A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.     

Increased abundance of snails and trematode parasites of <Emphasis Type="Italic">Fundulus heteroclitus</Emphasis> (L.) in restored New Jersey wetlands

The Hackensack Meadowlands District is a large heavily degraded, brackish marsh system in the urbanized northeastern region of New Jersey, USA. Six study sites were used, three of which were restored (Mill Creek, Skeetkill Creek and Vince Lombardi), and three others were unrestored (Richard DeKorte Park, Cedar Creek and Kingsland Creek). Highly significant differences were found with respect to snail abundance and gill parasite abundance. In the three restored sites, significantly more Littoridinops tenuipes were found, and Fundulus heteroclitus had significantly more digenean trematode metacercariae gill infections than at unrestored sites. As habitat quality improves following restoration, the number of suitable digenean trematode parasite hosts multiplies as substrate for benthic invertebrates (first intermediate host) increases and usage by other species, such as Fundulus spp. (second intermediate host), is encouraged, which then attracts more wading birds (definitive host). Though the restoration process enhances trophic complexity, including primary consumers (gastropods), secondary consumers (fish) and tertiary consumers (wading birds), and ultimately parasite diversity, restoration also helps facilitate parasite life cycles.     

Priming in Oxytocin Cells and in Gonadotrophs

At proestrous, the sensitivity of gonadotrophs to gonadotrophin-releasing hormone (GnRH) increases with repeated exposure to GnRH, a process known as self-priming. An apparently similar phenomenon can also occur in peptidergic neurons; activity-dependent oxytocin release from dendrites can be potentiated by oxytocin itself. In the brain, such priming actions have the potential to alter the strength of communication between neuronal populations for a very prolonged period. In the case of both oxytocin neurons and gonadotrophs, priming appears to involve an augmentation of a readily releasable pool of vesicles. Special issue article in honor of George Fink.     

Growth,survival, and metal content of marsh invertebrates fed diets of detritus from Spartina alterniflora Loisel. and Phragmites australis Cav. Trin. ex Steud. from metal-contaminated and clean sites

Marsh vegetation plays an important role in trophic ecology of estuaries. Once broken down to detritus, it is an important food source for manyorganisms. In Atlantic Coast marshes, the reed Phragmites australis hasbeen invading many areas once dominated by smooth cordgrass, Spartina alterniflora. In this study we evaluated the growth of and trophictransfer of metals to estuarine invertebrates when fed diets of detritus fromthese different plant species. Decaying leaves from populations of Phragmites, natural Spartina, and restored Spartina from boththe Hackensack Meadowlands, New Jersey, and the more pristineAccabonac Harbor of East Hampton, New York, were collected from themarsh surface in the spring. Decaying leaves were pureed and fed to thefiddler crabs Uca pugnax and U. pugilator, and to the grassshrimp Palaemonetes pugio. In fiddler crabs we monitored limbregeneration, molting and weight. U. pugilator regenerated limbs andmolted equally well on all six diets. Most of the U. pugnax arrestedgrowth midway through regeneration on all 6 diets. A repeat experimentwith smaller crabs, which did complete the process, found no consistentdifferences among the six diets and control food, although control food andPhragmites detritus had higher N concentrations than the Spartinadetritus. Grass shrimp fed all six diets did not survive beyond 3 weeks. Inanother experiment using HM sediments from each vegetation type(containing detritus, meiofauna, and microflora), survival was equally highamong treatments and the shrimp fed sediments from the restored Spartina site or control food grew better than those fed sediments fromthe Phragmites or natural Spartina sites. Although metalconcentrations in detritus varied between sites and plant species, the crabsof each group did not differ in metal concentrations after the feedingexperiment. Our data do not support the general assumption that Phragmites leaf detritus is of poorer nutritional quality than Spartinaalterniflora leaf detritus to estuarine consumers.     

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.     

New perspectives in radiation oncology: Young radiation oncologist point of view and challenges

AimTo assess the role of the young radiation oncologist in the context of important recent advancements in the field of radiation oncology, and to explore new perspectives and competencies of the young radiation oncologist.BackgroundRadiation oncology is a field that has rapidly advanced over the last century. It holds a rich tradition of clinical care and evidence-based practice, and more recently has advanced with revolutionary innovations in technology and computer science, as well as pharmacology and molecular biology.Materials and methodsSeveral young radiation oncologists from different countries evaluated the current status and future directions of radiation oncology.ResultsFor young radiation oncologists, it is important to reflect on the current practice and future directions of the specialty as it relates to the role of the radiation oncologist in the comprehensive management of cancer patients. Radiation oncologists are responsible for the radiation treatment provided to patients and its subsequent impact on patients’ quality of life. Young radiation oncologists must proactively master new clinical, biological and technical information, as well as lead radiation oncology teams consisting of physicists, dosimetrists, nurses and technicians.ConclusionsThe role of the young radiation oncologist in the field of oncology should be proactive in developing new competencies. Above all, it is important to remember that we are dealing with the family members and loved ones of many individuals during the most difficult part of their lives.