Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle

We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

Plagiorchis muris infection in Apodemus agrarius from northern Gyeonggi-do (Province) near the demilitarized zone

The small intestines of 6 species of rodents and 1 species of insectivore were examined seasonally for Plagiorchis muris infection in 3 different localities in northern Gyeonggi-do (Province), near the demilitarized zone (DMZ). A total of 1,496 animals, including 1,366 Apodemus agrarius, 54 Crocidura lasiura (insectivore), 32 Mus musculus, 28 Micronytus fortis, 9 Eothenomys regulus, 6 Micronys minutus, and 3 Cricetulus triton, were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804) and Pocheon-gun (343) at 3-mo intervals from December 2004 to September 2005. A total of 1,647 P. muris were collected from 72 (5.3%) A. agrarius. The infection rate was the highest in Pocheon-gun (8.2%), followed by Yeoncheon-gun (5.0%) and Paju-shi (4.2%). A higher infection rate was observed in A. agrarius captured during September (19.4%) than those captured during December (3.0%), June (2.6%), or April (0%). However, the worm burden was the highest in June (av. 32.1/animal), followed by September (24.7), December (4.0), and April (0). None of the other animal species were found infected with P. muris. The results reveal that A. agrarius is a natural definitive host for P. muris, and infection rates and worm burdens vary seasonally and geographically.     

Plant stem cell research is uncovering the secrets of longevity and persistent growth

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project ‘Principles of pluripotent stem cells underlying plant vitality’ was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Spectroscopic investigations of Er(3+) :CdO-Bi(2) O(3) -B(2) O(3) glasses

This article reports on the optical properties of Er³⁺ ions doped CdO–Bi₂O₃–B₂O₃ (CdBiB) glasses. The materials were characterized by optical absorption and emission spectra. By using Judd–Ofelt theory, the intensity parameters Ω_λ (λ = 2, 4, 6) and also oscillatory strengths were calculated from the absorption spectra. The results were used to compute the radiative properties of Er³⁺:CdBiB glasses. The concentration quenching and energy transfer from Yb³⁺–Er³⁺ were explained. The stimulated emission cross‐section, full width at half maximum (FWHM) and FWHM × values are also calculated for all the Er³⁺:CdBiB glasses. Copyright © 2011 John Wiley & Sons, Ltd.     

The Guts of Herbivorous Invertebrates: Promising Sources of Diverse Microorganisms with Unique Hemicellulolytic Systems

Invertebrates including insects are heterotrophic organisms and widely distributed in ecosystems. Due to their superior ability to digest various types of plant biomass taken as foods, some herbivorous invertebrates have attracted a great deal of industrial attention because such organisms include diverse cellulolytic and hemicellulolytic symbionts in their gut. Recent studies have shown that some of gut microorganisms of herbivores possess one or more extracellular fibrolytic enzymes with unique functions, which can be exploited as useful biocatalysts in various bioindustrial fields. Specifically, microbial hemicellulases with favorable biocatalytic activities are expected to be used for the development of excellent animal feed additives, production of prebiotics such as xylo‐ and mannooligosaccharides, and pretreatment of lignocellulosic biomass for the preparation of fermentable sugars. Here, we review our recent studies accomplished on several hemicellulolytic bacteria isolated from the guts of invertebrates and their glycoside hydrolases such as endo‐β‐1,4‐xylanases and endo‐β‐1,4‐mannanases.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

Peripheral Afferent Mechanisms Underlying Acupuncture Inhibition of Cocaine Behavioral Effects in Rats

Administration of cocaine increases locomotor activity by enhancing dopamine transmission. To explore the peripheral mechanisms underlying acupuncture treatment for drug addiction, we developed a novel mechanical acupuncture instrument (MAI) for objective mechanical stimulation. The aim of this study was to evaluate whether acupuncture inhibition of cocaine-induced locomotor activity is mediated through specific peripheral nerves, the afferents from superficial or deep tissues, or specific groups of nerve fibers. Mechanical stimulation of acupuncture point HT7 with MAI suppressed cocaine-induced locomotor activity in a stimulus time-dependent manner, which was blocked by severing the ulnar nerve or by local anesthesia. Suppression of cocaine-induced locomotor activity was elicited after HT7 stimulation at frequencies of either 50 (for Meissner corpuscles) or 200 (for Pacinian corpuscles) Hz and was not affected by block of C/Aδ-fibers in the ulnar nerve with resiniferatoxin, nor generated by direct stimulation of C/Aδ-fiber afferents with capsaicin. These findings suggest that HT7 inhibition of cocaine-induced locomotor activity is mediated by A-fiber activation of ulnar nerve that originates in superficial and deep tissue.     

Biochemical characterization of cultivated Cordyceps bassiana mycelia and fruiting bodies by 1H nuclear magnetic resonance spectroscopy

In this study, nuclear magnetic resonance techniques coupled with multivariate data analysis were used for the metabolic profiling of mycelia and fruiting bodies of the entomopathogenic fungi, Cordyceps bassiana according to developmental stages. A direct extraction method using two deuterated solvents of D₂O and CDCl₃ was used to investigate the relative levels of identified metabolites in each extraction condition in the mycelium and fruiting body formation stages. There was a clear separation among mycelia and fruiting bodies with various developmental stages in partial least-squares discriminant analysis (PLS-DA) derived score plots. During the transition from mycelia to fruiting bodies, the major metabolic change observed was the conversion of glucose to mannitol, and beauvericin to phenylalanine and 1-hydroxyisovaleric acid. In the developmental stages of fruiting bodies studied, there was a clear separation between stage 3 and the other stages in PLS-DA derived score plots. Nineteen compounds including 13 amino acids, 2 nucleosides, 3 organic acids, and glucose showed the highest levels in stage 3 fruiting bodies. The flavonoid content in the fruiting bodies showed similar levels during stages 1, 2, and 3, whereas the level at stage 4 was significantly decreased compared to the other stages. Results suggest that the fruiting body of C. bassiana is richer in natural resources at stage 3 compared to the other fruiting body stages due to its high abundance of compounds including total flavonoids. The metabolome information acquired in this study can be useful criteria for the quality control of commercial use of C. bassiana.