The Evolutionary Relationship between Microbial Rhodopsins and Metazoan Rhodopsins

Rhodopsins are photoreceptive proteins with seven-transmembrane alpha-helices and a covalently bound retinal. Based on their protein sequences, rhodopsins can be classified into microbial rhodopsins and metazoan rhodopsins. Because there is no clearly detectable sequence identity between these two groups, their evolutionary relationship was difficult to decide. Through ancestral state inference, we found that microbial rhodopsins and metazoan rhodopsins are divergently related in their seven-transmembrane domains. Our result proposes that they are homologous proteins and metazoan rhodopsins originated from microbial rhodopsins. Structure alignment shows that microbial rhodopsins and metazoan rhodopsins share a remarkable structural homology while the position of retinal-binding lysine is different between them. It suggests that the function of photoreception was once lost during the evolution of rhodopsin genes. This result explains why there is no clearly detectable sequence similarity between the two rhodopsin groups: after losing the photoreception function, rhodopsin gene was freed from the functional constraint and the process of divergence could quickly change its original sequence beyond recognition

Shallow water marine sediment bacterial community shifts along a natural CO2 gradient in the Mediterranean Sea off Vulcano, Italy.

The effects of increasing atmospheric CO(2) on ocean ecosystems are a major environmental concern, as rapid shoaling of the carbonate saturation horizon is exposing vast areas of marine sediments to corrosive waters worldwide. Natural CO(2) gradients off Vulcano, Italy, have revealed profound ecosystem changes along rocky shore habitats as carbonate saturation levels decrease, but no investigations have yet been made of the sedimentary habitat. Here, we sampled the upper 2 cm of volcanic sand in three zones, ambient (median pCO(2) 419 μatm, minimum Ω(arag) 3.77), moderately CO(2)-enriched (median pCO(2) 592 μatm, minimum Ω(arag) 2.96), and highly CO(2)-enriched (median pCO(2) 1611 μatm, minimum Ω(arag) 0.35). We tested the hypothesis that increasing levels of seawater pCO(2) would cause significant shifts in sediment bacterial community composition, as shown recently in epilithic biofilms at the study site. In this study, 454 pyrosequencing of the V1 to V3 region of the 16S rRNA gene revealed a shift in community composition with increasing pCO(2). The relative abundances of most of the dominant genera were unaffected by the pCO(2) gradient, although there were significant differences for some 5 % of the genera present (viz. Georgenia, Lutibacter, Photobacterium, Acinetobacter, and Paenibacillus), and Shannon Diversity was greatest in sediments subject to long-term acidification (>100 years). Overall, this supports the view that globally increased ocean pCO(2) will be associated with changes in sediment bacterial community composition but that most of these organisms are resilient. However, further work is required to assess whether these results apply to other types of coastal sediments and whether the changes in relative abundance of bacterial taxa that we observed can significantly alter the biogeochemical functions of marine sediments

Differential Expression of miRNAs in Response to Topping in Flue-Cured Tobacco (Nicotiana tabacum) Roots

Topping is an important cultivating measure for flue-cured tobacco, and many genes had been found to be differentially expressed in response to topping. But it is still unclear how these genes are regulated. MiRNAs play a critical role in post-transcriptional gene regulation, so we sequenced two sRNA libraries from tobacco roots before and after topping, with a view to exploring transcriptional differences in miRNAs.Two sRNA libraries were generated from tobacco roots before and after topping. Solexa high-throughput sequencing of tobacco small RNAs revealed a total of 12,104,207 and 11,292,018 reads representing 3,633,398 and 3,084,102 distinct sequences before and after topping. The expressions of 136 conserved miRNAs (belonging to 32 families) and 126 new miRNAs (belonging to 77 families) were determined. There were three major conserved miRNAs families (nta-miR156, nta-miR172 and nta-miR171) and two major new miRNAs families (nta-miRn2 and nta-miRn26). All of these identified miRNAs can be folded into characteristic miRNA stem-loop secondary hairpin structures, and qRT-PCR was adopted to validate and measure the expression of miRNAs. Putative targets were identified for 133 out of 136 conserved miRNAs and 126 new miRNAs. Of these miRNAs whose targets had been identified, the miRNAs which change markedly (>2 folds) belong to 53 families and their targets have different biological functions including development, response to stress, response to hormone, N metabolism, C metabolism, signal transduction, nucleic acid metabolism and other metabolism. Some interesting targets for miRNAs had been determined.The differential expression profiles of miRNAs were shown in flue-cured tobacco roots before and after topping, which can be expected to regulate transcripts distinctly involved in response to topping. Further identification of these differentially expressed miRNAs and their targets would allow better understanding of the regulatory mechanisms for flue-cured tobacco response to topping

Measurement of intracellular Ca²⁺, [Ca²⁺]_i, levels showing strong cross-talk between TRPV4 and the KCa channels in mCCDcl1.

<p>All traces are representative examples as indicated. Addition of GSK101 or the blockers is indicated by the length of the “bar” below each symbol in the individual tracings. <b>A.</b> In the absence of TRPV4 stimulation, [Ca²⁺]_i levels remain relative low in all cells. <b>B.</b> Upon activation of TPRV4 with the selective agonist, GSK101, [Ca²⁺]_i displays a biphasic response, rising to a peak [Ca²⁺]_i level with the first few minutes, then partial relaxes to a steady-state plateau which is above the basal [Ca²⁺]_i levels. <b>C.</b> The average changes in [Ca²⁺]_i are given showing the basal (GSK-) and peak (GSK+) [Ca²⁺]_i levels upon stimulation with GSK101 (3 nM). <b>D.</b> Effect of addition of a cocktail of KCa channel inhibitors (Apamin, 300 nM, IbTX, 100 nM, TRAM-34, 300 nM, and TQ, 5 nM) on [Ca²⁺]_i prior to activation of TRPV4 with GSK101. No response is apparent. <b>E.</b> Effect of addition of the same cocktail of KCa blockers as in panel <b>D</b>, but added after GSK101 activation of TRPV4. The cocktail of blockers markedly depressed the TRPV4-mediated [Ca²⁺]_i levels. <b>F.</b> Summary of the average decrease in [Ca²⁺]_i upon addition of the cocktail of KCa blockers (4 Blockers) before after activation of TRPV4. Addition of the 4 Blockers had not affect in the absence of GSK101 (-), averaging 0.4 ± 2.5 nM (n = 24). In the presence of GSK101 (+), the 4 Blockers induce a marked decrease in [Ca²⁺]_i of 477 ± 45 nM (n = 24, P<0.001) with the 4 Blockers, demonstrates a strong functional cross-talk between TRPV4, [Ca²⁺]_I, and KCa channels. <b>G.</b> Effect of selective inhibition of BK (IbTX, 100 nM) or ROMK (TQ, 5 nM) on GSK101-induced [Ca²⁺]_i levels showing a significant reduction in [Ca²⁺]_i with either blocker. <b>H.</b> Effect of selective inhibition of IK1 (TRAM-34, 300 nM) or SKs (Apamin, 300 nM) on GSK101-induced [Ca²⁺]_i levels showing a significant reduction in [Ca²⁺]_i with either blocker. <b>I.</b> Summary results of the actions of each of the KCa blockers on GSK101-induced [Ca²⁺]_i levels showing a pronounced decrease in [Ca²⁺]_i levels with each of the blockers on TRPV4-mediated Ca2+ influx. The reduction in [Ca²⁺]_i averaged 236 ± 45 nM (n = 24) for IbTX, 175 ± 18 nM (n = 24) for TQ, 193 ± 22 nM (n = 24) for TRAM-34, and 208 ± 14 nM (n = 24) for Apamin (P<0.001 for each case). The individual responses do not differ among blockers. In contrast, the effect of the individual blockers is significantly less than that observed for the 4 Blocker combination in 8F (P<0.01).</p

Differential expression of ion channels between principal cells (PC) and intercalated cells (IC) in the mouse kidney CCD.

<p>AQP2 staining was used to identify PC, the AQP2-positive cells, and IC, the APQ2-negative cells. Bar graphs give the mean ± SEM for the normalized intensities for each KCa channel for both PC and IC. The number of PC and IC cells analyzed, n, is indicated on the bar graphs. The images of CCD show representative immunofluorescence examples for each KCa channel and AQP2 (40X) where one or two PC (AQP2-positive) and IC (AQP2-negative) cells are labelled as “PC” or “IC.” The outer border of the tubule in each image is indicated by the dashed white line (indicating the basal side or anti-luminal side of tubular cells). “L” identifies the tubular lumen. The immunofluorescence intensity was determined for each KCa channel using ImageJ and normalized to the intensity levels of AQP2 expression in PC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155006#sec002" target="_blank">Methods</a>). Each example gives staining for AQP2 (red), the KCa channel (green), and the merged image (AQP2, KCa channel) that includes DAPI staining to identify nuclei. <b>A.</b> Expression of SK1 in PC and IC showing dominant relative expression in IC over PC (P<0.001). SK1 expression was apparent within the cytosol and along the luminal border of both PC and IC as apparent in the representative images. Light staining along the basolateral border is also apparent in some cells. <b>B.</b> Expression of SK3 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). SK3 expression was apparent within the cytosol with light expression along the basal aspect of some cells and more dominant expression along the luminal border of PC. <b>C.</b> Expression of IK1 in PC and IC showing relatively more dominant expression in PC over IC (P<0.001). IK1 expression was apparent within the cytosol, especially of IC, but also apparent along the luminal border of PC with light staining along the basal aspect of the cells. <b>D.</b> Expression of BKα in PC and IC showing strong staining along the luminal border of both PC and IC. However, the intensity of staining between PC and IC was found to vary depending on the primary antibody used for the immunostaining. In our hands, the Alomone APC-151 BKα antibody staining was most apparent in PC over IC (P<0.001, compare bar graph labeled APC-151 with APC-107). In contrast, the Alomone APC-107 BKα antibody staining was most apparent in IC over PC. Since the antibodies were made to different epitopes of the BKα channel, such variations can be anticipated (see text).</p

Confocal immunocytochemistry fluorescence images showing results of staining for ion channels in mCCDcl1 cells grown on Transwell^™ Permeable Supports.

<p>Results show immunostaining for the ion channel of interest and for nuclei (DAPI). <b>A.</b> AQP2 staining of cells (green, 488 nm) shows prominent staining along the plasma membrane with low staining within the cytosol. <b>B.</b> SK1 staining of cells (green, 488 nm) shows clear staining along the plasma membrane borders and modest staining within the cytosol. <b>C.</b> SK3 staining of cells (red, 594 nm) shows significant staining along the cell borders and within the cytosol. Modest staining of the nuclei is apparent (SK3, Merged). <b>D.</b> IK1 staining (green, 488 nm) is modest along the plasma membrane borders and within the cytosol. <b>E.</b> BKα staining of cells (green, 488 nm) with prominent staining of the cell borders and low staining of the cytosol and nuclei. <b>F.</b> TRPV4 staining of cells (green, 488 nm) with prominent staining of the cell plasma membrane and modest staining of the cytosol and nuclei. All fluorescence images were obtained at 100x (oil) with a Nikon A1R Confocal Laser Microscope.</p