Investigation of the chiral recognition ability of human carboxylesterase 1 using indomethacin esters

Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation of the chiral recognition ability of hCES1 was attempted using indomethacin esters in which various chiral alcohols were introduced. Indomethacin was condensed with various chiral alcohols to synthesize indomethacin esters. The synthesized esters were hydrolyzed with a human liver microsome (HLM) solution and a human intestine microsome (HIM) solution. High hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis reaction in the HLM solution but not in the HIM solution, and these indomethacin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates of the esters were calculated. The stereoselectivity confirmed in HLM solution was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters in which a phenyl group is bonded next to the ester, the V_max value of the (R) form was 10 times larger than that of the (S) form.     

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a ’shiro’. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

Isolation of Ciliatine (2-Aminoethylphosphonic Acid) from the Bile of the Bovine

To elucidate the formation mechanism of N,N′-dialkylpyrazine cation radical during browning reaction of sugars with amino compounds, main products in an early stage of the reaction were determined quantitatively by TLC and GLC. It was shown that the Schiff base, two-carbon fragmental product of sugar, the free radical and deoxyosone were successively produced prior to the browning. Polarographical measurements indicated that the radical formation was induced by the production of some reducing substances in the reaction mixture. These results suggest that the free radical was formed by the reduction of N,N′-dialkylpyrazinium; a compound, which demonstrated to have a strong activity to browning, might be formed by condensation of two-carbon enaminol followed by oxidation.     

Bone Inner Structure Suggests Increasing Aquatic Adaptations in Desmostylia (Mammalia,Afrotheria)

Background

The paleoecology of desmostylians has been discussed controversially with a general consensus that desmostylians were aquatic or semi-aquatic to some extent. Bone microanatomy can be used as a powerful tool to infer habitat preference of extinct animals. However, bone microanatomical studies of desmostylians are extremely scarce.

Methodology/Principal Findings

We analyzed the histology and microanatomy of several desmostylians using thin-sections and CT scans of ribs, humeri, femora and vertebrae. Comparisons with extant mammals allowed us to better understand the mode of life and evolutionary history of these taxa. Desmostylian ribs and long bones generally lack a medullary cavity. This trait has been interpreted as an aquatic adaptation among amniotes. Behemotops and Paleoparadoxia show osteosclerosis (i.e. increase in bone compactness), and Ashoroa pachyosteosclerosis (i.e. combined increase in bone volume and compactness). Conversely, Desmostylus differs from these desmostylians in displaying an osteoporotic-like pattern.

Conclusions/Significance

In living taxa, bone mass increase provides hydrostatic buoyancy and body trim control suitable for poorly efficient swimmers, while wholly spongy bones are associated with hydrodynamic buoyancy control in active swimmers. Our study suggests that all desmostylians had achieved an essentially, if not exclusively, aquatic lifestyle. Behemotops, Paleoparadoxia and Ashoroa are interpreted as shallow water swimmers, either hovering slowly at a preferred depth, or walking on the bottom, and Desmostylus as a more active swimmer with a peculiar habitat and feeding strategy within Desmostylia. Therefore, desmostylians are, with cetaceans, the second mammal group showing a shift from bone mass increase to a spongy inner organization of bones in their evolutionary history.     

MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.     

Bird carcass detection from integrated trials at multiple wind farms

It is often necessary to estimate the number of wind turbine collision fatalities to assess impacts to birds following construction of wind farms. Detection of bird carcasses at wind turbines in the field is affected by carcass persistence and searcher detection rate. Integrated detection trials, which integrate carcass persistence and searcher detection trials into the periodic fatality search, have been proposed as an effective method for estimating these parameters. The purpose of our study was to test whether and how environmental factors affect integrated detection trial outcomes at multiple wind farms. We conducted this study at 10 wind farms in various environments of Japan. Binary data on trial outcomes in open versus forested areas served as our response variable in a generalized additive mixed model informed by days into trial, carcass body mass, season, whether snow covered the ground, and precipitation. For both ground cover types, days into trial and body mass were included in all the top models, suggesting that these factors most influenced bird carcass detection probability in integrated trials. The best model in open areas included days into trial, body mass, snow, and precipitation, and the best model in forested areas included days into trial, body mass, snow, precipitation, and season. Values of area under the curve indicated high accuracy of the best model for both ground cover types. The survey design needs to be appropriate to the size of the target species and to the environment in which the impacts will occur, such as the site's seasonality, its ground cover, and whether snow will cover the ground. Frequency of post-construction fatality monitoring should also be set cautiously, especially at wind farms located on small-bird migration routes, at wind farms in open areas, in areas with snow-covered ground in winter, or in forested areas during spring and summer because detection probabilities decline fastest under such conditions.     

Bacterial translocation can increase plasma corticosterone and brain catecholamine and indoleamine metabolism

The potential contribution of stress-induced bacterial translocation to the activation of the hypothalamo-pituitary-adrenocortical (HPA) axis and brain biogenic amines was assessed. Mice were restrained for various periods, and brain concentrations of tryptophan, catecholamines, serotonin, and their metabolites, plasma corticosterone, and the translocation of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes, spleen, and liver were measured. Restraint induced the translocation of indigenous gram-positive bacteria in only a small proportion of animals, but translocation of gram-negative bacteria did not occur. Restraint induced short-lived increases in plasma corticosterone and brain amine metabolism, whereas bacterial translocation was slower and persisted long after the HPA axis and neurochemical responses had dissipated. When mice were infected with Salmonella typhimurium, spontaneous translocation occurred and plasma corticosterone, interleukin-6 concentrations, and brain catecholamine and indoleamine metabolism were elevated. These findings indicate that the translocation of indigenous gastrointestinal bacteria did not contribute to the HPA axis and neurochemical changes induced by restraint. However, translocation of nonindigenous S. typhimurium with or without restraint did induce HPA and neurochemical responses.     

Genomic Evidence for Speciation with Gene Flow in Broadcast Spawning Marine Invertebrates

How early stages of speciation in free-spawning marine invertebrates proceed is poorly understood. The Western Pacific abalones, Haliotis discus, H. madaka, and H. gigantea, occur in sympatry with shared breeding season and are capable of producing viable F₁ hybrids in spite of being ecologically differentiated. Population genomic analyses revealed that although the three species are genetically distinct, there is evidence for historical and ongoing gene flow among these species. Evidence from demographic modeling suggests that reproductive isolation among the three species started to build in allopatry and has proceeded with gene flow, possibly driven by ecological selection. We identified 27 differentiation islands between the closely related H. discus and H. madaka characterized by high F_ST and d_A, but not high d_XY values, as well as high genetic diversity in one H. madaka population. These genomic signatures suggest differentiation driven by recent ecological divergent selection in presence of gene flow outside of the genomic islands of differentiation. The differentiation islands showed low polymorphism in H. gigantea, and both high F_ST, d_XY, and d_A values between H. discus and H. gigantea, as well as between H. madaka and H. gigantea. Collectively, the Western Pacific abalones appear to occupy the early stages speciation continuum, and the differentiation islands associated with ecological divergence among the abalones do not appear to have acted as barrier loci to gene flow in the younger divergences but appear to do so in older divergences.     

Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL-IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19922-19927). We have now isolated two other pyruvylated galactose-containing phosphonoglycosphingolipids, named FGL-V and FGL-IIa, from the nervous tissue of Aplysia, and characterized them as [3,4-O-(S-1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3[6'-O-(2- aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6) Gal beta 1----4Glc beta 1----1 ceramide and [3,4,O-(S-1-carboxyethylidene)] Gal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 11----ceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.