Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia  coli -expressed glutathione- S -transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein–GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.     

Inhibition of mutation and combating the evolution of antibiotic resistance

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.     

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.     

Differential regulation of somatodendritic and nerve terminal dopamine release by serotonergic innervation of substantia nigra

Nigrostriatal dopaminergic neurons release dopamine from dendrites in substantia nigra and axon terminals in striatum. The cellular mechanisms for somatodendritic and axonal dopamine release are similar, but somatodendritic and nerve terminal dopamine release may not always occur in parallel. The current studies used in vivo microdialysis to simultaneously measure changes in dendritic and nerve terminal dopamine efflux in substantia nigra and ipsilateral striatum respectively, following intranigral application of various drugs by reverse dialysis through the nigral probe. The serotonin releasers (+/-)-fenfluramine (100 micro m) and (+)-fenfluramine (100 micro m) significantly increased dendritic dopamine efflux without affecting extracellular dopamine in striatum. The non-selective serotonin receptor agonist 1-(m-chlorophenyl)-piperazine (100 micro m) elicited a similar pattern of dopamine release in substantia nigra and striatum. NMDA (33 micro m) produced an increase in nigral dopamine of a similar magnitude to mCPP or either fenfluramine drug. However, NMDA also induced a concurrent increase in striatal dopamine. The D2 agonist quinpirole (100 micro m) had a parallel inhibitory effect on dopamine release from dendritic and terminal sites as well. Taken together, these data suggest that serotonergic afferents to substantia nigra may evoke dendritic dopamine release through a mechanism that is uncoupled from the impulse-dependent control of nerve terminal dopamine release.     

Decline in American marten occupancy rates at Sagehen Experimental Forest,California

We compared the distribution and frequency of American marten (Martes americana) detections during historic surveys and a recent survey on the Sagehen Experimental Forest (SEF) in the Sierra Nevada Mountains, California. This area has been the location of 9 previous marten surveys during 1980–1993, each involving a systematic detection/non-detection survey on the same grid. These data are a time series of information on the occupancy of martens that can be related to habitat change in the study area. Our objectives were to 1) resurvey martens in SEF using methodology similar to previous studies to assess current marten occupancy; 2) evaluate changes in marten occupancy during the period 1980–2008; and 3) examine associations between marten occurence and changes in habitat and landscape metrics. Current marten occupancy was estimated using surveys conducted in summer 2007, winter 2007–2008, and summer 2008. From 1978 to 2007 there was a decrease in predicted habitat patch size, core area, and total amount of marten habitat in the study area, as well as an increase in distance between patches. Marten detections in 2007–2008 were approximately 60% lower than in surveys in the 1980s. We detected no martens in the summers of 2007 and 2008, and 10 detections in winter 2007–2008 were limited to higher elevations in the southwestern portion of SEF. No martens were detected in the lower elevations where most of the recent forest management activity occurred. We suggest that the marten population at SEF has been negatively affected by the loss and fragmentation of habitat. We recommend that future management of forests in the Sagehen basin focus on restoring and connecting residual marten habitat to improve habitat quality for martens. © 2011 The Wildlife Society.     

Population status,habitat dependence,and reproductive ecology of Bahama Orioles: a critically endangered synanthropic species

ABSTRACT Recent elevation of critically endangered Bahama Orioles (Icterus northropi) to species status prompted us to evaluate their population status, habitat use, and breeding ecology. From surveys, we estimated that at least 141 to 254 individuals remain globally, with 90 to 162, 24 to 44, and 27 to 48 individuals remaining on North Andros Island, Mangrove Cay, and South Andros Island, The Bahamas, respectively. Orioles were observed nesting exclusively in anthropogenic habitat (residential and agricultural land), but home ranges also included nearby pine forest and coppice (dry broadleaf forest). Most nests (40 of 46, or 87%) were in nonnative coconut palm (Cocos nucifera), with native Sabal palmetto and Thrinax morrisii, and an introduced Brassaia actinophylla also used. Trees selected by orioles for nesting were significantly taller, less likely to have shrubs underneath, further from cover, and had more palm trees nearby than randomly selected palm trees. Three of eight nests with known contents were parasitized by Shiny Cowbirds (Molothrus bonariensis). Lethal yellowing disease recently devastated coconut palms and reduced the number of orioles on North Andros, but palms on Mangrove Cay and South Andros remain healthy. The juxtaposition of anthropogenic habitat to suitable native habitats may be more important than any single factor for Bahama Orioles, especially for breeding adults and fledged young. Conservation of coppice habitat, at high risk for agricultural and residential development, is crucial for survival of this critically endangered synanthropic species.     

Regulation of matrix metalloproteinase (MMP) activity by the low-density lipoprotein receptor-related protein (LRP). A new function for an "old friend"

Matrix metalloproteinases (MMPs) are essential contributors to a microenvironment that promotes tumour progression. During the two last decades, inhibition of MMPs has become the focus of considerable interest for cancer therapy, and numerous synthetic metalloproteinase inhibitors have been developed by the pharmaceutical industry. However, clinical trials have shown disappointing efficacy or unexpected toxicity and new targets are thus eagerly awaited. The identification of endocytic clearance of several MMPs by the low-density lipoprotein receptor-related protein (LRP) might provide insight into novel strategies for controlling MMP level during malignant processes. This review attempts to summarize recent aspects on the cellular and molecular basis of LRP-mediated endocytic disposal of MMPs.     

Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus

Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37 degrees C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a DeltacgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the DeltacgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although DeltacgrA conidia germinated normally at 22 degrees C, they swelled excessively when incubated at 37 degrees C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.