Asymmetric trehalose analogues to probe disaccharide processing pathways in mycobacteria

The uptake and metabolism of the disaccharide trehalose by Mycobacterium tuberculosis is essential for the virulence of this pathogen. Here we describe the chemoenzymatic synthesis of new azido-functionalised asymmetric trehalose probes that resist degradation by mycobacterial enzymes and are used to probe trehalose processing pathways in mycobacteria

MF2294

The information in this document has been funded wholly or in part by the United States Environmental Protection Agency.Jennifer R. Weins, Christopher S. Guy & Charles D. Lee, Streambank revetment, Kansas State University, September 1997

Phosphorylation in intrinsically disordered regions regulates the activity of Neurogenin2.

BACKGROUND: Neuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear. RESULTS: Here we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks). CONCLUSIONS: Phosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation.This work was supported by MRC Research Grant G0700758 (AP), a Cancer Research UK Studentship (CH) and an MRC DTA Studentship (GM). Support was also received (IL) from the TGE RMN THC (FR-3050, France). We acknowledge support for international collaboration by a BQR fellowship from Lille North of France University. The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and the University of Sciences and Technologies of Lille 1.This is the final version. It was first published by BioMed Central at http://www.biomedcentral.com/1471-2091/15/2

Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection

Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRP ML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell

An Evaluation of Lake Trout Suppression in Yellowstone Lake, Yellowstone National Park

Introduced lake trout (Salvelinus namaycush) threaten to extirpate native Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) from Yellowstone Lake, Yellowstone National Park. A National Park Service gill netting program has removed nearly 400,000 lake trout from Yellowstone Lake since 1995. Lake trout population size has not been estimated; therefore, it is difficult to determine the proportion that has been removed. Our objectives were to (1) examine catch as a function of effort to determine if the suppression program has caused lake trout abundance to decline, (2) determine if certain population metrics have changed over time as a function of harvest, and (3) develop age-structured models to determine the level of mortality required to cause population growth rate to decline below 1.0 (replacement). Catch has continued to increase as a function of effort, indicating lake trout abundance is increasing. Population metrics were not clearly indicative of a response to harvest, but were comparable to North American lake trout populations where harvest has occurred. Results from an age-structured matrix model determined the rate of population growth was 1.1 given the current rate of fishing mortality and that population growth rate would be 1.3 in the absence of fishing mortality. The current rate of population growth is positive; however, it is slower than it would be in the absence of lake trout suppression. Fishing mortality needs to increase by at least 10 percent to reduce population growth rate below 1.0 in the future

Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA)

The (Mtb) pathogen encodes an -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from (MSNagA) and (MMNagA), close relatives of Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria

Movements of Spawning and Non-Spawning Shovelnose Sturgeon in the Missouri River Above Fort Peck Reservoir

During the last 40 yrs there has been a lack of pallid sturgeon (Scaphirhynchus albus) recruitment in the upper Missouri River (UMR). However, shovelnose sturgeon (Scaphirhynchus platorynchus) continue to exhibit recruitment in the UMR. Understanding the recruitment dichotomy between species is receiving much attention throughout their range. The objectives of this study were to identify the effects of varying discharge on spawning locations and spawning movements for pallid and shovelnose sturgeon. Two female pallid sturgeon, 32 gravid female shovelnose sturgeon, and 32 non-reproductively active female shovelnose sturgeon were radio tagged at three locations and tracked from 1 May to 5 July 2009. Unfortunately, no data are available for spawning pallid sturgeon movements because fish were not reproductively active. Upstream movement by gravid shovelnose sturgeon varied from 20 percent of the fish tagged at Judith Landing to 56 percent of the fish tagged at Coal Banks Recreation Area (CBRA). Mean maximum upstream movement of gravid shovelnose sturgeon varied from 35.7 km at CBRA to 87.9 km at Fred Robinson Bridge (FRB), mean maximum downstream movement varied from 24.9 km at FRB to 80.3 km at CBRA. Reproductively inactive shovelnose sturgeon exhibited lower mean maximum movements than reproductively active fish (mean maximum distance 5.7 km). Shovelnose sturgeon in the UMR exhibit both upstream and downstream movements prior to spawning and are using several spawning areas. Thus, maintaining spawning habitat throughout a regulated river is important with regard to shovelnose sturgeon conservation

Reproductive Health of Bass in the Potomac, USA, Drainage: Part 1. Exploring the Effects of Proximity to Wastewater Treatment Plant Discharge

Intersex (specifically, testicular oocytes) has been observed in male smallmouth bass (SMB; Micropterus dolomieu) and other centrarchids in the South Branch of the Potomac River, USA, and forks of the Shenandoah River, USA, during the past five years. This condition often is associated with exposure to estrogenic endocrine-disrupting chemicals in some fish species, but such chemicals and their sources have yet to be identified in the Potomac. In an attempt to better understand the plausible causes of this condition, we investigated the reproductive health of bass sampled up- and downstream of wastewater treatment plant (WWTP) effluent point sources on the Potomac River in Maryland, USA. Smallmouth bass were sampled from the Conococheague Creek and the Monocacy River, and largemouth bass (LMB; Micropterus salmoides) were collected near the Blue Plains WWTP on the mainstem of the Potomac River. Chemical analyses of compounds captured in passive samplers at these locations also were conducted. A high prevalence of intersex (82–100%) was identified in male SMB at all sites regardless of collection area. A lower prevalence of intersex (23%) was identified in male LMB collected at the Blue Plains site. When up- and downstream fish were compared, significant differences were noted only in fish from the Conococheague. Differences included condition factor, gonadosomatic index, plasma vitellogenin concentration, and estrogen to testosterone ratio. In general, chemicals associated with wastewater effluent, storm-water runoff, and agriculture were more prevalent at the downstream sampling sites. An exception was atrazine and its associated metabolites, which were present in greater concentrations at the upstream sites. It appears that proximity to effluent from WWTPs may influence the reproductive health of bass in the Potomac watershed, but inputs from other sources likely contribute to the widespread, high incidence of testicular oocytes

Spatial Drift Dynamics of Shovelnose Sturgeon and Pallid Sturgeon Prelarvae in the Transition Zone of Ft. Peck Reservoir

Habitats in reservoir headwaters may cause high mortality of sturgeon prelarvae. Short inter-reservoir reaches export drifting prelarvae from hatch locations into reservoirs. However, flooded vegetation could entrain prelarvae. We used 2 day post hatch (dph) shovelnose sturgeon (Scaphirhynchus platorynchus) and 1-dph pallid sturgeon (Scaphirhynchus albus) to determine the spatial dynamics of drifting prelarvae.We released 220,000 2-dph shovelnose sturgeon 4 km upstream of Ft. Peck Reservoir and 135,000 1-dph pallid sturgeon 2.5 km upstream of the reservoir the following day. We recaptured shovelnose sturgeon prelarvae with nets deployed along three transects of the transition zone and within the headwaters of the reservoir.We sampled 5148.2 m3 of water and recaptured 323 prelarval shovelnose sturgeon for a recapture rate of 0.14 percent. Fifty-nine percent of recaptured prelarvae were recaptured from the thalweg, 12 percent from the flooded vegetation-main channel interface, 9 percent from the channel border, and 19 percent from the zero-velocity area of Ft. Peck Reservoir. We recaptured pallid sturgeon prelarvae with nets deployed along one transect of the transition zone and within the headwaters of the reservoir. We sampled 6608.5 m3 of water and recaptured 397 pallid sturgeon prelarvae for a recapture rate of 0.29 percent. Twenty one percent of prelarvae were recaptured within the thalweg, 0.25 percent were recaptured along the channel margins, and 79 percent from the zero-velocity area of Ft. Peck Reservoir. Although recapture rates were low, the majority of prelarvae were captured in the thalweg and transported to the headwaters of Ft. Peck Reservoir. The drift dynamics observed in this study provide a springboard for further research