Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

In theXenopus oocyte heterologous expressionsystem, the electrophysiological characteristics of rabbit ClC-2current and its contribution to volume regulation were examined.Expressed currents on oocytes were recorded with a two-electrodevoltage-clamp technique. Oocyte volume was assessed by taking picturesof oocytes with a magnification of ×40. Rabbit ClC-2 currentsexhibited inward rectification and had a halide anion permeabilitysequence of Cl  Br  I  F. ClC-2 currents wereinhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid(9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similarto those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of ratClC-2. During a 30-min perfusion with hyposmolar solution, currentamplitude at 160 mV and oocyte diameter were compared amongthree groups: oocytes injected with distilled water, oocytes injectedwith ClC-2 cRNA, and oocytes injected with ClC-2NT cRNA (an openchannel mutant with NH₂-terminaltruncation). Maximum inward current was largest in ClC-2NT-injectedoocytes (5.9 ± 0.4 µA), followed by ClC-2-injected oocytes(4.3 ± 0.6 µA), and smallest in water-injected oocytes(0.2 ± 0.2 µA), whereas the order of increase in oocytediameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2NT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling wassmallest in oocytes with the largest expressed currents suggest thatClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to ahyposmolar environment.

     

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-l-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.     

Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca²⁺ imaging of cells loaded with the Ca²⁺ indicator Fluo-4 enabled us to examine intracellular Ca²⁺ handling properties, and we found a significantly higher incidence of irregular Ca²⁺ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca²⁺ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na^+/Ca²⁺exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca²⁺ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (I_NCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of I_NCX.     

Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the cockroach Cryptocercus

Cryptocercus cockroaches and lower termites harbour obligate, diverse and unique symbiotic cellulolytic flagellates in their hindgut that are considered critical in the development of social behaviour in their hosts. However, there has been controversy concerning the origin of these symbiotic flagellates. Here, molecular sequences encoding small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase were identified in the symbiotic flagellates of the order Trichonymphida (phylum Parabasalia) in the gut of Cryptocercus punctulatus and compared phylogenetically to the corresponding species in termites. In each of the monophyletic lineages that represent family-level groups in Trichonymphida, the symbionts of Cryptocercus were robustly sister to those of termites. Together with the recent evidence for the sister-group relationship of the host insects, this first comprehensive study comparing symbiont molecular phylogeny strongly suggests that a set of symbiotic flagellates representative of extant diversity was already established in an ancestor common to Cryptocercus and termites, was vertically transmitted to their offspring, and subsequently became diversified to distinct levels, depending on both the host and the symbiont lineages.     

Induction of SCEs in CHL cells by dichlorobiphenyl derivative water pollutants, 2-phenylbenzotriazole (PBTA) congeners and river water concentrates

We recently identified dichlorobiphenyl (DCB) derivatives and 2-phenylbenzotriazole (PBTA) congeners as major mutagenic constituents of the waters of the Waka River and the Yodo River system in Japan, respectively. In this study we examined sister chromatid exchange (SCE) induction by two dichlorobiphenyl derivatives, 3,3′-dichlorobenzidine (DCB, 4,4′-diamino-3,3′-dichlorobiphenyl) and 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl (5-nitro-DCB); three PBTA congeners, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1), 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), and 2-[2-(acetylamino)amino]-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6); and water concentrates from the Waka River in Chinese hamster lung (CHL) cells. Concentration-dependent induction of SCE was found for all DCBs and PBTAs examined in the presence of S9 mix, and statistically significant increases of SCEs were detected at 2 μg per ml of medium or higher concentrations. SCE induction of MeIQx was examined to compare genotoxic activities of these water pollutants. According to the results, a ranking of the SCE-inducing potency of these compounds is the following: 5-nitro-DCB ≈ MeIQx > PBTA6 > PBTA-1 ≈ PBTA-2 > DCB.Water samples collected at a site at the Waka River showed concentration-related increases in SCEs at 6.25–18.75 ml-equivalent of river water per ml of medium with S9 mix. The concentrations of 5-nitro-DCB and DCB in the river water samples were from 2.5 to 19.4 ng/l and from 4100 to 18,900 ng/l, respectively. However, these chemicals showed only small contribution to SCE induction by the Waka River water.     

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia

Background

Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)₄-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4.

Methodology/Principal Findings

PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity.

Conclusions/Significance

Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.     

Anti-HIV-1 activities and pharmacokinetics of new arylpiperazinyl fluoroquinolones

Anti-HIV-1 activities and pharmacokinetics of a series of novel arylpiperazinyl fluoroquinolones are reported. Modification at the C-8 position with a trifluoromethyl group was superior to that with a difluoromethoxy group to achieve higher anti-HIV-1 activity. Two compounds studied exhibited quite high anti-HIV-1 activities (IC(50)<50 nM) in vitro and high bioavailabilities (BA>90%) in monkeys.