GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.     

Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum

Protein Nε‐acylation is emerging as a ubiquitous post‐translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of l ‐glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction was characterized. It was shown that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation‐mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine‐incorporated PEPC protein, we verified that K653‐acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin‐type deacetylase, deacetylated K653‐acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin‐type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate‐producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate‐producing conditions, supporting the hypothesis that PEPC is responsible for a large carbon flux change under glutamate‐producing conditions.     

Species‐dependent microarchitectural traits of iridescent scales in the triad taxa of Ornithoptera birdwing butterflies

Ornithoptera birdwing butterflies have blue, green, or orange iridescent scales in different species or subspecies. To understand the species‐ or subspecies‐dependent scale color differences, we performed comparative morphometric analyses of iridescent scales from three closely related taxa: O. priamus priamus (green), O. priamus urvillianus (blue), and O. croesus (orange). The three types of Ornithoptera wings exhibited reversible color changes to longer wavelengths with different kinetics upon immersion in methanol, suggesting that their color differences are at least partly based on differences in the size of air cavities made by nanostructures. Cover scales of all three color types were visually semi‐transparent glass scales that exhibited color when placed on a dark background. The dorsoventral differences in coloration were observed in single scales, suggesting the optical importance of scale surfaces. Scanning electron microscopy of cover scales in cross section revealed that all color types exhibited finely sculpted tapered ridges and thick, irregular basal multilayers containing tandemly clustered granular objects and air cavities. Scale thickness, ridge height, and multilayer thickness were significantly different among the three color types, and granular object size was significantly different between orange scales and blue and green scales. We conclude that each of the three taxa of Ornithoptera butterflies possesses unique quantitative size values on tapered ridges and irregular multilayers with granular objects and air cavities to express unique structural color. These species‐ or subspecies‐dependent structural colors might have evolved via quantitative shifts in these microarchitectural traits rather than via changes in the basic developmental or architectural plan for color expression.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Reverse genetics system for human rotaviruses

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.     

Effect of naftopidil,an alpha1D/A-adrenoceptor antagonist,on the urinary bladder in rats with spinal cord injury

AimsAlpha_1D-adrenoceptors (α_1D-ARs) located in the spinal cord are involved in the control of lower urinary tract function. In order to clarify the effect of α_1D-ARs on storage function in the spinal cord, we examined the effect of oral administration and intrathecal injection of the α_1D/A-AR antagonist, naftopidil, on bladder activity, as well as the effect of naftopidil on bladder wall histology, in female rats with spinal cord injury (SCI).Main methodsAdult female Sprague–Dawley rats with Th9–10 spinal cord transection were used. In SCI rats with or without 5 mg/day of naftopidil for 4 weeks, bladder activity was examined via continuous cystometry. In other SCI rats, bladder activity was examined before and after intrathecal injection of naftopidil. In addition, bladder wall histology was compared between SCI rats with or without oral administration of naftopidil for 4 weeks.Key findingsOral administration of naftopidil decreased the number of non-voiding contractions (NVCs). Intrathecal injection of naftopidil prolonged the interval between voiding contractions, decreased the maximum voiding contraction pressure and the number of NVCs, and increased bladder capacity without affecting the residual urine volume. Oral administration of naftopidil also decreased bladder wall fibrosis.SignificanceThe α_1D/A-AR antagonist naftopidil might act on the bladder and spinal cord to improve detrusor hyperreflexia in the storage state in SCI female rats. Naftopidil also suppressed bladder wall fibrosis, suggesting that it may be effective for the treatment of neurogenic lower urinary tract dysfunction after SCI.     

Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.     

Indirect female choice mediated by sex pheromones in the hermit crab Pagurus filholi

Males of the hermit crab Pagurus filholi perform precopulatory guarding behavior, and solitary males often show aggressive behavior to take away guarded females. Males behave coercively while guarding females, so direct mate choice by females seems difficult in such a situation. By performing several experiments we examined possible indirect female choice of hermit crab. Males were attached to a shell by their left cheliped to look like guarding pairs (fake guarding pairs). The shells were filled with cotton containing either seawater or pheromone water. The fake guarding pair with only seawater caused male–male combat in 60% of trials whereas with pheromone water combats occurred in 88% of trials. Mean duration of male–male combat was significantly longer in trials with drops of seawater containing pheromones than in those without pheromones. These results suggest guarding pairs themselves cause male–male combat by visual stimulation, that female sex pheromones have further significant function in the recognition of guarding pairs and intensification of male–male combat, and that females release sex pheromones while they are guarded. As a result of the combat, the larger male ended up guarding a female. This strongly suggests that females choose males indirectly by exploiting male–male competition induced by sex pheromones under male coercive behavior.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.