Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates.

The ability of ruminal microbes to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ovine whole rumen fluid (WRF) and as 24 bacterial isolates was examined under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography analysis, followed by liquid chromatography-tandem mass spectrometry identification of metabolites. Organisms in WRF microcosms degraded 180 µM RDX within 4 h. Nitroso-intermediates hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were present as early as 0.25 h and were detected throughout the 24-h incubation period, representing one reductive pathway of ring cleavage. Following reduction to MNX, peaks consistent with m/z 193 and 174 were also produced, which were unstable and resulted in rapid ring cleavage to a common metabolite consistent with an m/z of 149. These represent two additional reductive pathways for RDX degradation in ovine WRF, which have not been previously reported. The 24 ruminal isolates degraded RDX with varying efficiencies (0-96 %) over 120 h. Of the most efficient degraders identified, Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans degraded RDX when medium was supplemented with both nitrogen and carbon, while Anaerovibrio lipolyticus, Prevotella ruminicola, and Streptococcus bovis IFO utilized RDX as a sole source of nitrogen. This study showed that organisms in whole rumen fluid, as well as several ruminal isolates, have the ability to degrade RDX in vitro and, for the first time, delineated the metabolic pathway for its biodegradation.

Ovine ruminal microbes are capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

Bioremediation is of great interest in the detoxification of soil contaminated with residues from explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although there are numerous forms of in situ and ex situ bioremediation, ruminants would provide the option of an in situ bioreactor that could be transported to the site of contamination. Bovine rumen fluid has been previously shown to transform 2,4,6-trinitrotoluene (TNT), a similar compound, in 4 h. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within 4 h. Whole ovine rumen fluid was then inoculated into five different types of media to select for archaeal and bacterial organisms capable of RDX biotransformation. Cultures containing 30 µg mL(-1) RDX were transferred each time the RDX concentration decreased to 5 µg mL(-1) or less. Time point samples were analyzed for RDX biotransformation by HPLC. The two fastest transforming enrichments were in methanogenic and low nitrogen basal media. After 21 days, DNA was extracted from all enrichments able to partially or completely transform RDX in 7 days or less. To understand microbial diversity, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted. Cloning and sequencing of partial 16S rRNA fragments were performed on both low nitrogen basal and methanogenic media enrichments. Phylogenetic analysis revealed similar homologies to eight different bacterial and one archaeal genera classified under the phyla Firmicutes, Actinobacteria, and Euryarchaeota. After continuing enrichment for RDX degraders for 1 year, two consortia remained: one that transformed RDX in 4 days and one which had slowed after 2 months of transfers without RDX. DGGE comparison of the slower transforming consortium to the faster one showed identical banding patterns except one band. Homology matches to clones from the two consortia identified the same uncultured Clostridia genus in both; Sporanaerobacter acetigenes was identified only in the consortia able to completely transform RDX. This is the first study to examine the rumen as a potential bioremediation tool for soils contaminated with RDX, as well as to discover S. acetigenes in the rumen and its potential ability to metabolize this energetic compound.

GKAP, a novel synaptic protein that interacts with the guanylate kinase-like domain of the PSD-95/SAP90 family of channel clustering molecules.

The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH2-terminal PDZ domains, with certain ion channels (NMDA receptors and K+ channels), thereby promoting the clustering of these proteins. Although the function of the NH2-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K+ channels/NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein-protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK).

Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons.

We describe here a novel effect of activity on the subcellular distribution of NMDA receptors in hippocampal neurons in culture. In spontaneously active neurons, NMDA receptors were clustered at a few synaptic and nonsynaptic sites. Chronic blockade of NMDA receptor activity induced a 380% increase in the number of NMDA receptor clusters and a shift to a more synaptic distribution. This effect was reversible. The distributions of the presynaptic marker synaptophysin, the AMPA-type glutamate receptor subunit GluR1, and the putative NMDA receptor clustering protein PSD-95 were not affected by blockade. Regulation of the synaptic localization of NMDA receptors by activity may define a novel mechanism by which input controls a neuron's ability to modify its synapses.

Axon/dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains.

The subcellular targeting of neurotransmitter receptors is vital in controlling polarized information flow in the brain. We show here that metabotropic glutamate receptors are differentially targeted when expressed from defective viral vectors in cultured hippocampal neurons; mGluR1a and mGluR2 are targeted to dendrites and excluded from axons, whereas mGluR7 is targeted to axons and dendrites. Chimeras and deletions revealed that axon exclusion of mGluR2 versus axon targeting of mGluR7 is mediated by their 60 amino acid C-terminal cytoplasmic domains. Addition of the mGluR7 C-terminal sequence to mGluR2 or to the unrelated somatodendritic protein telencephalin (tln) induced axon targeting, indicating dominance of the axonal signal. These mGluR sorting signals represent novel plasma membrane axon/dendrite targeting signals.

The distribution of glutamate receptors in cultured rat hippocampal neurons: postsynaptic clustering of AMPA-selective subunits.

The distribution of several glutamate receptor subunits was investigated in cultured rat hippocampal neurons by in situ hybridization and immunocytochemistry. The AMPA/kainate-selective receptors GluR1-6 exhibited two patterns of mRNA expression: most neurons expressed GluR1, R2, and R6, whereas only about 20% expressed significant levels of GluR3, R4, and R5. By immunocytochemistry, the metabotropic glutamate receptor mGluR1 alpha was detectable only in a subpopulation of GABAergic interneurons. GluR1 and GluR2/3 segregated to the somatodendritic domain within the first week in culture, even in the absence of synaptogenesis. Glutamate receptor-enriched spines developed later and were present only on presumptive pyramidal cells, not on GABAergic interneurons. Clusters of GluR1 and GluR2/3 completely colocalized and were restricted to a subset of postsynaptic sites. Thus, glutamate receptor subunits exhibit both a cell type-specific expression and a selective subcellular localization.

Postsynaptically silent synapses in single neuron cultures.

We have used the synapses that isolated hippocampal cells in culture form onto themselves (autapses) to determine if some synapses lack functional AMPA receptors (AMPARs). A comparison of the synaptic variability of the AMPAR- and NMDAR-mediated evoked responses, as well as of miniature synaptic responses, indicates that a population of events exists that only contains an NMDAR component. Spillover of glutamate from adjacent synapses cannot explain these results because in single cell cultures all synaptic events mediated by AMPARs should be detected. Immunocytochemical analysis of these cultures clearly reveals a population of synapses with puncta for NR1 (NMDAR) but not for GluR1 (AMPAR). These results provide strong anatomical and physiological evidence for the existence of postsynaptically silent synapses.

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site.

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.

CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD-95/SAP90.

The synaptic protein PSD-95/SAP90 binds to and clusters a variety of membrane proteins via its two N-terminal PDZ domains. We report a novel protein, CRIPT, which is highly conserved from mammals to plants and binds selectively to the third PDZ domain (PDZ3) of PSD-95 via its C terminus. While conforming to the consensus PDZ-binding C-terminal sequence (X-S/T-X-V-COOH), residues at the -1 position and upstream of the last four amino acids of CRIPT determine its specificity for PDZ3. In heterologous cells, CRIPT causes a redistribution of PSD-95 to microtubules. In brain, CRIPT colocalizes with PSD-95 in the postsynaptic density and can be coimmunoprecipitated with PSD-95 and tubulin. These findings suggest that CRIPT may regulate PSD-95 interaction with a tubulin-based cytoskeleton in excitatory synapses.

Getting a bead on receptor movements.

Studies of populations of receptor proteins suggest that their number and location are highly regulated. Single-particle tracking of glycine receptors now reveals the direct movement of receptors between different clusters of the anchoring protein gephyrin.

Molecular heterogeneity of central synapses: afferent and target regulation.

Electrophysiological recordings show a functional spectrum even within a single class of synapse, with individual synapses ranging widely in fundamental properties, including release probability, unitary response and effects of previous stimulation on subsequent response. Molecular and cellular biological approaches have shown a corresponding diversity in the complement of ion channels, receptors, scaffolds and signal transducing proteins that make up individual synapses. Indeed, we believe that each individual synapse is unique, a function of presynaptic cell type, postsynaptic cell type, environment, developmental stage and history of activity. We review here the molecular diversity of glutamatergic and GABAergic synapses in the mammalian brain in the context of potential cell biological mechanisms that may explain how individual cells develop and maintain such a mosaic of synaptic connections.

Neuronal polarity.

The axonal and somatodendritic domains of neurons differ in their cytoskeletal and membrane composition, complement of organelles, and capacity for macromolecular synthesis. Recently there has been progress in elucidating the cellular mechanisms that underlie the establishment and maintenance of neuronal polarity, including microtubule organization and the sorting, transport, and anchoring of membrane proteins.

Detection of lysergic acid in ruminal fluid, urine, and in endophyte-infected tall fescue using high-performance liquid chromatography.

Ergot alkaloids present in endophyte-infected tall fescue induce fescue toxicosis in livestock consuming the plant. The lysergic acid (LA) ring structure is a common moiety among the ergot alkaloids. Little is known about the bioavailability of LA because of limitations in available analytical protocols. Thus, a high-performance liquid chromatography procedure was developed to analyze biological matrices for LA. The biological matrices of interest were tall fescue straw and seed, and ruminant feces, urine, and ruminal fluid. Lysergic acid was added to each matrix at a high (150 ng/ml) or low (30 ng/ml) level. Using the high-level addition, the greatest recovery of LA was obtained from ruminal fluid, feces, and urine (P < 0.05), with an average 85.1% recovered. At the low level, a greater recovery of added LA was observed in the ruminal fluid, urine, and feces (82.1%; P < 0.05) than that in the other 2 matrices (62.6%). The limit of quantitation (LOQ) in ruminal fluid and urine was 5.5 and 18.4 ng/ml, respectively. Seed, straw, and feces had higher LOQ (24.2, 14.5, and 36.0 ng/g, respectively). Limit of detection (LOD) was 1.64, 10.80, 4.35, 5.52, and 7.26 ng/g for ruminal fluid, feces, urine, seed, and straw, respectively. To test the assay in vivo, samples of ruminal fluid and urine were collected from steers consuming a diet containing 400 ng of ergovaline/g and 30 ng of LA/g. All matrices sampled resulted in levels above the LOD and LOQ for the assay, indicating that this assay is sufficiently sensitive for use in assessing the bioavailability of LA.

Reduced malignancy of ras-transformed NIH 3T3 cells expressing antisense osteopontin RNA.

Osteopontin (OPN) is a secreted, calcium-binding phosphoprotein that frequently has been associated with the transformed phenotype. To clarify the function of OPN in tumor cells, we designed experiments to: (a) express antisense OPN RNA in murine PAP2 cells (metastatic, ras-transformed NIH 3T3 cells) and (b) examine the effects of antisense OPN expression on the tumorigenic and metastatic properties of the cells. PAP2 cells were transfected with pNMH-asOPN, an inducible, mammalian expression vector that can generate antisense OPN RNA complementary to the OPN mRNA. Two clones have been identified that expressed antisense OPN RNA in vitro. While reduced OPN protein secretion was not detected when the cells were grown in vitro, the in vivo expression of antisense OPN RNA was associated with reduced tumorigenicity. Tumors that did arise, with greatly extended lag time, had lost expression of antisense OPN RNA in vivo, suggesting that antisense OPN RNA expression was associated with reduced tumorigenicity of these cells.

Mismatched appositions of presynaptic and postsynaptic components in isolated hippocampal neurons.

To determine whether presynaptic input is necessary for postsynaptic differentiation, we isolated hippocampal neurons in microisland culture and thus deprived pyramidal cells of GABA input and GABAergic neurons of glutamate input. We find that glutamate input is necessary for clustering the AMPA-type glutamate receptor but not for clustering the NMDA receptor or the associated PSD-95 family scaffold in GABAergic cells; GABA input is not necessary for clustering the GABA(A) receptor or gephyrin in pyramidal cells. Isolated neurons showed a surprising mismatch of presynaptic and postsynaptic components. For example, in isolated pyramidal neurons, although GABA(A) receptor clusters covered <4% of the dendritic surface and presynaptic boutons covered <12%, a full two-thirds of the GABA(A) receptor clusters were localized inappropriately opposite the non-GABAergic, presumed glutamatergic, terminals. Furthermore, inhibitory and excitatory postsynaptic components were segregated into separate clusters in isolated cells and apposed to separate boutons of a single axon. Thus, GABA(A) receptors were clustered opposite some terminals, whereas NMDA receptors were clustered opposite other terminals of a single axon. These results suggest the involvement of a synaptogenic signal common to glutamate and GABA synapses that permits experimentally induced mismatching of presynaptic and postsynaptic components in isolated neurons, as well as a second specificity-conferring signal that mediates appropriate matching in mixed cultures.

cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors.

Chronic activity blockade increases synaptic levels of NMDA receptor immunoreactivity in hippocampal neurons. We show here that blockade-induced synaptic NMDA receptors are functional and mediate enhanced excitotoxicity in response to synaptically released glutamate. Activity blockade increased the cell surface association of NMDA receptors. Blockade-induced synaptic targeting of NMDA receptors did not require protein synthesis but required phosphorylation and specifically cAMP-dependent protein kinase (PKA). Furthermore, activation of PKA was sufficient to induce synaptic targeting of NMDA receptors regardless of receptor activity status. These results implicate PKA activity downstream of receptor blockade as a mediator of enhanced synaptic transport or stabilization of NMDA receptors. Synaptic clustering of NR1-green fluorescent protein was observed in living neurons in response to NMDA receptor and cAMP phosphodiesterase antagonists and occurred gradually over the course of a day. This pathway represents a cellular mechanism for synaptic homeostasis and is likely to function in metaplasticity, long-term regulation of the ability of a synapse to undergo potentiation or depression.

Market structure and conduct in the pharmaceutical industry.

The pharmaceutical industry offers the world's population alleviation and cure from a variety of medical conditions, while also contributing to the economic performance of many countries. The potential importance of these characteristics have made the industry an area of intense debate, criticism and praise. This review aims to clarify some of the arguments surrounding the industry on both sides of the fence, and by doing so, to provide a balanced introduction on which opinions may be made.

The Canadian National EMS Research Agenda: Impact and Feasibility of Implementation of Previously Generated Recommendations.

BACKGROUND: A recent mixed-methods study on the state of emergency medical services (EMS) research in Canada led to the generation of nineteen actionable recommendations. As part of the dissemination plan, a survey was distributed to EMS stakeholders to determine the anticipated impact and feasibility of implementing these recommendations in Canadian systems. METHODS: An online survey explored both the implementation impact and feasibility for each recommendation using a five-point scale. The sample consisted of participants from the Canadian National EMS Research Agenda study (published in 2013) and additional EMS research stakeholders identified through snowball sampling. Responses were analysed descriptively using median and plotted on a matrix. Participants reported any planned or ongoing initiatives related to the recommendations, and required or anticipated resources. Free text responses were analysed with simple content analysis, collated by recommendation. RESULTS: The survey was sent to 131 people, 94 (71.8%) of whom responded: 30 EMS managers/regulators (31.9%), 22 researchers (23.4%), 15 physicians (16.0%), 13 educators (13.8%), and 5 EMS providers (5.3%). Two recommendations (11%) had a median impact score of 4 (of 5) and feasibility score of 4 (of 5). Eight recommendations (42%) had an impact score of 5, with a feasibility score of 3. Nine recommendations (47%) had an impact score of 4 and a feasibility score of 3. CONCLUSIONS: For most recommendations, participants scored the anticipated impact higher than the feasibility to implement. Ongoing or planned initiatives exist pertaining to all recommendations except one. All of the recommendations will require additional resources to implement.

The murine gene encoding secreted phosphoprotein 1 (osteopontin): promoter structure, activity, and induction in vivo by estrogen and progesterone.

Secreted phosphoprotein 1 (Spp-1) is a 41.5-kDa bone sialoprotein presumed to be important in the development and functioning of a number of mammalian organs and possibly also in the progression of malignancies. We report here the isolation of a phage lambda genomic clone of the murine Spp-1 gene containing the promoter and first six exons (4.6 kb of the 5.7-kb gene). We have found another exon located 5' to the 'exon 1' reported by Miyazaki et al. [J. Biol. Chem. 265 (1990) 14432-14438]. The DNA upstream from this 5' exon functions as a promoter in epidermal fibroblast and osteoblast-like cells, as demonstrated by transient transfection assays, S1 mapping of the transcription start point, and sequence analysis revealing TATA-like (TTTAAA) and CAAT (its inverse complement) boxes. A small region of the promoter (nt -253 to +79) was able to direct high-level expression of a fused cat reporter gene in JB6 mouse epidermal cells. The transient transfection assays indicated the presence of a positive transcription element between nt -543 and -253 and a negative transcription element between nt -777 and -543. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in a 1.5-3-fold induction of transcription, depending on the promoter construct and the TPA concentration. The Spp-1 mRNA was localized in several tissues, consistent with previous reports, and to novel sites in ovary, and in the skin and ventral fatty tissue of pregnant and lactating mice. The induction of Spp-1 mRNA was partially mimicked by painting beta-estradiol or progesterone on the skin of nonpregnant females.