Photo-antagonism of the GABAA receptor

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation

Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

Copyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program

Antihypertensive Drug Guanabenz Is Active In Vivo against both Yeast and Mammalian Prions

Background: Prion-based diseases are incurable transmissible neurodegenerative disorders affecting animals and humans. [br/] Methodology/Principal Findings: Here we report the discovery of the in vivo antiprion activity of Guanabenz (GA), an agonist of a2-adrenergic receptors routinely used in human medicine as an antihypertensive drug. We isolated GA in a screen for drugs active in vivo against two different yeast prions using a previously described yeast-based two steps assay. GA was then shown to promote ovine PrPSc clearance in a cell-based assay. These effects are very specific as evidenced by the lack of activity of some GA analogues that we generated. GA antiprion activity does not involve its agonist activity on a2-adrenergic receptors as other chemically close anti-hypertensive agents possessing related mechanism of action were found inactive against prions. Finally, GA showed activity in a transgenic mouse-based in vivo assay for ovine prion propagation, prolonging slightly but significantly the survival of treated animals. [br/] Conclusion/Significance: GA thus adds to the short list of compounds active in vivo in animal models for the treatment of prion-based diseases. Because it has been administrated for many years to treat hypertension on a daily basis, without major side-effects, our results suggest that it could be evaluated in human as a potential treatment for prion-based diseases

Distinct genes related to drug response identified in ER positive and ER negative breast cancer cell lines

Breast cancer patients have different responses to chemotherapeutic treatments. Genes associated with drug response can provide insight to understand the mechanisms of drug resistance, identify promising therapeutic opportunities, and facilitate personalized treatment. Estrogen receptor (ER) positive and ER negative breast cancer have distinct clinical behavior and molecular properties. However, to date, few studies have rigorously assessed drug response genes in them. In this study, our goal was to systematically identify genes associated with multidrug response in ER positive and ER negative breast cancer cell lines. We tested 27 human breast cell lines for response to seven chemotherapeutic agents (cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, and paclitaxel). We integrated publicly available gene expression profiles of these cell lines with their in vitro drug response patterns, then applied meta-analysis to identify genes related to multidrug response in ER positive and ER negative cells separately. One hundred eighty-eight genes were identified as related to multidrug response in ER positive and 32 genes in ER negative breast cell lines. Of these, only three genes (DBI, TOP2A, and PMVK) were common to both cell types. TOP2A was positively associated with drug response, and DBI was negatively associated with drug response. Interestingly, PMVK was positively associated with drug response in ER positive cells and negatively in ER negative cells. Functional analysis showed that while cell cycle affects drug response in both ER positive and negative cells, most biological processes that are involved in drug response are distinct. A number of signaling pathways that are uniquely enriched in ER positive cells have complex cross talk with ER signaling, while in ER negative cells, enriched pathways are related to metabolic functions. Taken together, our analysis indicates that distinct mechanisms are involved in multidrug response in ER positive and ER negative breast cells. © 2012 Shen et al

Use and commercialization of Podocnemis expansa (Schweiger 1812) (Testudines: Podocnemididae) for medicinal purposes in two communities in North of Brazil

<p>Abstract</p> <p>Background</p> <p>Throughout Brazil a large number of people seek out reptiles for their meat, leather, ornamental value and supposed medicinal importance. However, there is a dearth of information on the use of reptiles in folk medicine. In North Brazil, the freshwater turtle, <it>Podocnemis expansa</it>, is one of the most frequently used species in traditional medicines. Many products derived from <it>P. expansa </it>are utilized in rural areas and also commercialized in outdoor markets as a cure or treatment for different diseases. Here we document the use and commercialization of <it>P. expansa </it>for medicinal purposes in the state of Pará, Northern Brazil.</p> <p>Methods</p> <p>Data were gathered through interview-questionnaires, with some questions left open-ended. Information was collected in two localities in Pará State, North of Brazil. In the City of Belém, data was collected through interviews with 23 herbs or root sellers (13 men and 10 women). Attempts were made to interview all animal merchants in the markets visited. In fishing community of the Pesqueiro Beach, interviews were done with 41 inhabitants (23 men and 18 women) and during the first contacts with the local population, we attempted to identify local people with a specialized knowledge of medicinal animal usage.</p> <p>Results</p> <p><it>P. expansa </it>was traded for use in traditional medicines and cosmetics. Fat and egg shells were used to treat 16 different diseases. Turtle fat was the main product sold. The demand for these products is unknown. However, the use of this species in folk medicine might have a considerable impact on wild population, and this must be taken into account for the conservation and management of this species.</p> <p>Conclusion</p> <p>Our results indicated that the use and commercialization of <it>P. expansa </it>products for medicinal purposes is common in North of Brazil. More studies regarding the use and commerce of Brazilian turtles are urgently needed in order to evaluate the real impact of such activities on natural populations. We hope that our findings about the trade and use of <it>P. expansa </it>in folk medicine will motivate further studies on the use of animals in folk medicine and its implications for conservation.</p

Limitations in a frataxin knockdown cell model for Friedreich ataxia in a high-throughput drug screen

<p>Abstract</p> <p>Background</p> <p>Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay.</p> <p>Methods</p> <p>Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA.</p> <p>Results</p> <p>The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model.</p> <p>Conclusion</p> <p>Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future.</p

Insights into the Ecology and Evolutionary Success of Crocodilians Revealed through Bite-Force and Tooth-Pressure Experimentation

BackgroundCrocodilians have dominated predatory niches at the water-land interface for over 85 million years. Like their ancestors, living species show substantial variation in their jaw proportions, dental form and body size. These differences are often assumed to reflect anatomical specialization related to feeding and niche occupation, but quantified data are scant. How these factors relate to biomechanical performance during feeding and their relevance to crocodilian evolutionary success are not known.Methodology/Principal FindingsWe measured adult bite forces and tooth pressures in all 23 extant crocodilian species and analyzed the results in ecological and phylogenetic contexts. We demonstrate that these reptiles generate the highest bite forces and tooth pressures known for any living animals. Bite forces strongly correlate with body size, and size changes are a major mechanism of feeding evolution in this group. Jaw shape demonstrates surprisingly little correlation to bite force and pressures. Bite forces can now be predicted in fossil crocodilians using the regression equations generated in this research.Conclusions/SignificanceCritical to crocodilian long-term success was the evolution of a high bite-force generating musculo-skeletal architecture. Once achieved, the relative force capacities of this system went essentially unmodified throughout subsequent diversification. Rampant changes in body size and concurrent changes in bite force served as a mechanism to allow access to differing prey types and sizes. Further access to the diversity of near-shore prey was gained primarily through changes in tooth pressure via the evolution of dental form and distributions of the teeth within the jaws. Rostral proportions changed substantially throughout crocodilian evolution, but not in correspondence with bite forces. The biomechanical and ecological ramifications of such changes need further examination

High-Affinity Inhibitors of Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase: Mechanisms of Inhibition and Structure-Activity Relationships

BACKGROUND: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. PRINCIPAL FINDINGS: To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing &gt;160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. CONCLUSIONS: Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2

Proteoglycan-4 Regulates Fibroblast to Myofibroblast Transition and Expression of Fibrotic Genes in the Synovium

Background: Synovial tissue fibrosis is common in advanced OA with features including the presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. Proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and is a major component of synovial fluid. PRG4 is a ligand of the CD44 receptor. Our objective was to examine the role of PRG4-CD44 interaction in regulating synovial tissue fibrosis in vitro and in vivo. Methods: OA synoviocytes were treated with TGF-β ± PRG4 for 24h and α-SMA content was determined using immunofluorescence. Rhodamine-labeled rhPRG4 was incubated with OA synoviocytes ± anti-CD44 or isotype control antibodies and cellular uptake of rhPRG4 was determined following a 30-min incubation and α-SMA expression following a 24-h incubation. HEK-TGF-β cells were treated with TGF-β ± rhPRG4 and Smad3 phosphorylation was determined using immunofluorescence and TGF-β/Smad pathway activation was determined colorimetrically. We probed for stress fibers and focal adhesions (FAs) in TGF-β-treated murine fibroblasts and fibroblast migration was quantified ± rhPRG4. Synovial expression of fibrotic markers: α-SMA, collagen type-I, and PLOD2 in Prg4 gene-trap (Prg4GT) and recombined Prg4GTR animals were studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-month-old Prg4GT/GT&Cd44−/− and Prg4GTR/GTR&Cd44−/− animals. Results: PRG4 reduced α-SMA content in OA synoviocytes (p \u3c 0.001). rhPRG4 was internalized by OA synoviocytes via CD44 and CD44 neutralization attenuated rhPRG4’s antifibrotic effect (p \u3c 0.05). rhPRG4 reduced pSmad3 signal in HEKTGF- β cells (p \u3c 0.001) and TGF-β/Smad pathway activation (p \u3c 0.001). rhPRG4 reduced the number of stress fiberpositive myofibroblasts, FAs mean size, and cell migration in TGF-β-treated NIH3T3 fibroblasts (p \u3c 0.05). rhPRG4 inhibited fibroblast migration in a macrophage and fibroblast co-culture model without altering active or total TGF-β levels. Synovial tissues of 9-month-old Prg4GT/GT animals had higher α-SMA, collagen type-I, and PLOD2 (p \u3c 0.001) content and Prg4 re-expression reduced these markers (p \u3c 0.01). Prg4 re-expression also reduced α-SMA and PLOD2 staining in CD44-deficient mice. Conclusion: PRG4 is an endogenous antifibrotic modulator in the joint and its effect on myofibroblast formation is partially mediated by CD44, but CD44 is not required to demonstrate an antifibrotic effect in vivo

A New Horned Crocodile from the Plio-Pleistocene Hominid Sites at Olduvai Gorge, Tanzania

BACKGROUND: The fossil record reveals surprising crocodile diversity in the Neogene of Africa, but relationships with their living relatives and the biogeographic origins of the modern African crocodylian fauna are poorly understood. A Plio-Pleistocene crocodile from Olduvai Gorge, Tanzania, represents a new extinct species and shows that high crocodylian diversity in Africa persisted after the Miocene. It had prominent triangular "horns" over the ears and a relatively deep snout, these resemble those of the recently extinct Malagasy crocodile Voay robustus, but the new species lacks features found among osteolaemines and shares derived similarities with living species of Crocodylus. METHODOLOGY/PRINCIPAL FINDINGS: The holotype consists of a partial skull and skeleton and was collected on the surface between two tuffs dated to approximately 1.84 million years (Ma), in the same interval near the type localities for the hominids Homo habilis and Australopithecus boisei. It was compared with previously-collected material from Olduvai Gorge referable to the same species. Phylogenetic analysis places the new form within or adjacent to crown Crocodylus. CONCLUSIONS/SIGNIFICANCE: The new crocodile species was the largest predator encountered by our ancestors at Olduvai Gorge, as indicated by hominid specimens preserving crocodile bite marks from these sites. The new species also reinforces the emerging view of high crocodylian diversity throughout the Neogene, and it represents one of the few extinct species referable to crown genus Crocodylus