Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.     

Ras guanyl nucleotide releasing protein 2 affects cell viability and cell–matrix adhesion in ECV304 endothelial cells

Ras guanyl nucleotide releasing proteins (RasGRPs) are guanine nucleotide exchange factors that activate Ras and Rap. We recently reported that xrasgrp2, which is a homolog of the human rasgrp2, plays a role in vasculogenesis and/or angiogenesis during early development of Xenopus embryos. However, the function of RasGRP2 in human vascular endothelium remains unknown. Therefore we aimed to analyze the function of human RasGRP2 in vascular endothelial cells. RasGRP2 overexpression did not increase Ras activation. However, it slightly increased Ras expression and increased proliferation in ECV304 cells. Furthermore, RasGRP2 overexpression increased Rap1 activation and cell–matrix adhesion in ECV304 cells. These data demonstrate that RasGRP2 increases cell viability and cell–matrix adhesion through increased Ras expression and Rap1 activation, respectively, in endothelial cells.     

Butyric acid retention in gingival tissue induces oxidative stress in jugular blood mitochondria

Butyric acid (BA) is a major extracellular metabolite produced by anaerobic periodontopathic bacteria and is commonly deposited in the gingival tissue. BA induces mitochondrial oxidative stress in vitro; however, its effects in vivo were never elucidated. Here, we determined the effects of butyric acid retention in the gingival tissues on oxidative stress induction in the jugular blood mitochondria. We established that BA injected in the rat gingival tissue has prolonged retention in gingival tissues. Blood taken at 0, 60, and 180 min after BA injection was used for further analysis. We isolated blood mitochondria, verified its purity, and measured hydrogen peroxide (H₂O₂), heme, superoxide (SOD), and catalase (CAT) to determine BA effects. We found that H₂O₂, heme, SOD, and CAT levels all increased after BA injection. This would insinuate that mitochondrial oxidative stress was induced ascribable to BA.     

Oral supplementation of Bifidobacterium longum strain BR-108 alters cecal microbiota by stimulating gut immune system in mice irrespectively of viability

Effect on cecal microbiota and gene expression of various cytokines in ileal Peyer’s patches and cecal tissues were compared between viable and heat-killed Bifidobacterium longum strain BR-108 (BR-108) using a mouse model. Irrespectively of viability, oral supplementation of BR-108 altered the cecal microbiota and stimulated gene expression of cytokines such as IL-6 and IL-10 in ileal Peyer’s patches and cecal tissue of mice. In addition, BR-108 supplementation significantly affected the relative abundance of bacterial genera and family, Oscillospira, Bacteroides and S24-7. The abundance of these bacterial genera and family strongly correlated with gene expression induced by BR-108. This study demonstrated that the effect of heat-killed BR-108 on the mouse cecal microbiota is similar to that of viable BR-108, most likely due to stimulation of the gut immune system by both heat-killed and viable BR-108 is also similar.     

Lake restoration by biomanipulation using piscivore and Daphnia stocking; results of the biomanipulation in Japan

Biomanipulation has been employed in numerous locations throughout the world as a means for reducing phytoplankton biomass; however, it has not been employed very often in Japan. A common approach involves the introduction of piscivorous fish to reduce the abundance of planktivorous fish. In our study, to first apply biomanipulation, we stocked Lake Shirakaba (a high-altitude, protected area in a park) in central Japan with rainbow trout fingerlings and cladoceran Daphnia (Daphnia galeata) in 2000. A “pre-biomanipulation” data set (1997–1999) and “a post-biomanipulation” data set (2000–2006) allowed us to evaluate the lake's response to biomanipulation. After the biomanipulation, zoo-planktivorous pond smelt disappeared and a large population of Daphnia had been established, which substantially reduced the number of the previously dominant small cladocerans and rotifers. Water transparency increased from about 2 m (before biomanipulation) to more than 4 m (after biomanipulation). Reductions in algal biomass and increased transparency led to expansion of the submerged macrophyte Elodea nuttallii. Total phosphorus concentrations declined as well over this time period. Based on these results, we concluded that biomanipulation using piscivore and Daphnia stocking succeeded in improving lake water quality by reducing algal abundance and providing favorable conditions for the establishment of rooted plants.     

Succinate accumulation in pig large intestine during antibiotic-associated diarrhea and the constitution of succinate-producing flora

Succinate was the major organic acid detected in the hindgut content of pigs suffering from antibiotic-associated diarrhea. Antibiotic-associated diarrhea was induced by an oral dose of polymyxin B sulfate (3,000,000 units/day) or an intramuscular injection of enrofloxacin (0.6 g enrofloxacin/day). In the large intestine of enrofloxacin-treated pigs, Gram-negative facultative anaerobic rods phylogenetically related to Escherichia coli and Gram-positive facultative anaerobic non-spore-forming rods phylogenetically related to Lactobacilli were isolated as succinate producers. Succinate-producing Lactobacilli were only isolated as the succinate producer in polymyxin B sulfate-treated pigs. In contrast to antibiotic-associated diarrhea pigs, bacteria belonging to Bacteroidaceae, Fusobacteria, and Enterobacteriaceae were detected as succinate producers in a non-treated pig. In antibiotic-associated diarrhea conditions, antibiotic-resistant Enterobacteria, E. coli in particular, and Lactobacilli may contribute to an abnormal succinate accumulation and may affect water absorption in the hindgut that relates to an expression of antibiotic-associated diarrhea.     

Temporal and Motor Representation of Rhythm in Fronto-Parietal Cortical Areas: An fMRI Study

When sounds occur with temporally structured patterns, we can feel a rhythm. To memorize a rhythm, perception of its temporal patterns and organization of them into a hierarchically structured sequence are necessary. On the other hand, rhythm perception can often cause unintentional body movements. Thus, we hypothesized that rhythm information can be manifested in two different ways; temporal and motor representations. The motor representation depends on effectors, such as the finger or foot, whereas the temporal representation is effector-independent. We tested our hypothesis with a working memory paradigm to elucidate neuronal correlates of temporal or motor representation of rhythm and to reveal the neural networks associated with these representations. We measured brain activity by fMRI while participants memorized rhythms and reproduced them by tapping with the right finger, left finger, or foot, or by articulation. The right inferior frontal gyrus and the inferior parietal lobule exhibited significant effector-independent activations during encoding and retrieval of rhythm information, whereas the left inferior parietal lobule and supplementary motor area (SMA) showed effector-dependent activations during retrieval. These results suggest that temporal sequences of rhythm are probably represented in the right fronto-parietal network, whereas motor sequences of rhythm can be represented in the SMA-parietal network.     

Dynamic intracellular reorganization of cytoskeletons and the vacuole in defense responses and hypersensitive cell death in plants

Plants have evolved various means for controlled and organized cell destruction, known as programmed cell death (PCD). In plant immune responses against microbial infection, hypersensitive cell death as a form of PCD is a crucial event to prevent the spread of biotrophic pathogens. Recent live cell imaging techniques have revealed dynamic features and significant roles of cytoskeletons and the vacuole during defense responses and the PCD. Actin microfilaments (MFs) focus on the infection sites and function as tracks for the polar transport of antimicrobial materials. To accomplish hypersensitive cell death, further dynamic changes in cytoskeletons are induced. MFs play a role in the structural and functional regulation of the vacuole, leading to execution of the PCD. We here overview spatiotemporal dynamic changes in the cytoskeletons and the vacuoles triggered by signals from pathogens, and propose a hypothetical model for MF-regulated vacuole-mediated PCD in plant immunity.     

Phosphorylation by Pho85 cyclin-dependent kinase acts as a signal for the down-regulation of the yeast sphingoid long-chain base kinase Lcb4 during the stationary phase

Sphingoid long-chain base 1-phosphates (LCBPs) act as bioactive lipid molecules in eukaryotic cells. In yeast, LCBPs are synthesized mainly by the long-chain base kinase Lcb4p. Until now, the regulatory mechanism for Lcb4p has been unclear. In the present study, we found that Lcb4p is post-translationally modified by phosphorylation. Using a protein kinase mutant yeast collection, we further demonstrated that the cyclin-dependent kinase Pho85p is involved in this phosphorylation. Pho85p functions in a number of cellular processes, especially in response to environmental changes. Two of 10 Pho85p cyclins, Pcl1p and Pcl2p had overlapping functions in the phosphorylation of Lcb4p. Site-directed mutagenesis identified the phosphorylation sites in Lcb4p as Ser(451) and Ser(455). Additionally, pulse-chase experiments revealed that Lcb4p is degraded via the ubiquitin-dependent pathway. The protein was stabilized in Deltapho85 cells, suggesting that phosphorylation acts as a signal for the degradation. Lcb4p is down-regulated in the stationary phase of cell growth, and both phosphorylation and ubiquitination appear to be important for this process. Moreover, we demonstrated that Lcb4p is delivered to the vacuole for degradation via the multivesicular body. Since forced accumulation of LCBPs results in prolonged growth during the stationary phase, down-regulation of Lcb4p may be physiologically important for proper cellular responses to nutrient deprivation.