Genetic composition of exotic and native teak (<Emphasis Type="Italic">Tectona</Emphasis><Emphasis Type="Italic">grandis</Emphasis>) in Myanmar as revealed by cpSNP and nrSSR markers

With the rapid fragmentation of tropical forests harboring valuable tree species, conservation of natural genetic resources is an important issue. In Myanmar, teak plantations have been established by Myanmar government since the 1700 s using local Myanmar teak. Commercial plantations have recently been established by the private sector using both exotic and Myanmar teak without consideration of their genetic make-up. If the genetic composition of commercial teak plantations is severely different from that of Myanmar teak, introgression of non-indigenous genes could damage the remaining natural populations. We investigated genetic compositions of commercial plantations using both exotic and Myanmar teak seeds with 10 nuclear simple sequence repeat and three chloroplast single nucleotide polymorphism markers. We then compared the genetic compositions of these populations with those of neighboring native teak forests. The genetic diversity and composition of one exotic plantation using Costa Rican seeds was similar to those of native populations. However, the diversity of the other three exotic plantations was low and their composition was markedly different from those of native populations. Our results suggest that exotic gene flow would cause serious genetic disturbance. Commercial plantations using Myanmar seeds were characterized by relatively high genetic diversity and by many genetic components. These results suggest that these plantations may be established using various seed sources in Myanmar. Given that native teak in Myanmar is geographically structured, native gene pools will be homogenized by gene flow from these commercial plantations. Seed transfer guidelines based on genetic information should be considered in future.     

Pollen dispersal patterns and population persistence in a small isolated population of <Emphasis Type="Italic">Fagus crenata</Emphasis>

The potential of long-distance pollen dispersal and the effects of small population size and population isolation on persistence of Fagus crenata populations were investigated in a small, severely isolated population (the Gofuku-ji population) and two other populations located within 7 km of this population (including 87 adult trees in total). Parentage analysis using 13 microsatellite loci showed that 94 of 100 seedlings derived from seeds collected from the Gofuku-ji population had parent pairs within this population, six had one parent within the population, and four of the six seedlings had alleles that were not detected in any of the three populations, indicating that some pollen is dispersed over distances exceeding 7 km. The estimated expected heterozygosity and effective population size were lower in the Gofuku-ji population than in previously examined large continuous populations. Therefore, levels of genetic diversity within the population may have been reduced by strong genetic drift and limitations of pollen- and seed-mediated gene flow associated with the small size and severe isolation. The contemporary mating pattern estimated at the seedling stage was biased toward outbreeding, which may be explained by possible processes: the level of inbreeding in the adult trees is increased; then, inbreeding frequently occurs but is rarely successful, while outbreeding successfully produces offspring. Additionally, high levels of significant linkage disequilibrium and higher numbers of alleles than expected under mutation–drift equilibrium from analyses of the populations’ evolutionary history suggest that the Gofuku-ji population may have experienced admixture before its severe isolation. Therefore, the persistence of the Gofuku-ji population is being adversely affected by the decrease in population size and severe isolation. Further studies of gene flow via pollen in other populations with various degrees of isolation could enhance our understanding of the effects of population isolation and long-distance pollen dispersal in F. crenata and similar species.     

A novel environmental DNA approach to quantify the cryptic invasion of non‐native genotypes

The invasion of non‐native species that are closely related to native species can lead to competitive elimination of the native species and/or genomic extinction through hybridization. Such invasions often become serious before they are detected, posing unprecedented threats to biodiversity. A Japanese native strain of common carp (Cyprinus carpio) has become endangered owing to the invasion of non‐native strains introduced from the Eurasian continent. Here, we propose a rapid environmental DNA‐based approach to quantitatively monitor the invasion of non‐native genotypes. Using this system, we developed a method to quantify the relative proportion of native and non‐native DNA based on a single‐nucleotide polymorphism using cycling probe technology in real‐time PCR. The efficiency of this method was confirmed in aquarium experiments, where the quantified proportion of native and non‐native DNA in the water was well correlated to the biomass ratio of native and non‐native genotypes. This method provided quantitative estimates for the proportion of native and non‐native DNA in natural rivers and reservoirs, which allowed us to estimate the degree of invasion of non‐native genotypes without catching and analysing individual fish. Our approach would dramatically facilitate the process of quantitatively monitoring the invasion of non‐native conspecifics in aquatic ecosystems, thus revealing a promising method for risk assessment and management in biodiversity conservation.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

Coordination of changes in expression and phosphorylation of eukaryotic elongation factor 2 (eEF2) and eEF2 kinase in hypertrophied cardiomyocytes

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is one of the Ca²⁺/calmodulin-dependent protein kinases. Activated eEF2K phosphorylates its specific substrate, eEF2, which results in inhibition of protein translation. We have recently shown that protein expression of eEF2K was specifically increased in hypertrophied left ventricles (LV) from spontaneously hypertensive rats (SHR). However, phosphorylation state of eEF2K and eEF2 in hypertrophied LV is not determined. In the present study, we examined expression and phosphorylation of eEF2K and eEF2 in LV from SHR as well as the pressure overload (transverse aortic constriction: TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy model. In LV from TAC mice, eEF2K expression was increased as determined by Western blotting. In LV from TAC mice and SHR, eEF2K phosphorylation at Ser366 (inactive site) was decreased. Consistently, eEF2 phosphorylation at Thr56 was increased. In LV from ISO rats, while eEF2K phosphorylation was decreased, eEF2K expression and eEF2 phosphorylation were not different as determined by Western blotting. In the results obtained from immunohistochemistry, however, total eEF2K and phosphorylated eEF2 (at Thr56) localized to cardiomyocytes were increased in LV cardiomyocytes from ISO rats. Accordingly, the increased expression and the decreased phosphorylation of eEF2K and the increased phosphorylation of eEF2 in hypertrophied LV were common to all models in this study. The present results thus suggest that cardiac hypertrophy may be regulated at least partly via eEF2K-eEF2 signaling pathway.     

A high-throughput sequence analysis of Japanese patients revealed 11 candidate genes associated with type 1 autoimmune pancreatitis susceptibility

The pathogenesis of autoimmune pancreatitis is unknown. In the present study we used high-throughput sequencing with next generation sequencing to identify the candidate genes associated with AIP. A total of 27 type 1 AIP patients and 30 healthy blood donors were recruited, and DNA samples were isolated from their mononuclear cells. A high-throughput sequencer with an original custom panel of 1031 genes was used to detect the genetic variants in each sample. Polymorphisms of CACNA1S (c.4642C>T), rs41554316, rs2231119, rs1042131, rs2838171, P2RX3 (c.195delG), rs75639061, SMAD7 (c.624delC) and TOP1 (c.2007delG), were identified as candidate genetic variants in patients with type 1 AIP. P2RX3 and TOP1 were significantly associated with AIP, even after adjusting bay means of Bonferroni's correction. In addition, we also identified eight candidate genetic variants that were associated with the relapse of type 1 AIP, namely: rs1143146, rs1050716, HLA-C (c.759_763delCCCCCinsTCCCG), rs1050451, rs4154112, rs1049069, CACNA1C (c.5996delC) and CXCR3 (c.630_631delGC). Finally polymorphisms of rs1050716 and rs111493987 were identified as candidate genetic variants associated with extra-pancreatic lesions in patients with type 1 AIP. These candidates might be used as markers of AIP susceptibility and could contribute to the pathogenesis of type 1 AIP.     

Lampteromyces bioluminescence : 3. Structure of lampteroflavin, the light emitter in the luminous mushroom, L. japonicus

The structure of lampteroflavin, the light emitter in the luminous mushroom (Lampteromyces japonicus), was determined to be a new riboflavin α- -riboside, its structure being elucidated on the basis of fast atom bombardment tandem mass spectrometry, NMR, and CD studies.     

GABA-gated chloride ion influx in brains of epileptic El mice

GABA-gated chloride ion influx was measured in brain microsac preparations of epileptic El mice. There was significantly greater sensitivity to GABA in stimulated El mice (which had 14–18 convulsions induced at weekly intervals) than in unstimulated El mice (which had not experienced convulsions) or ddY mice. GABA-gated chloride ion influx was significantly decreased 20 min after a single convulsion, and returned to the preconvulsion level 60 min after a convulsion. These findings suggest that the functional state of GABA-gated chloride channel in El mice is changed secondarily by single or repeated convulsions.