Fluorescent glycosidase inhibiting 1,5-dideoxy-1,5-iminoalditols

1,5-Dideoxy-1,5-iminoalditols of various configurations as well as isofagomine were N-alkylated with non-polar straight chain spacer-arms by a set of simple standard procedures. The spacer-arms' terminal functional groups, primary amines, were employed to introduce fluorescent tags such as dansyl and dapoxyl moieties. Resulting derivatives in the D-xylo, D-gluco, D-galacto as well as GlcNAc series showed distinctly improved glycosidase inhibitory activities compared to parent compounds and are designed to be useful analytical tools.     

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom,HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.

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Molecular and Cellular Mechanisms of Lamina-specific Axon Targeting

The specificity of synaptic connections is directly related to the functional integrity of neural circuits. Long-range axon guidance and topographic mapping mechanisms bring axons into spatial proximity of target cells and thus limit the number of potential synaptic partners. Synaptic specificity is then achieved by extracellular short-range guidance cues and cell-surface recognition cues. Neural activity may enhance the precision and strength of specific circuit connections. Here, we focus on one of the final steps of synaptic matchmaking: the targeting of synaptic layers and the mutual recognition of axons and dendrites within these layers.Perception and behavior are critically dependent on synaptic communication between specific neurons. Understanding how neurons achieve such “synaptic specificity” is therefore one of the most fundamental issues in developmental neuroscience. Langley’s notion of “chemical relations” between synaptically connected neurons (Langley 1892) and Sperry’s “chemoaffinity” hypothesis (Sperry 1963) provided a conceptual framework for the development of precise synaptic connections in the central nervous system. Sperry postulated that molecular interactions between neurons and their extracellular environment (including between and amongst axons and dendrites) ensure that connections form only between “appropriate” synaptic partners (Sperry 1963). This hypothesis has been confirmed by experimental work over the last four decades, most importantly by the identification of molecular cues that provide synaptic specificity (see Sanes and Yamagata 2009 for a recent comprehensive review). However, within this broad framework, a number of alternate mechanisms have been shown or proposed to play roles in specific aspects of such targeting processes. Here, we focus on mechanisms that underlie the formation of synaptic layers, a prominent anatomical feature of the visual system as well as many other areas of the CNS.As reviewed previously (O''Leary 2010), the chemoaffinity principle underlies the developmental process of topographic mapping. Indeed, the precision with which neurons preserve the spatial relationships between the visual world and its representation in the brain is remarkable: Across animals ranging from flies to vertebrates, axons that bear signals from adjacent points in visual space invariably choose adjacent targets in the brain (Braitenberg 1967; Lemke and Reber 2005; Sperry 1963). Thus, position-dependent guidance of axons ensures that a visuotopic map develops. However, position in space is just one attribute of a visual stimulus; others include color, brightness, edge detection, and movement. If position in visual space is encoded by localized activation within a two-dimensional field of neurons, then these other features are encoded by local circuits that act both in series and in parallel and are reiterated many times across the field (Fig. 1). These local circuit modules are often envisioned as “columns” that lie orthogonal to the topographic map, with each column corresponding to a pixel in visual space and each level of the column representing a different, specific visual feature within that pixel, such as brightness, color, etc. (Fig. 1). How these columns acquire their laminated structure represents a developmental challenge of extraordinary scale. Although long-range axon guidance and topographic mapping no doubt contribute to restricting the astronomical number of potential synaptic partners, these mechanisms are clearly not sufficient; additional mechanisms must (and do) exist that act on a local scale to provide an additional level of positional information and cell-type-specific “chemoaffinity.”Open in a separate windowFigure 1.Laminae are a fundamental organizing unit of neural circuits. Each column corresponds to a single topographic position (e.g., location on the retina). Within each column, different cell types (shown type A: blue, and type B: red) respond to different features in the visual world, such as motion or luminance. These pixels are repeated many times over and thus cover all of visual space. A simple rule of “Cell type A connects to Layer A, etc.” ensures that functional segregation is maintained in the connections from the retina to the target (parallel processing). Each pixel P1, P2, and P3 connects to a single column (C1, C2, and C3), establishing serial processing. Within each column, there are local circuits that, too, are layer-specific. Thus, laminae ensure functional specificity of both afferent-target connections and local circuit connections.A prominent principle, which guides the formation of connections between specific cell types and is a characteristic feature of CNS architecture, is the concentration of synapses in small areas. These synapse clusters can take the form of planar layers or spherical glomeruli. Although glomeruli are a specialization that appears most prominent in the olfactory system, layers, or laminae, are an almost ubiquitous feature of central nervous system architecture. Indeed, even crude histological stains reveal that axons and dendrites often accumulate in neuropil (cell-body-free areas). Cell-type-specific or single-cell labeling has shown that, within individual neuropil layers, neurites and synapses are not distributed randomly. Rather, synaptic connections arising between neurons with the same or similar functional properties are localized to particular sublaminae that distinguish synapses with different properties (Fig. 1). The structural underpinnings of this functional principle are provided by mechanisms that ensure the lamina-specific branching of the corresponding neurites. How this enormous precision is achieved is the subject of intense investigations in the Drosophila, zebrafish, chick, and mouse visual systems. We will begin by describing three anatomical regions in these model organisms. Then, we will discuss three broad principles of layer-specific targeting in the visual system, namely cell–cell recognition, guidance by matrix cues, and activity-dependent sorting of axon terminals.     

Male hosts drive infracommunity structure of ectoparasites

We studied the co-occurrence of flea species in infracommunities of 16 rodents from four regions (South Africa, Tanzania, central Europe and western Siberia) using null models, and predicted that flea co-occurrences will be expressed more strongly in male than in female hosts. We examined patterns of co-occurrence (measured as the C score) in infracommunities of fleas that are parasitic on male and female hosts by comparing co-occurrence frequencies with those expected by chance. When a significant degree of nonrandomness in flea co-occurrences was detected, it indicated aggregative infracommunity structure. In Tanzanian rodents, no significant flea co-occurrences were detected in either male or female hosts. In a South African rodent, significant flea co-occurrences were not detected in males, but were found in females in some localities. In Palaearctic rodents, significant nonrandomness was detected either equally for males and females or more frequently in males than in females. Meta-analyses demonstrated that the frequency of the detection of nonrandomness in flea co-occurrences was significantly higher in male than in female hosts. The values of the standardized effect size (SES) for the C score differed significantly among host species, but not between host genders. When the Palaearctic hosts were analyzed separately, the effects of both host gender and species appeared to be significant, with the SES values for the C score in males being smaller than those in females. The strength of the gender difference in the manifestation of flea community structure increased with increasing gender difference in flea species richness, and with decreasing gender difference in flea prevalence for the Palaearctic hosts. We conclude that male hosts are the main drivers of flea infracommunity structure. However, the manifestation of gender bias in flea community structure varies among host species, and is likely determined by the pattern of species-specific spatial behavior.     

Geographical trends in the yolk carotenoid composition of the pied flycatcher (Ficedula hypoleuca)

Carotenoids in the egg yolks of birds are considered to be important antioxidants and immune stimulants during the rapid growth of embryos. Yolk carotenoid composition is strongly affected by the carotenoid composition of the female??s diet at the time of egg formation. Spatial and temporal differences in carotenoid availability may thus be reflected in yolk concentrations. To assess whether yolk carotenoid concentrations or carotenoid profiles show any large-scale geographical trends or differences among habitats, we collected yolk samples from 16 European populations of the pied flycatcher, Ficedula hypoleuca. We found that the concentrations and proportions of lutein and some other xanthophylls in the egg yolks decreased from Central Europe northwards. The most southern population (which is also the one found at the highest altitude) also showed relatively low carotenoid levels. Concentrations of ??-carotene and zeaxanthin did not show any obvious geographical gradients. Egg yolks also contained proportionally more lutein and other xanthophylls in deciduous than in mixed or coniferous habitats. We suggest that latitudinal gradients in lutein and xanthophylls reflect the lower availability of lutein-rich food items in the northern F. hypoleuca populations and in montane southern populations, which start egg-laying earlier relative to tree phenology than the Central European populations. Similarly, among-habitat variation is likely to reflect the better availability of lutein-rich food in deciduous forests. Our study is the first to indicate that the concentration and profile of yolk carotenoids may show large-scale spatial variation among populations in different parts of the species?? geographical range. Further studies are needed to test the fitness effects of this geographical variation.     

Insertion Sequence Element Single Nucleotide Polymorphism Typing Provides Insights into the Population Structure and Evolution of Mycobacterium ulcerans across Africa

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the “pan-African clade” were found to be widespread throughout Africa, while the ISE-SNP types of the “Gabonese/Cameroonian clade” were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.     

The Root Extract of the Medicinal Plant Pelargonium sidoides Is a Potent HIV-1 Attachment Inhibitor

Global HIV-1 treatment would benefit greatly from safe herbal medicines with scientifically validated novel anti-HIV-1 activities. The root extract from the medicinal plant Pelargonium sidoides (PS) is licensed in Germany as the herbal medicine EPs®7630, with numerous clinical trials supporting its safety in humans. Here we provide evidence from multiple cell culture experiments that PS extract displays potent anti-HIV-1 activity. We show that PS extract protects peripheral blood mononuclear cells and macrophages from infection with various X4 and R5 tropic HIV-1 strains, including clinical isolates. Functional studies revealed that the extract from PS has a novel mode-of-action. It interferes directly with viral infectivity and blocks the attachment of HIV-1 particles to target cells, protecting them from virus entry. Analysis of the chemical footprint of anti-HIV activity indicates that HIV-1 inhibition is mediated by multiple polyphenolic compounds with low cytotoxicity and can be separated from other extract components with higher cytotoxicity. Based on our data and its excellent safety profile, we propose that PS extract represents a lead candidate for the development of a scientifically validated herbal medicine for anti-HIV-1 therapy with a mode-of-action different from and complementary to current single-molecule drugs.     

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.

Methodology/Principal Findings

We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).

Conclusion/Significance

This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.