Expression pattern of the type 1 sigma receptor in the brain and identity of critical anionic amino acid residues in the ligand-binding domain of the receptor

The type 1 sigma receptor (sigmaR1) has been shown to participate in a variety of functions in the central nervous system. To identify the specific regions of the brain that are involved in sigmaR1 function, we analyzed the expression pattern of the receptor mRNA in the mouse brain by in situ hybridization. SigmaR1 mRNA was detectable primarily in the cerebral cortex, hippocampus, and Purkinje cells of cerebellum. To identify the critical anionic amino acid residues in the ligand-binding domain of sigmaR1, we employed two different approaches: chemical modification of anionic amino acid residues and site-directed mutagenesis. Chemical modification of anionic amino acids in sigmaR1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide reduced the ligand-binding activity markedly. Since it is known that a splice variant of this receptor which lacks exon 3 does not have the ability to bind sigma ligands, the ligand-binding domain with its critical anionic amino acid residues is likely to be present in or around the region coded by exon 3. Therefore, each of the anionic amino acids in this region was mutated individually and the influence of each mutation on ligand binding was assessed. These studies have identified two anionic amino acids, D126 and E172, that are obligatory for ligand binding. Even though the ligand-binding function was abolished by these two mutations, the expression of these mutants was normal at the protein level. These results show that sigmaR1 is expressed at high levels in specific areas of the brain that are involved in memory, emotion and motor functions. The results also provide important information on the chemical nature of the ligand-binding site of sigmaR1 that may be of use in the design of sigmaR1-specific ligands with potential for modulation of sigmaR1-related brain functions.     

Morphological selection in an extreme flow environment: body shape and waterfall-climbing success in the Hawaiian stream fish Sicyopterus stimpsoni

Flow characteristics are a prominent factor determining body shapes in aquatic organisms, and correlations between body shape and ambient flow regimes have been established for many fish species. In this study, we investigated the potential for a brief period of extreme flow to exert selection on the body shape of juvenile climbing Hawaiian gobiid fishes. Because of an amphidromous life history, juvenile gobies that complete an oceanic larval phase return to freshwater habitats, where they become adults. Returning juveniles often must scale waterfalls (typically with the use of a ventral sucker) in order to reach the habitats they will use as adults, thereby exposing these animals to brief periods of extreme velocities of flow. Hydrodynamic theory predicts that bodies with larger suckers and with lower heights that reduce drag would have improved climbing success and, thus, be well suited to meet the demands of the flows in waterfalls. To test the potential for the flow environment of waterfalls to impose selection that could contribute to differences in body shape between islands, we subjected juvenile Sicyopterus stimpsoni to climbing trials up artificial waterfalls (～100 body lengths) and measured differences in body shape between successful and unsuccessful climbers. Waterfalls appear to represent a significant selective barrier to these fishes, as nearly 30% failed our climbing test. However, the effects of selection on morphology were not straightforward, as significant differences in shape between successful and unsuccessful climbers did not always match hydrodynamic predictions. In both selection experiments and in adult fish collected from habitats with different prevailing conditions of flow (the islands of Hawai'i versus Kaua'i), lower head heights were associated with exposure to high-flow regimes, as predicted by hydrodynamic theory. Thus, a premium appears to be placed on the reduction of drag via head morphology throughout the ontogeny of this species. The congruence of phenotypic selection patterns observed in our experiments, with morphological character divergence documented among adult fish from Hawai'i and Kaua'i, suggests that differences in morphology between subpopulations of adult climbing gobies may result, at least in part, from the selective pressures of high-velocity flows encountered by migrating juveniles.     

UNC-45 is required for NMY-2 contractile function in early embryonic polarity establishment and germline cellularization in C. elegans

The Caenorhabditis elegans UNC-45 protein is required for proper body wall muscle assembly and acts as a molecular co-chaperone for type II myosins. In contrast to other body wall muscle components, UNC-45 is also abundant in the germline and embryo. We show that maternally provided UNC-45 acts with non-muscle myosin II (NMY-2) during embryonic polarity establishment, cytokinesis and germline cellularization. In embryos depleted for UNC-45, myosin contractility is eliminated resulting in embryonic defects in polar body extrusion, cytokinesis and establishment of polarity. Despite a lack of contractility in an unc-45(RNAi) embryo, NMY-2::GFP localizes to the cortex and accumulates at the presumptive cytokinetic furrow indicating that UNC-45 is not required for cortical localization. UNC-45 and NMY-2 are also required for fertility since the lack of either component results in complete sterility due to failed initiation of the cellularization furrows that separate syncytial nuclei into germ cells. In the absence of UNC-45, the actomyosin cytoskeleton does not contract despite non-functional myosin still directly binding actin. UNC-45 has been previously suggested to be required for the folding of the myosin head, and our results refine this hypothesis suggesting that UNC-45 is not required to fold or maintain the actin binding domain but is still required for myosin function.     

Does fumagillin control the recently detected invasive parasite Nosema ceranae in western honey bees (Apis mellifera)?

Western honey bee (Apis mellifera) colonies in Nova Scotia, Canada were sampled in spring and late summer 2007 to evaluate efficacy of fumagillin dicyclohexylammonium (hereafter, fumagillin) against Nosema ceranae. Colonies treated with fumagillin in September 2006 (n = 94) had significantly lower Nosema intensity in spring 2007 than did colonies that received no treatment (n = 51), but by late summer 2007 no difference existed between groups. Molecular sequencing of 15 infected colonies identified N. ceranae in 93.3% of cases, suggesting that fumagillin is successful at temporarily reducing this recent invasive parasite in western honey bees.     

The phenology and clutch size of UK Blue Tits does not differ with woodland composition

The deciduous tree-herbivorous caterpillar-insectivorous bird food chain is a well-studied system for investigating the impacts of climate change across trophic levels. To date, across Europe, most attention has focused on the impacts of increasing spring temperature on changes to phenology in Oak-dominated (Quercus spp.) woodlands. Paridae species and Pied Flycatcher Ficedula hypoleuca are the most studied secondary consumers, all of which demonstrate an advancement in reproductive phenology with increases in spring temperature. Shifts in climate and phenology may also impact on reproductive investment in clutch size, and the effects of climate on phenology and clutch size may vary depending on woodland composition. To date, the effects of among-habitat variation in phenology and reproductive investment have received little attention. Insectivorous birds inhabiting woodlands that differ in tree composition may differ in the timing of breeding, due to local tree leafing phenology acting as a cue for egg-laying date and/or clutch size. Moreover, for most insectivorous birds, woodland composition within a territory is likely to be the main determinant of food availability for both adults and chicks. Consequently, if warming springs affect the temporal patterns of food availability differently across different woodland compositions, this may affect the optimal average local phenology for nesting birds. Here, using data from 34 long-term (mean 15 years) nest monitoring sites across the UK, we investigate the effect of woodland tree composition and temperature on Blue Tit Cyanistes caeruleus first egg date (FED) and clutch size. We supplemented the nest monitoring data by quantifying woodland composition, at a site level, through modified point counts. We predict that birds breeding in woodlands with greater proportions of late-leafing species, such as Oak and Ash Fraxinus excelsior, will breed later than those breeding in woodlands with greater proportions of early-leafing species, such as Birch Betula spp. and Beech Fagus sylvatica. We found no evidence for differences in Blue Tit FED or clutch size in relation to the proportion of any of the tree species investigated, after controlling for temperature and latitude (FED: −3.4 and 2.2, clutch size: −0.4 and − 0.2 eggs for one-unit increase in temperature and latitude, respectively). In recent decades and across all sites, clutch size has decreased as spring temperatures have increased, a strategy which could allow birds flexibly to adjust their breeding phenology such that nestling demand coincides with peak food availability. The lack of an effect of woodland composition on Blue Tit phenology suggests Blue Tits do not fine-tune their reproductive phenology to the local tree composition. Whether this lack of evidence for phenological divergence is due to an absence of divergent selection on breeding phenology and clutch size or to gene flow is not clear.     

Mössbauer spectroscopy on respiratory complex I: the iron-sulfur cluster ensemble in the NADH-reduced enzyme is partially oxidized

In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-cluster chain extending from the flavin) are (at least partially) reduced by NADH. The other three clusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyme: they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use M?ssbauer spectroscopy on (57)Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent clusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential clusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential clusters. The implications for intramolecular electron transfer along the extended chain of cofactors in complex I are discussed.     

Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.     

Hyaluronic acid production in Bacillus subtilis

The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.     

Impact of agricultural practices on the Zea mays L. endophytic community

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.