Receptor-mediated changes at the myristoylated amino terminus of Galpha(il) proteins

G protein-coupled receptors (GPCRs) catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to aid in anchoring G i proteins to the membrane, the role of myristoylation with regard to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of Galpha proteins in solution [Medkova, M. (2002) Biochemistry 41, 9963-9972; Preininger, A. M. (2003) Biochemistry 42, 7931-7941], but changes in the environment of specific residues within the Galpha i1 amino terminus during receptor-mediated G i activation have not been reported. Using site-specific fluorescence labeling of individual residues along a stretch of the Galpha il amino terminus, we found specific changes in the environment of these residues upon interaction with the activated receptor and following GTPgammaS binding. These changes map to a distinct surface of the amino-terminal helix opposite the Gbetagamma binding interface. The receptor-dependent fluorescence changes are consistent with a myristoylated amino terminus in the proximity of the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTPgammaS binding). This work demonstrates that the myristoylated amino terminus of Galpha il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling.     

Building up Analgesia in Humans via the Endogenous μ-Opioid System by Combining Placebo and Active tDCS: A Preliminary Report

Transcranial Direct Current Stimulation (tDCS) is a method of non-invasive brain stimulation that has been frequently used in experimental and clinical pain studies. However, the molecular mechanisms underlying tDCS-mediated pain control, and most important its placebo component, are not completely established. In this pilot study, we investigated in vivo the involvement of the endogenous μ-opioid system in the global tDCS-analgesia experience. Nine healthy volunteers went through positron emission tomography (PET) scans with [¹¹C]carfentanil, a selective μ-opioid receptor (MOR) radiotracer, to measure the central MOR activity during tDCS in vivo (non-displaceable binding potential, BP_ND) - one of the main analgesic mechanisms in the brain. Placebo and real anodal primary motor cortex (M1/2mA) tDCS were delivered sequentially for 20 minutes each during the PET scan. The initial placebo tDCS phase induced a decrease in MOR BP_ND in the periaqueductal gray matter (PAG), precuneus, and thalamus, indicating activation of endogenous μ-opioid neurotransmission, even before the active tDCS. The subsequent real tDCS also induced MOR activation in the PAG and precuneus, which were positively correlated to the changes observed with placebo tDCS. Nonetheless, real tDCS had an additional MOR activation in the left prefrontal cortex. Although significant changes in the MOR BP_ND occurred with both placebo and real tDCS, significant analgesic effects, measured by improvements in the heat and cold pain thresholds, were only observed after real tDCS, not the placebo tDCS. This study gives preliminary evidence that the analgesic effects reported with M1-tDCS, can be in part related to the recruitment of the same endogenous MOR mechanisms induced by placebo, and that such effects can be purposely optimized by real tDCS.     

A revision of the genus Arenivaga (Rehn) (Blattodea,Corydiidae), with descriptions of new species and key to the males of the genus

The cockroach genus Arenivaga is revised. Forty-eight Arenivaga species are recognized with nine previously known species and 39 described as new including the following: A. pagana sp. n., A. grandiscanyonensis sp. n., A. haringtoni sp. n., A. hopkinsorum sp. n., A. umbratilis sp. n., A. tenax sp. n., A. impensa sp. n., A. trypheros sp. n., A. darwini sp. n., A. nalepae sp. n., A. sequoia sp. n., A. mckittrickae sp. n., A. gaiophanes sp. n., A. belli sp. n., A. estelleae sp. n., A. delicata sp. n., A. mortisvallisensis sp. n., A. milleri sp. n., A. pratchetti sp. n., A. gumperzae sp. n., A. rothi sp. n., A. ricei sp. n., A. adamsi sp. n., A. nicklei sp. n., A. akanthikos sp. n., A. moctezuma sp. n., A. paradoxa sp. n., A. apaeninsula sp. n., A. hebardi sp. n., A. dnopheros sp. n., A. aquila sp. n., A. florilega sp. n., A. galeana sp. n., A. gurneyi sp. n., A. pumila sp. n., A. hypogaios sp. n., A. diaphana sp. n., A. nocturna sp. n., A. alichenas sp. n. All species are described or redescribed, major morphological features are illustrated, distributions are characterized, and the biology of the species is reviewed. A neotype series is designated for A. investigata Friauf & Edney.     

Migratory flexibility in native Hawai'ian amphidromous fishes

We assessed the prevalence of life history variation across four of the five native amphidromous Hawai'ian gobioids to determine whether some or all exhibit evidence of partial migration. Analysis of otolith Sr.: Ca concentrations affirmed that all are amphidromous and revealed evidence of partial migration in three of the four species. We found that 25% of Lentipes concolor (n = 8), 40% of Eleotris sandwicensis (n = 20) and 29% of Stenogobius hawaiiensis (n = 24) did not exhibit a migratory life-history. In contrast, all individuals of Sicyopterus stimpsoni (n = 55) included in the study went to sea as larvae. Lentipes concolor exhibited the shortest mean larval duration (LD) at 87 days, successively followed by E. sandwicensis (mean LD = 102 days), S. hawaiiensis (mean LD = 114 days) and S. stimpsoni (mean LD = 120 days). These findings offer a fresh perspective on migratory life histories that can help improve efforts to conserve and protect all of these and other at-risk amphidromous species that are subject to escalating anthropogenic pressures in both freshwater and marine environments.     

Late Pleistocene and Holocene environments of the Southeastern United States from the stratigraphy and pollen content of a peat deposit on the Georgia Coastal Plain

A 5.4 m peat core from the Sandy Run Creek (SRC) valley in the upper Coastal Plain of Georgia shows that before 30 ka the valley had an aggradational sandy floor with shallow swales and low ridges of 1–2 m amplitude suggesting a braided stream pattern and a low water table. The climate from 30–25 ka was cool and dry and the vegetation open grassland with stands of pine and spruce that produced few fires. At ca. 16 ka a warmer, wetter climate caused SRC to meander and incise the valley fill removing some previously deposited sediment at the site of our peat core so that sediments of 13 ka rest directly on sediments deposited at 25 ka. After ca. 13 ka higher groundwater levels initiated vertical accretion of floodplains allowing peat accumulation in shallow depressions across the valley. Pollen from the Younger Dryas period (ca. 13–11 ka) indicates a cool, moist environment of open oak woodland, mesic trees, riparian populations of alder, and reduced levels of pine. By the early to mid Holocene, tupelo and oak dominated over pine indicating wetter conditions, while sand stringers in peat record periodic heavy rains, an elevated water table, and intervals of substantial runoff. Cooling after ca. 4.5 ka brought drier and more variable conditions. Fires increased and southern pine replaced tupelo and oak. The absence of sand stringers in peat younger than 4.5 ka indicates fewer intense rainfall events.     

Further optimization of mouse spermatozoa evaporative drying techniques

It has been shown in the past that mouse spermatozoa could be dried under a stream of nitrogen gas at ambient temperature and stored at 4 °C or 22 °C for up to 3 months and was capable of generating live-born offspring. In previous desiccation work, dried sperm were stored in a vacuum-sealed plastic bag placed in a vacuum-packed Mylar bag. However, dried specimens stored in this way often lost moisture, particularly in samples stored at higher temperatures (22 °C) compared to lower temperatures (4 °C). The present report describes a method which minimizes this water loss from the dried sperm samples. Its use is described in a preliminary study on the effect of supplementing the trehalose with glycerol. The results have demonstrated that mouse sperm can be stored at 4 °C over saturated NaBr without the uptake of water which occurs when they are stored in Mylar packages. In addition, we were able to get some survival of sperm (9–15%) at room temperature storage after 3 months. The addition of glycerol to trehalose had little effect on the survival of dried mouse sperm stored over NaBr for 1 and 3 months.     

[18F](R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2,5-dimethyl phenylamino)pyrazin-2(1H)-one: Introduction of N3-phenylpyrazinones as potential CRF-R1 PET imaging agents

Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [¹⁸F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.     

Linkage of High Rates of Sulfate Reduction in Yellowstone Hot Springs to Unique Sequence Types in the Dissimilatory Sulfate Respiration Pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.