In situ expression of the mitochondrial ATPase6 gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles on embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to identify genes expressed at its beginning. Adenosine triphosphate synthase subunit a (Atpase6) is one of the highly expressed genes in the E12.0 mandible including tooth germs. In situ hybridization was conducted using the mouse mandibular first molar from E10.5 to E18.0 to determine the precise expression patterns of Atpase6 mRNA in the developing tooth germ. Atpase6 mRNA was strongly expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0 and E13.0. Strong in situ signals were observed in the epithelium at E14.0, and in the enamel organ excluded the area of the primary enamel knot at E15.0. Atpase6 was strongly expressed in the inner enamel epithelium, the adjacent stratum intermedium, and the outer enamel epithelium in the cervical loops from E16.0 to E18.0. In addition, strong Atpase6 signals were coincidently demonstrated in various developing cranio-facial organs. These results suggest that Atpase6 participates in the high energy-utilizing functions of the cells related to the initiation and the development of the tooth germ as well as those of the other cranio-facial organs.     

Genetically-engineered pig-to-baboon liver xenotransplantation: histopathology of xenografts and native organs

Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (α1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4–7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4–7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.     

Testosterone deficiency accelerates neuronal and vascular aging of SAMP8 mice: protective role of eNOS and SIRT1

Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8. Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD(+)-dependent deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in the prevention of vascular and neuronal aging.     

Cytokine and chemokine responses in pediatric patients with severe pneumonia associated with pandemic A/H1N1/2009 influenza virus

Severe pneumonia and leukocytosis are characteristic, frequently observed, clinical findings in pediatric patients with pandemic A/H1N1/2009 influenza virus infection. The aim of this study was to elucidate the role of cytokines and chemokines in complicating pneumonia and leukocytosis in patients with pandemic A/H1N1/2009 influenza virus infection. Forty‐seven patients with pandemic A/H1N1/2009 influenza virus infection were enrolled in this study. Expression of interleukin (IL)‐10 (P = 0.027) and IL‐5 (P = 0.014) was significantly greater in patients with pneumonia than in those without pneumonia. Additionally, serum concentrations of interferon‐γ (P = 0.009), tumor necrosis factor‐α (P = 0.01), IL‐4 (P = 0.024), and IL‐2 (P = 0.012) were significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without neutrophilic leukocytosis. Of the five serum chemokine concentrations assessed, only IL‐8 was significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without leukocytosis (P = 0.001). These cytokines and chemokines may play important roles in the pathogenesis of childhood pneumonia associated with A/H1N1/2009 influenza virus infection.     

Water channel proteins in the inner ear and their link to hearing impairment and deafness

The inner ear is a fluid-filled sensory organ that transforms mechanical stimuli into the senses of hearing and balance. These neurosensory functions depend on the strict regulation of the volume of the two major extracellular fluid domains of the inner ear, the perilymph and the endolymph. Water channel proteins, or aquaporins (AQPs), are molecular candidates for the precise regulation of perilymph and endolymph volume. Eight AQP subtypes have been identified in the membranous labyrinth of the inner ear. Similar AQP subtypes are also expressed in the kidney, where they function in whole-body water regulation. In the inner ear, AQP subtypes are ubiquitously expressed in distinct cell types, suggesting that AQPs have an important physiological role in the volume regulation of perilymph and endolymph. Furthermore, disturbed AQP function may have pathophysiological relevance and may turn AQPs into therapeutic targets for the treatment of inner ear diseases. In this review, we present the currently available knowledge regarding the expression and function of AQPs in the inner ear. We give special consideration to AQP subtypes AQP2, AQP4 and AQP5, which have been studied most extensively. The potential functions of AQP2 and AQP5 in the resorption and secretion of endolymph and of AQP4 in the equilibration of cell volume are described. The pathophysiological implications of these AQP subtypes for inner ear diseases, that appear to involve impaired fluid regulation, such as Menière's disease and Sj?gren's syndrome, are discussed.     

Novel use of burrow casting as a research tool in deep-sea ecology

Although the deep sea is the largest ecosystem on Earth, its infaunal ecology remains poorly understood because of the logistical challenges. Here we report the morphology of relatively large burrows obtained by in situ burrow casting at a hydrocarbon-seep site and a non-seep site at water depths of 1173 and 1455 m, respectively. Deep and complex burrows are abundant at both sites, indicating that the burrows introduce oxygen-rich sea water into the deep reducing substrate, thereby influencing benthic metabolism and nutrient fluxes, and providing an oxic microhabitat for small organisms. Burrow castings reveal that the solemyid bivalve Acharax johnsoni mines sulphide from the sediment, as documented for related shallow-water species. To our knowledge, this is the first study to examine in situ burrow morphology in the deep sea by means of burrow casting, providing detailed information on burrow structure which will aid the interpretation of seabed processes in the deep sea.     

Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca2+ and reactive oxygen species

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network.     

Chiral HPLC Separation,Absolute Structural Elucidation,and Determination of Stereochemical Stability of trans‐Bis[2‐(2‐pyridinyl)aminophenolato] Cyclotriphosphazene

Chiral high‐performance liquid chromatography (HPLC) separation of trans‐bis[2‐(2‐pyridyl)aminophenolato] dichlorocyclotriphosphazene 1 was achieved and the absolute configuration of (+)-1 was assigned to be S,S by single‐crystal X‐ray structural analysis. The optically pure 1,2‐diphenyl‐1,2‐ethanediolate derivatives (+)‐ 2a and (?)‐ 2b were synthesized by the reactions of (+)-1 and (-)-1 with (R,R)‐hydrobenzoin, respectively, in refluxing toluene in the presence of an excess amount of triethylamine and a catalytic amount of 4‐(dimethylamino)pyridine. The racemization of the enantiomers of 1 and the epimerization of diastereomers of 2 were not observed in refluxing toluene neither under acidic nor basic conditions. The stereochemistry of (+)-1 was confirmed by the crystal structure of (+)‐ 2a and bis[(4‐methyl‐2‐pyridyl)oxy]cyclotriphosphazene (+)-3 derived from (+)-1 . Chirality 28:556–561, 2016. © 2016 Wiley Periodicals, Inc.     

Mismatch base pair detection by fluorescence spectral change upon addition of metal cation--toward efficient analysis of single nucleotide polymorphism

Addition of mercury (II) cation to fluorescent-labeled duplex involving a T:T mismatch base pair and silver (I) cation to fluorescent-labeled duplex involving a C:C mismatch base pair significantly changed the fluorescence intensity, but no significant change in the fluorescence intensity was observed for duplexes involving the other base pairs. The fluorescence spectral change upon addition of the metal cation can discriminate T:T and C:C mismatch base pairs from the other base pairs. Our results certainly support the idea that the fluorescence spectral change upon addition of the metal cation could be a convenient strategy for the mismatch base pair detection by the heteroduplex analysis, and may eventually lead to progress in single nucleotide polymorphism genotyping.