An Improved Fluorometric High-Performance Liquid Chromatography Method for Sialic Acid Determination: An Internal Standard Method and Its Application to Sialic Acid Analysis of Human Apolipoprotein E

An improved fluorometric HPLC method for sialic acid determination was developed by employing synthetic N-propionylneuraminic acid (NPNA) as an internal standard. A fixed amount of NPNA was added to a sialoglycoconjugate sample. After hydrolyzing sialioglycoconjugates with diluted sulfuric acid, the released sialic acids and NPNA were derivatized with a fluorogenic compound, 1,2-diamino-4,5-(methylenedioxy)benzene (DMB), followed by fluorometric HPLC. The fluorescent derivative of NPNA was separated from those of N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid, and 2-keto-3-deoxyoctanoate on HPLC. The separation of NPNA derivative on HPLC was not interfered by components of biological samples such as human sera. Using this internal standard method, low amounts of NANA (0.15-1.0 ng) were quantified with the coefficient of variation values below 4%. Using this method, the sialic acid content of human apolipoprotein E was successfully determined. The present method is useful for sensitive and accurate quantification of sialic acids of different molecular species in biological samples.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.     

Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.     

A novel anti-inflammatory role for spleen-derived interleukin-10 in obesity-induced hypothalamic inflammation

Obesity can be associated with systemic low-grade inflammation that contributes to obesity-related metabolic disorders. Recent studies raise the possibility that hypothalamic inflammation contributes to the pathogenesis of diet-induced obesity (DIO), while another study reported that obesity decreases the expression of pro-inflammatory cytokines in spleen. The following study examines the hypothesis that obesity suppresses the splenic synthesis of the anti-inflammatory cytokine, interleukin (IL)-10, thereby resulting in chronic hypothalamic inflammation. The results showed that due to oxidative stress or apoptosis, the synthesis of splenic IL-10 was decreased in DIO when compared with non-obesity rats. Splenectomy (SPX) accelerated DIO-induced inflammatory responses in the hypothalamus. Interestingly, SPX suppressed the DIO-induced increases in food intake and body weight and led to a hypothalamic pro-inflammatory state that was similar to that produced by DIO, indicating that hypothalamic inflammation exerts a dual effect on energy metabolism. These SPX-induced changes were inhibited by the systemic administration of IL-10. Moreover, SPX had no effect on hypothalamic inflammatory responses in IL-10-deficient mice. These data suggest that spleen-derived IL-10 plays an important role in the prevention of hypothalamic inflammation and may be a therapeutic target for the treatment of obesity and hypothalamic inflammation.     

Environmental DNA enables detection of terrestrial mammals from forest pond water

Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues. The results suggested that MiMammal primers are capable of amplifying and distinguishing a diverse group of mammalian species. In addition, analyses of water samples from zoo cages of mammals with known species composition suggested that MiMammal primers could successfully detect mammalian species from water samples in the field. Then, we performed an experiment to detect mammals from natural ecosystems by collecting five 500‐ml water samples from ponds in two cool‐temperate forests in Hokkaido, northern Japan. MiMammal amplicon libraries were constructed using eDNA extracted from water samples, and sequences generated by Illumina MiSeq were subjected to data processing and taxonomic assignment. We thereby detected multiple species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Many previous applications of the eDNA metabarcoding approach have been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even for forest mammal biodiversity surveys.     

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 ± 0.33 × 10⁻¹⁵ mol s⁻¹, 1.38 ± 0.58 × 10⁻¹⁵ mol s⁻¹, and 3.44 ± 2.07 × 10⁻¹⁵ mol s⁻¹, respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).     

Efficient establishment of pure cultures of yellow chanterelle Cantharellus anzutake from ectomycorrhizal root tips,and morphological characteristics of ectomycorrhizae and cultured mycelium

Species of fleshy yellow Cantharellus are known as chanterelles, which are among the most popular wild edible mycorrhizal mushrooms in the world. However, pure culture isolates of Cantharellus are rare. We report an efficient isolation technique of the Japanese golden chanterelle, Cantharellus anzutake, from its ectomycorrhizal root tips. Field-sampled fresh ectomycorrhizal root tips of C. anzutake on various hosts such as pines, spruce, and oaks were vortexed with 0.005% Tween 80 solution, surface sterilized with 1% calcium hypochlorite solution, rinsed with sterilized distilled water, and placed on modified Norkrans’ C (MNC) agar plate medium. Most ectomycorrhizal root tips of C. anzutake produced yellowish mycelial colonies within a few months. In contrast, tissue isolation from basidiomata provided limited cultures of C. anzutake but much contamination of bacteria and molds, even on media that contained antibiotics. The established C. anzutake cultures had clamp connections on the hyphae and contained intracellular oily droplets. These cultured isolates were identified as C. anzutake by sequence analysis of the rRNA internal transcribed spacer (ITS) region and translation elongation factor EF1-alpha (tef-1) genes.     

Overexpression of a rice gene encoding a small C2 domain protein OsSMCP1 increases tolerance to abiotic and biotic stresses in transgenic Arabidopsis

Plant growth and crop production are limited by environmental stress. We used a large population of transgenic Arabidopsis expressing rice full-length cDNAs to isolate the rice genes that improve the tolerance of plants to environmental stress. By sowing T2 seeds of the transgenic lines under conditions of salinity stress, the salt-tolerant line R07047 was isolated. It expressed a rice gene, OsSMCP1, which encodes a small protein with a single C2 domain, a Ca²⁺-dependent membrane-targeting domain. Retransformation of wild-type Arabidopsis revealed that OsSMCP1 is responsible for conferring the salt tolerance. It is particularly interesting that R07047 and newly constructed OsSMCP1-overexpressing Arabidopsis showed enhanced tolerance not only to high salinity but also to osmotic, dehydrative, and oxidative stresses. Furthermore, R07047 showed improved resistance to Pseudomonas syringae. The OsSMCP1 expression in rice is constitutive. Particle-bombardment-mediated transient expression analysis revealed that OsSMCP1 is targeted to plastids in rice epidermal cells. It induced overexpression of several nuclear encoded genes, including the stress-associated genes, in transgenic Arabidopsis. No marked morphological change or growth retardation was observed in R07047 or retransformants. For molecular breeding to improve the tolerance of crops against environmental stress, OsSMCP1 is a promising candidate.     

Characterization of the Replication, Maintenance, and Transfer Features of the IncP-7 Plasmid pCAR1, Which Carries Genes Involved in Carbazole and Dioxin Degradation

Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 × 10⁻¹ and 3 × 10⁻³ per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.