Extraction of arbutin and its comparative content in branches,leaves, stems,and fruits of Japanese pear Pyrus pyrifolia cv. Kousui

Arbutin is a tyrosinase inhibitor and is extensively used as a human skin-whitening agent. This study investigated the optimum conditions for extracting arbutin by ultrasonic homogenization from discarded branches pruned from Japanese pear (Pyrus pyrifolia cv. Kousui) trees. The arbutin content was measured in the branches and also in the leaves, stems, fruit peel, and fruit flesh.     

MK-801 alters diaphragmatic activities in unanesthetized rats differently between normoxia and hypoxia

This study was designed to examine how systemic administration of an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, altered respiratory timing in unanesthetized rats under normoxia and hypoxia. To detect fine changes in inspiratory time (TI) and expiratory time (TE), and cycle duration (TTOT), we prepared a diaphragmatic electromyogram (EMGdia). Diaphragm electrodes and arterial and venous catheters were inserted into Wistar rats (n = 8) under pentobarbital anesthesia. The next day, EMGdia was recorded before and after intravenous administration of MK-801 (3 mg/kg) under normoxia and hypoxia (12% O2) without anesthesia, and the respiratory timing (TI, TE, TTOT), respiratory frequency (fR), and amplitude of the integrated EMGdia were measured. Arterial blood gases (ABGs), mean arterial pressure (MAP), and heart rate (fH) were also measured with the EMGdia. Under normoxia, MK-801 increased fR owing to a significant decrease in TE, and elevated both MAP and fH. Under hypoxia, MK-801 suppressed an increase in fR owing to a significant increase in TI, and did not accelerate fH. In both gaseous conditions, on ABGs, MK-801 did not alter partial pressure of O2 (PaO2) or CO2 (PaCO2), and slightly decreased pH (but not less than 7.4). MK-801 significantly decreased hypoxic response (%change from normoxia) in fR, and increased that in EMGdia amplitude, and did not alter a total ventilatory index (fRxEMGdia amplitude). The results suggest that an NMDA receptor-mediated mechanism partially determines fR through significant alterations in respiratory timing, particularly in which the hypoxic ventilatory response was obtained in unanesthetized rats.     

Evidence for multiple retroposition events and gene evolution in the ADP/ATP translocase gene family in Ranid frogs

Analysis of partial ADP/ATP Translocase gene (Aat) sequences from 11 species of Ranid frogs (Amphibia: Ranidae) identified multiple Aat gene copies. All frog species examined had at least one of the 2 Aat gene copies differing mainly by presence or absence of an approximately 85-bp intron sequence. The sequence data suggest that the gene variant with intron is the ancestral Aat form, whereas the other variants are retroposed copies. Surprisingly, a phylogenetic analysis suggests that multiple reptroposition events have taken place in the Ranid frogs. An analysis including sequences from Drosophila, zebra fish, Xenopus, mouse, and human indicated that all the frog Aat sequences form a monophyletic group with mammalian X-chromosomal Ant2 as a sister unit. Furthermore, the selection test suggested that different variants of the Aat gene appear to have evolved under different modes of evolution (viz., neutral, purifying, and positive selection), and the evolutionary history of different AAT variants appears to differ even among different species.     

Kinetic proofreading of ligand-FcepsilonRI interactions may persist beyond LAT phosphorylation

Cells may discriminate among ligands with different dwell times for receptor binding through a mechanism called kinetic proofreading in which the formation of an activated receptor complex requires a progression of events that is aborted if the ligand dissociates before completion. This mechanism explains how, at equivalent levels of receptor occupancy, a rapidly dissociating ligand can be less effective than a more slowly dissociating analog at generating distal cellular responses. Simple mathematical models predict that kinetic proofreading is limited to the initial complex; once the signal passes to second messengers, the dwell time no longer regulates the signal. This suggests that an assay for kinetic proofreading might be used to determine which activation events occur within the initial signaling complex. In signaling through the high affinity IgE receptor FcepsilonRI, the transmembrane adaptor called linker for activation of T cells (LAT) is thought to nucleate a distinct secondary complex. Experiments in which the concentrations of two ligands with different dwell times are adjusted to equalize the level of LAT phosphorylation in rat basophilic leukemia 2H3 cells show that Erk2 phosphorylation, intracellular Ca(2+), and degranulation exhibit kinetic proofreading downstream of LAT phosphorylation. These results suggest that ligand-bound FcepsilonRI and LAT form a complex that is required for effective signal transmission.     

Natural genetic variation in Arabidopsis: tools, traits and prospects for evolutionary ecology

BACKGROUND: The model plant Arabidopsis thaliana (Arabidopsis) shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, the potential of Arabidopsis for molecular genetic analysis of this natural variation has increased dramatically in recent years. SCOPE: Advanced genomics has accelerated molecular phylogenetic analysis and gene identification by quantitative trait loci (QTL) mapping and/or association mapping in Arabidopsis. In particular, QTL mapping utilizing natural accessions is now becoming a major strategy of gene isolation, offering an alternative to artificial mutant lines. Furthermore, the genomic information is used by researchers to uncover the signature of natural selection acting on the genes that contribute to phenotypic variation. The evolutionary significance of such genes has been evaluated in traits such as disease resistance and flowering time. However, although molecular hallmarks of selection have been found for the genes in question, a corresponding ecological scenario of adaptive evolution has been difficult to prove. Ecological strategies, including reciprocal transplant experiments and competition experiments, and utilizing near-isogenic lines of alleles of interest will be a powerful tool to measure the relative fitness of phenotypic and/or allelic variants. CONCLUSIONS: As the plant model organism, Arabidopsis provides a wealth of molecular background information for evolutionary genetics. Because genetic diversity between and within Arabidopsis populations is much higher than anticipated, combining this background information with ecological approaches might well establish Arabidopsis as a model organism for plant evolutionary ecology.     

Induction of beating by imposed bending or mechanical pulse in demembranated, motionless sea urchin sperm flagella at very low ATP concentrations

A basic feature of the movement of eukaryotic flagella is oscillation. Although flagellar oscillation is thought to be regulated by a self-regulatory feedback system including the mechanical signal of bending itself, the mechanism regulating the dynein motile activity to produce oscillation is not well understood. To elucidate the mechanism, we developed a new experimental system which allowed us to analyze the conditions necessary for the induction of oscillation. When a mechanical signal of bending or a pulse was applied by micromanipulation to a demembranated motionless sea urchin sperm flagellar axoneme at very low ATP concentrations (1-3 microM), a localized pair of bends was induced. The bend formation was often followed by further responses including propagation of the distal bend of paired bends, growth and propagation of the paired bends, and cyclical beating. The beating was induced at 2.0 microM or higher concentrations of ATP, but appeared even at 1.5 microM ATP if a few muM of ADP was also present. When the proximal half of a flagellum was attached to a microneedle, beating could not be induced in the distal free region at 2 microM ATP. These results suggest that mechanical signal is involved in the mechanism regulating the motile activity of dynein to produce oscillation. Our results also showed that the presence of a small amount of ADP and the axial difference along the flagellum are factors essential for the induction of flagellar oscillation.     

Analysis of tissue-specific differentially methylated regions (TDMs) in humans

Alterations in DNA methylation have been implicated in mammalian development. Hence, the identification of tissue-specific differentially methylated regions (TDMs) is indispensable for understanding its role. Using restriction landmark genomic scanning of six mouse tissues, 150 putative TDMs were identified and 14 were further analyzed. The DNA sequences of the 14 mouse TDMs are analyzed in this study. Six of the human homologous regions show TDMs to both mouse and human and genes in five of these regions have conserved tissue-specific expression: preferential expression in testis. A TDM, DDX4, is further analyzed in nine testis tissues. An increase in methylation of the promoter region is significantly associated with a marked reduction of the gene expression and defects in spermatogenesis, suggesting that hypomethylation of the DDX4 promoter region regulates DDX4 gene expression in spermatogenic cells. Our results indicate that some genomic regions with tissue-specific methylation and expression are conserved between mouse and human and suggest that DNA methylation may have an important role in regulating differentiation and tissue-/cell-specific gene expression of some genes.     

Sox5 Functions as a Fate Switch in Medaka Pigment Cell Development

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).