Greenhouse Gas Budget of a Cool-Temperate Deciduous Broad-Leaved Forest in Japan Estimated Using a Process-Based Model

A terrestrial ecosystem model, called the Vegetation Integrative Simulator for Trace gases model (VISIT), which fully integrates biogeochemical carbon and nitrogen cycles, was developed to simulate atmosphere–ecosystem exchanges of greenhouse gases (CO₂, CH₄, and N₂O), and to determine the global warming potential (GWP) taking into account the radiative forcing effect of each gas. The model was then applied to a cool-temperate deciduous broad-leaved forest in Takayama, central Japan (36°08′N, 137°25′E, 1420 m above sea level). Simulations were conducted at a daily time step from 1948 to 2008, using time-series meteorological and nitrogen deposition data. VISIT accurately captured the carbon and nitrogen cycles of this typical Japanese forest, as validated by tower and chamber flux measurements. During the last 10 years of the simulation, the model estimated that the forest was a net greenhouse gas sink, having a GWP equivalent of 1025.7 g CO₂ m⁻² y⁻¹, most of which (1016.9 g CO₂ m⁻² y⁻¹) was accounted for by net CO₂ sequestration into forest biomass regrowth. CH₄ oxidation by the forest soil made a small contribution to the net sink (11.9 g CO₂-eq. m⁻² y⁻¹), whereas N₂O emissions were a very small source (3.2 g CO₂-eq. m⁻² y⁻¹), as expected for a volcanic soil in a humid climate. Analysis of the sensitivity of GWP to changes in temperature, precipitation, and nitrogen deposition indicated that warming temperatures would decrease the size of the sink, mainly as a result of increased CO₂ release due to increased ecosystem respiration.     

Direct assay for alpha-amylase using fluorophore-modified cyclodextrins

A simple assay method for alpha-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae alpha-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4gammaCD, having a gamma-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the alpha-amylase assay. The initial rate of hydrolysis of NC4gammaCD displayed a liner correlation to the concentration of the alpha-amylase. NC4gammaCD was sensitive to the alpha-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.     

Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.Polyamines (PAs) are low-molecular mass aliphatic amines that are present in almost all living organisms. Cellular PA concentrations are governed primarily by the balance between biosynthesis and catabolism. In plants, the major PAs are the diamine putrescine (Put), the triamine spermidine (Spd), and the tetraamines spermine (Spm) and thermospermine (T-Spm; Kusano et al., 2008; Alcázar et al., 2010; Mattoo et al., 2010; Takahashi and Kakehi, 2010; Tiburcio et al., 2014). Put is synthesized from Orn by Orn decarboxylase and/or from Arg by three sequential reactions catalyzed by Arg decarboxylase (ADC), agmatine iminohydrolase, and N-carbamoylputrescine amidohydrolase. Arabidopsis (Arabidopsis thaliana) does not contain an ORNITHINE DECARBOXYLASE gene (Hanfrey et al., 2001) and synthesizes Put from Arg via the ADC pathway. Put is further converted to Spd via an aminopropyltransferase reaction catalyzed by spermidine synthase (SPDS). In this reaction, an aminopropyl residue is transferred to Put from decarboxylated S-adenosyl-Met, which is synthesized by S-adenosyl-Met decarboxylase (SAMDC; Kusano et al., 2008). Spd is then converted to Spm or T-Spm, reactions catalyzed in Arabidopsis by spermine synthase (SPMS; encoded by SPMS) or thermospermine synthase (encoded by Acaulis5 [ACL5]), respectively (Hanzawa et al., 2000; Knott et al., 2007; Kakehi et al., 2008; Naka et al., 2010). A recent review reports that T-Spm is ubiquitously present in the plant kingdom (Takano et al., 2012).The PA catabolic pathway has been extensively studied in mammals. Spm and Spd acetylation by Spd/Spm-N¹-acetyltransferase (Enzyme Commission no. 2.3.1.57) precedes the catabolism of PAs and is a rate-limiting step in the catabolic pathway (Wallace et al., 2003). A mammalian polyamine oxidase (PAO), which requires FAD as a cofactor, oxidizes N¹-acetyl Spm and N¹-acetyl Spd at the carbon on the exo-side of the N⁴-nitrogen to produce Spd and Put, respectively (Wang et al., 2001; Vujcic et al., 2003; Wu et al., 2003; Cona et al., 2006). Mammalian spermine oxidases (SMOs) perform oxidation of the carbon on the exo-side of the N⁴-nitrogen to produce Spd, 3-aminopropanal, and hydrogen peroxide (Vujcic et al., 2002; Cervelli et al., 2003; Wang et al., 2003). Thus, mammalian PAOs and SMOs are classified as back-conversion (BC)-type PAOs.In plants, Spm, T-Spm, and Spd are catabolized by PAO. Plant PAOs derived from maize (Zea mays) and barley (Hordeum vulgare) catalyze terminal catabolism (TC)-type reactions (Tavladoraki et al., 1998). TC-type PAOs oxidize the carbon at the endo-side of the N⁴-nitrogen of Spm and Spd to produce N-(3-aminopropyl)-4-aminobutanal and 4-aminobutanal, respectively, plus 1,3-diaminopropane and hydrogen peroxide (Cona et al., 2006; Angelini et al., 2008, 2010). The Arabidopsis genome contains five PAO genes, designated as AtPAO1 to AtPAO5. Four recombinant AtPAOs, AtPAO1 to AtPAO4, have been homogenously purified and characterized (Tavladoraki et al., 2006; Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012). AtPAO1 to AtPAO4 possess activities that convert Spm (or T-Spm) to Spd, called partial BC, or they convert Spm (or T-Spm) first to Spd and subsequently to Put, called full BC. Ahou et al. (2014) report that recombinant AtPAO5 also catalyzes a BC-type reaction. Therefore, all Arabidopsis PAOs are BC-type enzymes (Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012; Ahou et al., 2014). Four of the seven PAOs in rice (Oryza sativa; OsPAO1, OsPAO3, OsPAO4, and OsPAO5) catalyze BC-type reactions (Ono et al., 2012; Liu et al., 2014a), whereas OsPAO7 catalyzes a TC-type reaction (Liu et al., 2014b). OsPAO2 and OsPAO6 remain to be characterized, but may catalyze TC-type reactions based on their structural similarity with OsPAO7. Therefore, plants possess both TC-type and BC-type PAOs.PAs are involved in plant growth and development. Recent molecular genetic analyses in Arabidopsis indicate that metabolic blocks at the ADC, SPDS, or SAMDC steps lead to embryo lethality (Imai et al., 2004; Urano et al., 2005; Ge et al., 2006). Potato (Solanum tuberosum) plants with suppressed SAMDC expression display abnormal phenotypes (Kumar et al., 1996). It was also reported that hydrogen peroxide derived from PA catabolism affects root development and xylem differentiation (Tisi et al., 2011). These studies indicate that flux through metabolic and catabolic PA pathways is required for growth and development. The Arabidopsis acl5 mutant, which lacks T-Spm synthase activity, displays excessive differentiation of xylem tissues and a dwarf phenotype, especially in stems (Hanzawa et al., 2000; Kakehi et al., 2008, 2010). An allelic ACL5 mutant (thickvein [tkv]) exhibits a similar phenotype as that of acl5 (Clay and Nelson, 2005). These results indicate that T-Spm plays an important role in Arabidopsis xylem differentiation (Vera-Sirera et al., 2010; Takano et al., 2012).Here, we demonstrate that Arabidopsis pao5 mutants contain 2-fold higher T-Spm levels and exhibit aerial tissue growth retardation approximately 50 d after sowing compared with that of wild-type plants. Growth inhibition of pao5 stems and leaves at an early stage of development is induced by growth on media containing low T-Spm concentrations. Complementation of pao5 with AtPAO5 rescues T-Spm-induced growth inhibition. We confirm that recombinant AtPAO5 catalyzes BC of T-Spm (or Spm) to Spd. Our data strongly suggest that endogenous T-Spm levels in Arabidopsis are fine tuned, and that AtPAO5 regulates T-Spm homeostasis through a T-Spm oxidation pathway.     

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain

The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM.     

SOD1 (copper/zinc superoxide dismutase) deficiency drives amyloid β protein oligomerization and memory loss in mouse model of Alzheimer disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(ε)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.     

Phylogenetic analysis of urease-positive thermophilic Campylobacter (UPTC) strains based on the molecular characterization of the flaA gene

Molecular cloning, nucleotide sequencing, and characterization of the flaA gene from additional isolates of urease-positive thermophilic Campylobacter (UPTC) were performed. These isolates were obtained from the natural environment in Northern Ireland (n?=?9 from mussels) and in England (n?=?1 from sea water). All isolates carried the shorter flaA gene, [open reading frames (ORFs), 1,461 to 1,503?base pairs], without any internal termination codons, and did not carry any flaA pseudogenes. The UPTC isolates were well discriminated by the neighbor joining (NJ) phylogenetic tree constructed based on the putative flaA genes ORFs nucleotide sequence information. In addition, the NJ tree constructed based on the flaA-short variable region sequence information discriminated the Campylobacter lari isolates with a similar degree of discrimination power.     

Glycosylphosphatidylinositol anchor-dependent stimulation pathway required for generation of baculovirus-derived recombinant scrapie prion protein

The pathogenic isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP(Sc) seed catalyzes the structural conversion of endogenous PrP(C) into nascent PrP(Sc) in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP(res)) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP(Sc) by PMCA. However, the biological properties of PrP(Sc) obtained by in vivo transmission of recPrP(res) are unique or different from those of PrP(Sc) used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP(Sc). In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP(C) substrate for amplification of the mouse scrapie prion strain Chandler, and PrP(Sc) that accumulated in mice inoculated with Bac-PrP(res) had biochemical and pathological properties very similar to those of the PrP(Sc) seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP(res), the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP(Sc). Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.     

Effects of Candesartan on Electrical Remodeling in the Hearts of Inherited Dilated Cardiomyopathy Model Mice

Inherited dilated cardiomyopathy (DCM) is characterized by dilatation and dysfunction of the ventricles, and often results in sudden death or heart failure (HF). Although angiotensin receptor blockers (ARBs) have been used for the treatment of HF, little is known about the effects on postulated electrical remodeling that occurs in inherited DCM. The aim of this study was to examine the effects of candesartan, one of the ARBs, on cardiac function and electrical remodeling in the hearts of inherited DCM model mice (TNNT2 ΔK210). DCM mice were treated with candesartan in drinking water for 2 months from 1 month of age. Control, non-treated DCM mice showed an enlargement of the heart with prolongation of QRS and QT intervals, and died at t_1/2 of 70 days. Candesartan dramatically extended the lifespan of DCM mice, suppressed cardiac dilatation, and improved the functional parameters of the myocardium. It also greatly suppressed prolongation of QRS and QT intervals and action potential duration (APD) in the left ventricular myocardium and occurrence of ventricular arrhythmia. Expression analysis revealed that down-regulation of Kv4.2 (I_to channel protein), KChIP2 (auxiliary subunit of Kv4.2), and Kv1.5 (I_Kur channel protein) in DCM was partially reversed by candesartan administration. Interestingly, non-treated DCM heart had both normal-sized myocytes with moderately decreased I_to and I_Kur and enlarged cells with greatly reduced K⁺ currents (I_to, I_Kur I_K1 and I_ss). Treatment with candesartan completely abrogated the emergence of the enlarged cells but did not reverse the I_to, and I_Kur in normal-sized cells in DCM hearts. Our results indicate that candesartan treatment suppresses structural remodeling to prevent severe electrical remodeling in inherited DCM.     

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.