnose ray Rhinobatos typus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spatial Heterogeneity as a Genetic Mixing Mechanism in Highly Philopatric Colonial Seabirds

How genetic diversity is maintained in philopatric colonial systems remains unclear, and understanding the dynamic balance of philopatry and dispersal at all spatial scales is essential to the study of the evolution of coloniality. In the King penguin, Aptenodytes patagonicus, return rates of post-fledging chicks to their natal sub-colony are remarkably high. Empirical studies have shown that adults return year after year to their previous breeding territories within a radius of a few meters. Yet, little reliable data are available on intra- and inter-colonial dispersal in this species. Here, we present the first fine-scale study of the genetic structure in a king penguin colony in the Crozet Archipelago. Samples were collected from individual chicks and analysed at 8 microsatellite loci. Precise geolocation data of hatching sites and selective pressures associated with habitat features were recorded for all sampling locations. We found that despite strong natal and breeding site fidelity, king penguins retain a high degree of panmixia and genetic diversity. Yet, genetic structure appears markedly heterogeneous across the colony, with higher-than-expected inbreeding levels, and local inbreeding and relatedness hotspots that overlap predicted higher-quality nesting locations. This points towards heterogeneous population structure at the sub-colony level, in which fine-scale environmental features drive local philopatric behaviour, while lower-quality patches may act as genetic mixing mechanisms at the colony level. These findings show how a lack of global genetic structuring can emerge from small-scale heterogeneity in ecological parameters, as opposed to the classical model of homogeneous dispersal. Our results also emphasize the importance of sampling design for estimation of population parameters in colonial seabirds, as at high spatial resolution, basic genetic features are shown to be location-dependent. Finally, this study stresses the importance of understanding intra-colonial dispersal and genetic mixing mechanisms in order to better estimate species-wide gene flows and population dynamics.