Molecular determinants for G protein betagamma modulation of ionotropic glycine receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.     

<Emphasis Type="Italic">Pinus contorta</Emphasis> invasion in the Chilean Patagonia: local patterns in a global context

Alien conifer invasions are affecting ecosystems across the globe, but until recently, reports of such invasions in South America were scarce. Pinus contorta was first established in Chilean Patagonia for erosion control caused by historical fires and cattle farming. Recently, the species has been planted over large areas for commercial purposes. It is well adapted to local conditions and is now spreading into natural areas. This study analyzes natural regeneration of Pinus contorta around Coyhaique city, Chile, to determine the spatial patterns of invasion. Five study sites were selected, four with grasslands dominated by exotic species and one site in the steppe. In each site, the plantation (seed source) was characterized using morphological attributes and density. Regeneration, density, height and age at different distances from the seed source were recorded, and ground cover was measured as an environmental factor influencing the invasion process. A comparative analysis was also conducted between the situation in Chile and other countries affected by P. contorta invasion. In Chile, P. contorta regeneration is significantly influenced by distance from the seed source. Higher densities are found close to the parent stand (up to 13,222 trees ha⁻¹), decreasing as distance from the seed source increases. Age and height structure indicate that the invasion process is at an early stage, and this offers a unique opportunity to study the process of invasion and to monitor it over time. In order to preserve the distinctiveness of Patagonian ecosystem, decisive action is required to control invasive conifers, with P. contorta as the number one priority. Relative to control, there is much that can be learnt from the experiences of other countries, such as New Zealand.     

Impact of different acclimation temperatures and duration on the chill coma temperature and oxygen consumption in the tenebrionid beetle Alphitobius diaperinus

When the ambient temperature is lowered to an insect's lower thermal limit, the insect enters into chill coma. Chill coma temperature and chill coma recovery can vary within species as a result of thermal acclimation, although the physiological basis of the onset of chill coma remains poorly understood. The present study investigates how the temperature of acclimation (0, 5, 10, 15 and 20 °C for 2 or 7 days) affects chill coma temperature and oxygen consumption in adult Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae). It is hypothesized that the threshold decline in metabolic rate corresponds to the entry into chill coma. Oxygen consumption (as a proxy of metabolism) is measured across the chill coma temperature threshold, and a strong decline in oxygen consumption is expected at entry into chill coma. The acclimation decreases the chill coma temperature significantly from 6.6 ± 1.1 °C in control insects to 3.1 ± 0.7 °C in those acclimated to 10 °C. The change in metabolic rate (Q₁₀) after acclimation to temperatures ranging from 10 to 20 °C is 3.7. Despite acclimation, the metabolic rate of A. diaperinus conforms to Arrhenius kinetics, suggesting that the response of this beetle does not show metabolic compensation. The data suggest the existence of a threshold decline in metabolic rate during cooling that coincides with the temperature at which an insect goes into chill coma.     

Cell therapy with bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma scaffolds

Background aimsCell therapy using bone marrow stromal cells (BMSCs) has been considered a promising strategy for neurologic sequelae after intracerebral hemorrhage (ICH). However, after intracerebral administration of BMSCs, most of the cells die, partly because of the absence of extracellular matrix. Intracerebral transplantation of BMSCs, supported in a platelet-rich plasma (PRP) scaffold, optimizes this type of cell therapy.MethodsICH was induced by stereotactic injection of 0.5 IU of collagenase type IV in the striatum of adult Wistar rats (n = 40). Two months later, the rats were subjected to intracerebral administration of 5 × 10⁶ allogeneic BMSCs embedded in a PRP scaffold (n = 10), 5 × 10⁶ allogeneic BMSCs in saline (n = 10), PRP-derived scaffold only (n = 10) or saline only (n = 10). Functional improvements in each group over the next 6 months were assessed using Rotarod and Video-Tracking-Box tests. Endogenous neurogenesis and survival of transplanted BMSCs were examined at the end of follow-up.ResultsOur study demonstrated neurologic improvement after BMSC transplantation and significantly better functional improvement for the group of animals that received BMSCs in the PRP-derived scaffold compared with the group that received BMSCs in saline. Histologic results showed that better functional outcome was associated with strong activation of endogenous neurogenesis. After intracerebral administration of BMSCs, donor cells were integrated in the injured tissue and showed phenotypic expression of glial fibrillary acidic protein and neuronal nucleus.ConclusionsPRP-derived scaffolds increase the viability and biologic activity of BMSCs and optimize functional recovery when this type of cell therapy is applied after ICH.     

Herbicides induce change in metabolic and genetic diversity of bacterial community from a cold oligotrophic lake

Pristine cold oligotrophic lakes show unique physical and chemical characteristics with permanent fluctuation in temperature and carbon source availability. Incorporation of organic toxic matters to these ecosystems could alter the bacterial community composition. Our goal was to assess the effects of simazine (Sz) and 2,4 dichlorophenoxyacetic acid (2,4-D) upon the metabolic and genetic diversity of the bacterial community in sediment samples from a pristine cold oligotrophic lake. Sediment samples were collected in winter and summer season, and microcosms were prepared using a ration 1:10 (sediments:water). The microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated for 20 days at 10 °C. Metabolic diversity was evaluated by using the Biolog Ecoplate? system and genetic diversity by 16S rDNA amplification followed by denaturing gradient gel electrophoresis analysis. Total bacterial counts and live/dead ratio were determined by epifluorescence microscopy. The control microcosms showed no significant differences (P > 0.05) in both metabolic and genetic diversity between summer and winter samples. On the other hand, the addition of 2,4-D or Sz to microcosms induces statistical significant differences (P < 0.05) in metabolic and genetic diversity showing the prevalence of Actinobacteria group which are usually not detected in the sediments of these non-contaminated lacustrine systems. The obtained results suggest that contaminations of cold pristine lakes with organic toxic compounds of anthropic origin alter their homeostasis by inhibiting specific susceptible bacterial groups. The concomitant increase of usually low representative bacterial groups modifies the bacterial composition commonly found in this pristine lake.     

Delayed setting of the photoperiodic response in recombinant clones of the aphid species Sitobion avenae

1. To explore the possible causes of apparent changes in reproductive mode from obligate to cyclical parthenogenesis over time in recombinant clones of the aphid Sitobion avenae Fabricius, all F1 progenies from various crosses were tested for several consecutive years for sexual morph production, after several weeks' exposure to a short photoperiod. 2. Variable proportions of the F1 progenies from selfing and outcrossing holocyclic clones did not produce mating females when induction was attempted in the year of hatching, but only after further induction, the following year or after. This ‘delayed setting of the photoperiodic response’ (DSPR) was much stronger in recombinants from crosses involving only clones from oceanic regions than in those involving one clone from a region with a continental climate. 3. F1 progenies resulting from crosses between one holocyclic and one intermediate clone did not show DSPR. It appeared again in the F2. 4. DSPR preferentially affected the latest hatched clones in a given progeny. 5. This phenomenon is neither an experimental artefact nor as a result of clone contamination. It appears to be because of a genetically controlled quantitative trait affecting the length of the ‘interval timer’, and may represent an adaptation of holocyclic aphid clones from oceanic regions to unpredictable winter climates.     

OBSERVATIONS OF AN UNIDENTIFIED BEAKED WHALE (MESOPLODON SP.) IN THE EASTERN TROPICAL PACIFIC

Multiple sightings of a distinctive but unidentified species of beaked whale have been made in the eastern tropical Pacific. The unidentified whale has two color morphs: a conspicuously marked black and white form (judged to be larger), and a uniformly gray-brown form. Maximum length estimates have been 5–5.5 m. Other features include a relatively flat head, with a small, distinct melon; a moderately long beak; and a low, wide-based, triangular dorsal fin. On most animals the trailing edge of the dorsal fin is only slightly falcate and often appears straight. On the black and white morph, a broad white or cream-colored swathe originates immediately posterior to the dorsal surface of the head and runs posterio-ventrally on either side of the animal. The prevalence of scarring on the black and white animals suggests sexual dimorphism and that these larger, more conspicuously marked animals are adult males, while the smaller, browner, unscarred animals are females and young. Possibilities for identification include: 1) a well-marked race of a known Mesoplodon sp., 2) Mesoplodon (Indopacetus) pacificus or 3) an undescribed species.     

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A₂ mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44^mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH₄) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O₂ ^•–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O₂ ^•– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH₄ level. Insulin and tempol blocked the high D-glucose–increased p42/44^mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O₂ ^•–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.