Primary succession of the warm-temperate broad-leaved forest on a volcanic island, Miyake-jima, Japan

The primary successional sere of a warm-temperate forest was inferred from an age sequence of four basaltic volcanic substrates (16-, 37- and 125-yr-old lava flows, and volcanic ejecta older than 800 years) at several altitudes (ranging from 30 to 550 m) on Miyake-jima Island. The DCA analysis of species composition indicated that the vegetation is patterned by substrate age and altitude. The vegetation successionally changed fromAlnus sieboldiana shrub, toMachilus thunbergii andPrunus speciosa forest, toCastanopsis sieboldii forest at all altitudes. The rate of succession seems to be slower at higher altitudes. The above-ground biomass was 20 kg/m² (160 m a.s.l.) and 12 kg/m² (300 m a.s.l.) on the 125-yr-old lava flow, and 54 kg/m² (100 m a.s.l.) and 16 kg/m² (350 m a.s.l.) on the old ejecta. However, the large difference of above-ground biomass between low versus high altitudes on the old ejecta might not only reflect climatic differences but also past human disturbances at the higher altitude. In spite of the colder climate of Miyake-jima the rate of above-ground biomass accumulation here was considerably faster than that of a Hawaiian montane tropical evergreen forest on the same basaltic lava substrate. We suggest that the faster above-ground biomass development is due to the facilitation effects of N-fixation byAlnus. The inorganic N soil concentration was extremely high on the 37- and 125-yr-old lava flows whereAlnus was dominant. The foliar N concentration inAlnus was 2% irrespective of substrate age. The deposition of N fromAlnus via litterfall would decrease the soil C/N ratio, which in turn facilitates net soil N mineralization and consequently provides an ample supply of inorganic N to plants. N limitation on vegetation development, which is prevalent during the early stage of succession on volcanic lava flows or similar substrates elsewhere, is thus alleviated.     

Structure-function analysis of VPS9-ankyrin-repeat protein (Varp) in the trafficking of tyrosinase-related protein 1 in melanocytes

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking.     

Molecular and genetic characterization of the gene family encoding the voltage-dependent anion channel in Arabidopsis

The voltage-dependent anion channel (VDAC), a major outer mitochondrial membrane protein, is thought to play an important role in energy production and apoptotic cell death in mammalian systems. However, the function of VDACs in plants is largely unknown. In order to determine the individual function of plant VDACs, molecular and genetic analysis was performed on four VDAC genes, VDAC1-VDAC4, found in Arabidopsis thaliana. VDAC1 and VDAC3 possess the eukaryotic mitochondrial porin signature (MPS) in their C-termini, while VDAC2 and VDAC4 do not. Localization analysis of VDAC-green fluorescent protein (GFP) fusions and their chimeric or mutated derivatives revealed that the MPS sequence is important for mitochondrial localization. Through the functional analysis of vdac knockout mutants due to T-DNA insertion, VDAC2 and VDAC4 which are expressed in the whole plant body are important for various physiological functions such as leaf development, the steady state of the mitochondrial membrane potential, and pollen development. Moreover, it was demonstrated that VDAC1 is not only necessary for normal growth but also important for disease resistance through regulation of hydrogen peroxide generation.     

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.     

Regulation of macrophage differentiation and polarization by group IVC phospholipase A₂

Although the cellular function of group IVC phospholipase A(2) (IVC-PLA(2)) remains to be understood, the expression of IVC-PLA(2) in human monocytic THP-1 cells was increased during phorbol ester-induced macrophage differentiation. We therefore examined the role of IVC-PLA(2) in macrophage differentiation using THP-1 cells. Two THP-1 cell lines stably expressing IVC-PLA(2)-specific shRNA were established. Differentiation and maturation into macrophages on treatment with phorbol ester were facilitated by knockdown of IVC-PLA(2) without the compensatory induction of mRNA expression for other group IV and VI PLA(2)s. Furthermore, the enhancement of macrophage differentiation by IVC-PLA(2)-knockdown were abolished by treatment with lysophosphatidylcholine, a metabolite of phospholipids generated by PLA(2)-mediated hydrolysis, indicating that PLA(2) activity is necessary for the inhibition of macrophage differentiation by IVC-PLA(2). Additionally, we found that the differentiation into classically activated M1 macrophage was superior in IVC-PLA(2)-knockdown cells, whereas the differentiation into alternatively activated M2 macrophage was suppressed by IVC-PLA(2)-knockdown. These findings suggest that IVC-PLA(2) is involved in regulations of macrophage differentiation and macrophage polarization.     

Microsomal prostaglandin E synthase-1 enhances bone cancer growth and bone cancer-related pain behaviors in mice

AimsNonsteroidal anti-inflammatory drugs are a therapeutic modality for chronic cancer pain arising from bone metastases. Chronic administration of a cyclooxygenase (COX)-2 inhibitor is effective to bone cancer-related pain. However, adverse cardiovascular effects have limited COX-2 inhibitor therapy, and elucidation of better targets for blocking prostaglandin (PG) biosynthesis is necessary. Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that catalyzes isomerization of the endoperoxide PGH₂ to PGE₂. To investigate the validity of mPGES-1 as a therapeutic target, we evaluated bone cancer pain-related behaviors in mPGES-1 knockout (PGES-1?/?) mice.Main methodsLewis lung carcinoma cells (LLCCs) were injected into the intramedullary space of the femur of wild-type (WT) and PGES-1?/? mice. Pain-related behaviors were evaluated.Key findingsPGES-1?/? mice exhibited reduced tumor growth in bone marrow compared to WT. The expression of pro-calcitonin gene-related peptide (CGPR) in the dorsal root ganglia of L_1–5 was significantly higher in WT mice at day 14, whereas it was unchanged in mPGES-1 mice. In the observation of pain-related behaviors, mPGES-1?/? mice exhibited significantly fewer spontaneous flinches and their onset was several days later than WT. The appearance of other pain-related behaviors in mPGES-1?/? mice was also delayed as compared to WT. LLCC-injected WT mice treated with a COX-2 inhibitor, celecoxib, exhibited similar temporal changes to mPGES1?/?.SignificanceThe present results suggest that mPGES-1 plays a crucial role in the enhancement of bone cancer growth and bone cancer pain, and that inhibition of mPGES-1 may have clinical utility in the management of bone cancer pain.     

Comprehensive sequence analysis of 24,783 barley full-length cDNAs derived from 12 clone libraries

Full-length cDNA (FLcDNA) libraries consisting of 172,000 clones were constructed from a two-row malting barley cultivar (Hordeum vulgare 'Haruna Nijo') under normal and stressed conditions. After sequencing the clones from both ends and clustering the sequences, a total of 24,783 complete sequences were produced. By removing duplicates between these and publicly available sequences, 22,651 representative sequences were obtained: 17,773 were novel barley FLcDNAs, and 1,699 were barley specific. Highly conserved genes were found in the barley FLcDNA sequences for 721 of 881 rice (Oryza sativa) trait genes with 50% or greater identity. These FLcDNA resources from our Haruna Nijo cDNA libraries and the full-length sequences of representative clones will improve our understanding of the biological functions of genes in barley, which is the cereal crop with the fourth highest production in the world, and will provide a powerful tool for annotating the barley genome sequences that will become available in the near future.     

A new insight into application for barley chromosome addition lines of common wheat: achievement of stigmasterol accumulation

Barley (Hordeum vulgare) has a much higher content of bioactive substances than wheat (Triticum aestivum). In order to investigate additive and/or synergistic effect(s) on the phytosterol content of barley chromosomes, we used a series of barley chromosome addition lines of common wheat that were produced by normal crossing. In determining the plant sterol levels in 2-week-old seedlings and dry seeds, we found that the level of stigmasterol in the barley chromosome 3 addition (3H) line in the seedlings was 1.5-fold higher than that in the original wheat line and in the other barley chromosome addition lines, but not in the seeds. Simultaneously, we determined the overall expression pattern of genes related to plant sterol biosynthesis in the seedlings of wheat and each addition line to assess the relative expression of each gene in the sterol pathway. Since we elucidated the CYP710A8 (cytochrome P450 subfamily)-encoding sterol C-22 desaturase as a key characteristic for the higher level of stigmasterol, full-length cDNAs of wheat and barley CYP710A8 genes were isolated. These CYP710A8 genes were mapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the 3H addition line, indicating that it is responsible for stigmasterol accumulation. Overexpression of the CYP710A8 genes in Arabidopsis increased the stigmasterol content but did not alter the total sterol level. Our results provide new insight into the accumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.     

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.