Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels

This work completes previous findings and, using cysteine scanning mutagenesis (CSM) and biochemical methods, provides detailed analysis of conformational changes of the S6 domain and C-linker during gating of CNGA1 channels. Specific residues between Phe375 and Val424 were mutated to a cysteine in the CNGA1 and CNGA1_cys-free background and the effect of intracellular Cd²⁺ or cross-linkers of different length in the open and closed state was studied. In the closed state, Cd²⁺ ions inhibited mutant channels A406C and Q409C and the longer cross-linker reagent M-4-M inhibited mutant channels A406C_cys-free and Q409C_cys-free. Cd²⁺ ions inhibited mutant channels D413C and Y418C in the open state, both constructed in a CNGA1 and CNGA1_cys-free background. Our results suggest that, in the closed state, residues from Phe375 to approximately Ala406 form a helical bundle with a three-dimensional (3D) structure similar to those of the KcsA; furthermore, in the open state, residues from Ser399 to Gln409 in homologous subunits move far apart, as expected from the gating in K⁺ channels; in contrast, residues from Asp413 to Tyr418 in homologous subunits become closer in the open state. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of the Na+-coupled glutamate transporter EAAT3 by PIKfyve

The Na⁺,glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end, EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I_glu) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I_glu. Coexpression of either, SGK1 or PIKfyve, significantly enhanced I_glu in EAAT3 expressing oocytes. The increased I_glu was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I_glu and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant ^K127NSGK1 did not significantly alter I_glu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on I_glu. The stimulating effect of PIKfyve on I_glu was abolished by replacement of the serine by alanine in the SGK consensus sequence (^S318APIKfyve). Moreover, additional coexpression of ^S318APIKfyve significantly blunted I_glu in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.     

Calcium signals activated by ghrelin and D-Lys-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys³-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca²⁺]_i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca²⁺ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys³-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca²⁺ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys³-GHRP-6 ghrelin antagonist features ghrelin independent activities.     

Acylated anthocyanins in inflorescence of spider flower (Cleome hassleriana)

Five anthocyanins, cyanidin 3-(2′′-(6′′′-caffeoyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, cyanidin 3-(2′′-(6′′′-E-sinapoyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, cyanidin 3-(2′′-(6′′′-feroyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, pelargonidin 3-(2′′-(6′′′-E-sinapoyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, and pelargonidin 3-(2′′-(6′′′-E-p-coumaroyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, together with five known anthocyanins have been identified in flowers of Cleome hassleriana Queen line. One monoacylated and four diacylated cyanidin 3-sophoroside-5-glucosides were identified as the main anthocyanins in flowers with mauve colouration, while a homologous glycosidic pattern based on pelargonidin was found in the five main anthocyanins from flowers with pink colouration. The anthocyanins identified in C. hassleriana share the same glycosidic pattern as anthocyanins isolated from the genera Raphanus, Brassica and Iberis in the sister family Brassicaceae.     

In vitro sperm penetration through the zona pellucida of immature and in vitro matured oocytes using fresh, chilled and frozen canine semen

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1-10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p<0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p<0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P<0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. There was a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.     

The role of platelet ice microalgae in seeding phytoplankton blooms in Terra Nova Bay (Ross Sea,Antarctica): a mesocosm experiment

The aim of this study was to assess the role of platelet ice microalgal communities in seeding pelagic blooms. Nutrient dynamics, microalgal biomass, photosynthetic parameters, cell densities and species succession were studied in two mesocosm experiments, designed to simulate the transition of microalgal communities from platelet ice habitat to pelagic conditions. The microalgal assemblages were dominated by diatoms, 70% of which were benthic species such as Amphiprora kufferathii, Nitzschia stellata, and Berkeleya adeliensis. Photoacclimation of benthic species was inadequate also at relatively low irradiances. Exceptional growth capacity at different light levels was observed for pelagic species such as Fragilariopsis cylindrus and Chaetoceros spp. which may be important in seeding blooms at ice breakup. Fragilariopsis cylindrus showed high growth rates both at 65 and 10% of incident light and in nutrient replete as well as in nutrient depleted conditions. Five days after inoculation, phytoplankton biomass increased and nutrient concentrations decreased in both light conditions. Nutrient uptake rates were up to 9.10 μmol L⁻¹ d⁻¹ of TIN in the high light tank and 6.18 μmol L⁻¹ d⁻¹ in the low light tank and nutrient depletion in the high light tank occurred 3 days prior to depletion in the low light tank. At nutrient depletion, biomass concentrations were similar in both tanks, 30 and 34 μg Chla L⁻¹. This article belongs to a special topic: Five articles on Sea-ice communities in Terra Nova Bay (Ross Sea), coordinated by L. Guglielmo and V. Saggiomo, appear in this issue of Polar Biology. The studies were conducted in the frame of the National Program of Research in Antarctica (PNRA) of Italy.     

Expression pattern of transglutaminases in the early differentiation stage of erupting rat incisor

Several studies demonstrated that transglutaminases play a key role in extracellular matrix stabilization needed for cell differentiation. We evaluated transglutaminase expression and activity in the pre-secretory stage of differentiation of the continuously erupting rat incisor. We observed that transglutaminase-mediated incorporation of monodansylcadaverine into protein substrates was specifically located in the apical loop, and along the basement membrane joining mesenchyme and inner dental epithelium in the odontogenic organ. Enzyme activity was associated with mRNAs for transglutaminase 1 and 2. Notably, labelling cells for these isoenzymes were observed in both mesenchymal and epithelial compartments, but not in the basement membrane, in the ameloblast facing pulp anterior region, where ameloblast and odontoblast differentiation begins. These findings demonstrate that transglutaminase 1 and transglutaminase 2 are expressed at a major extent in the pre-secretory stage of regenerating rat incisor, where they probably play complementary roles in cell signalling between mesenchyme and epithelium and extracellular matrix.