Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.     

A novel flow cytometry-based technique to measure adult neurogenesis in the brain

The stimulation of neurogenesis is an exciting novel therapeutic option for diseases of the central nervous system, ranging from depression to neurodegeneration. One major bottleneck in screening approaches for neurogenesis-inducing compounds is the very demanding in vivo quantification of newborn neurons based on stereological techniques. To effectively develop compounds in this area, novel fast and reliable techniques for quantification of in vivo neurogenesis are needed. In this study, we introduce a flow cytometry-based method for quantifying newly generated neurons in the brain based on the counting of cell nuclei from dissected brain regions. Important steps involve density sedimentation of the cell nuclei, and staining for the proliferation marker bromodeoxy uridine and nuclear cell type markers such as NeuN. We demonstrate the ability of the technique to detect increased neurogenesis in the hippocampus of animals which underwent physical exercise and received fluoxetine treatment.     

Population structure and comparative phylogeography of jack species (Caranx ignobilis and C. melampygus) in the high Hawaiian Islands

Members of the family Carangidae are top-level predators and highly prized food and sport fishes. Although ecologically and economically important, little is known about the biology of numerous species in the family. This is particularly true of the jacks Caranx ignobilis and C. melampygus, which have experienced recent population reductions around the high Hawaiian Islands due to overfishing. Previous studies have documented territorial tendencies as well as cases of long-distance excursions in both species, suggesting populations may exhibit a range of structure at the genetic level. To explore this possibility, mitochondrial DNA ATPase6 and ATPase8 gene sequence variation was assessed from 91 individuals (33 C. ignobilis and 58 C. melampygus) spanning the islands of Kaua'i, O'ahu, Moloka'i, Maui, and Hawai'i. Although a total of 20 distinct haplotypes (8 for C. ignobilis; 12 for C. melampygus) were recovered, no evidence of population structure was found for either species across the examined geographic range. However, distinct demographic patterns were identified, implying differing evolutionary histories and/or population dynamics. Additionally, ～ 6% of the examined C. ignobilis were C. ignobilis × C. melampygus hybrids because they harbored mitochondrial haplotypes typical of C. melampygus. These hybrids contribute to measurable gene flow between the species and may play a significant role in the evolution of the genus.     

Ecological Assessment and Geological Significance of Microbial Communities from Cesspool Cave,Virginia

Microbial mats from hydrogen sulfide-rich waters and cave-wall biofilms were investigated from Cesspool Cave, Virginia, to determine community composition and potential geomicrobiological functioning of acid-producing bacteria. Rates of microbial mat chemoautotrophic productivity were estimated using [ 14 C]-bicarbonate incorporations and microbial heterotrophy was determined using [ 14 C]-leucine incubations. Chemoautotrophic fixation was measured at 30.4 - 12.0 ng C mg dry wt -1 h -1 , whereas heterotrophic productivity was significantly less at 0.17 - 0.02 ng C mg dry wt -1 h -1 . The carbon to nitrogen ratios of the microbial mats averaged 13.5, indicating that the mats are not a high quality food source for higher trophic levels. Ribosomal RNA-based methods were used to examine bacterial diversity in the microbial mats, revealing the presence of at least five strains of bacteria. The identity of some of the strains could be resolved to the genus Thiothrix and the Flexibacter - Cytophaga - Bacteriodes phylum, and the identity of the remaining strains was to either the Helicobacter or Thiovulum group. Two of 10 sulfur-oxidizing, chemoautotrophic pure cultures of Thiobacillus spp. (syn. Thiomonas gen. nov.) demonstrated the ability to corrode calcium carbonate, suggesting that the colonization and metabolic activity of these bacteria may be enhancing cave enlargement.     

Septin9 is involved in T-cell development and CD8+ T-cell homeostasis

SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8⁺ T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8⁺ central memory T-cells.     

Prostaglandin E(2) and I(2) facilitate noxious heat-induced spike discharge but not iCGRP release from rat cutaneous nociceptors

The bradykinin-induced sensitization of cutaneous nociceptors to heat was previously shown to be abolished by cyclooxygenase blockade suggesting that endogenous prostaglandins exerted a heat-sensitizing action. The present study aimed at investigating the effects of exogenous prostaglandin E(2) (PGE(2)) and I(2) (PGI(2)) on noxious heat-evoked responses of rat cutaneous nociceptors. As neuropeptides including calcitonin gene-related peptide (CGRP) can be released from the peptidergic subset of heat-sensitive nociceptors, both the spike-generating (afferent) and CGRP-releasing (efferent) responses to heat stimulation were assessed by recording action potentials from single cutaneous C-fibers and measuring immunoreactive CGRP (iCGRP) release from isolated skin flaps, respectively. A combination of PGE(2) and PGI(2) (100 microM for both) unlike 10 microM PGE(2) or PGI(2) increased the number of spikes discharged during a noxious heat stimulus whereas the heat threshold remained unchanged. In contrast, 100 microM PGE(2) plus PGI(2) failed to increase the iCGRP release induced by noxious heat (47 degrees C) from the isolated rat skin. PGE(2) (100 microM), however, augmented the iCGRP-releasing effect of protons (pH 5.7). The adenylyl cyclase activator forskolin and the protein kinase C activator phorbol ester (PMA, 10 microM for both) facilitated heat-induced iCGRP release whereas increasing the intracellular Ca(2+) concentration by 10 microM ionomycin produced a desensitization of the response. In conclusion, PGE(2) plus PGI(2) can sensitize the afferent function of nociceptors in the rat skin, by increasing heat-induced spike discharge, but not the heat-induced efferent response i.e. iCGRP release. This discrepancy might reflect the differences between mechanisms of Na(+) channel-dependent spike generation and Ca(2+)-dependent neuropeptide release.     

A SIMPLIFIED,RAPID METHOD FOR IDENTIFYING MATING TYPE IN ALGAE: RESULTS WITH THE PANDORINA MORUM (CHLOROPHYCEAE) SPECIES COMPLEX1

As a collection expands, increasing numbers of test crosses are required to identify new isolates in algal species containing numerous pairs of mating types. A short-cut is described that utilizes a reduced number of preliminary test crosses. The method was used successfully with the colonial green flagellate, Pandorina morum Bory de St. Vincent, to identify a new pair of milling types (syngen) from Japan and five new isolates from China of a previously known syngen. When tested on the 20 previously described syngens, it revealed one unexpected multi-clone effect on mating. The method should be valuable for identifying newly collected clones, examining potential, interactions among incompatible mating types, and for screening other highly specific inter-organism interactions such as host-parasite infections.