Genetic Differentiation and Spatial Structure of Phellinus noxius,the Causal Agent of Brown Root Rot of Woody Plants in Japan

Phellinus noxius is a pathogenic fungus that causes brown root rot disease in a variety of tree species. This fungus is distributed in tropical and sub-tropical regions of Southeast and East Asia, Oceania, Australia, Central America and Africa. In Japan, it was first discovered on Ishigaki Island in Okinawa Prefecture in 1988; since then, it has been found on several of the Ryukyu Islands. Recently, this fungus was identified from the Ogasawara (Bonin) Islands, where it has killed trees, including rare endemic tree species. For effective control or quarantine methods, it is important to clarify whether the Japanese populations of P. noxius are indigenous to the area or if they have been introduced from other areas. We developed 20 microsatellite markers from genome assembly of P. noxius and genotyped 128 isolates from 12 of the Ryukyu Islands and 3 of the Ogasawara Islands. All isolates had unique genotypes, indicating that basidiospore infection is a primary dissemination method for the formation of new disease foci. Genetic structure analyses strongly supported genetic differentiation between the Ryukyu populations and the Ogasawara populations of P. noxius. High polymorphism of microsatellite loci suggests that Japanese populations are indigenous or were introduced a very long time ago. We discuss differences in invasion patterns between the Ryukyu Islands and the Ogasawara Islands.     

Quantitative analysis of age-associated accumulation of mitochondrial DNA with deletion in human hearts

We have demonstrated that myocardial mitochondrial DNA (mtDNA) with a 7,436 base-pair deletion existed in all subjects that were over 70 years old. Since each mitochondrion has two or three copies of its own DNA, quantitative analysis is required for the evaluation of the role of mtDNA with deletions in the age-related deterioration of cardiac performance. For this purpose, the kinetic polymerase chain reaction (PCR) method developed in our laboratory was used in this investigation to determine myocardial mtDNA with this 7,436 base-pair deletion in human cadavers of various ages. The mtDNA population with this deletion increased exponentially with age [log f (% of deleted mtDNA) = -3.136 + 0.0454 x age, r = 0.95, P less than 0.01)], and was estimated at 3% and 9% in subjects of age 80 and 90, respectively. The deleted portion encodes 7 subunits of the mitochondrial ATP production system, and a population of mtDNA with this deletion over a certain threshold might induce a significant deterioration of cardiac energy metabolism. Cardiac function is known to deteriorate with age, and an increase in the population of mtDNA with deletion is likely to be an important contributing factor to aged heart (presbycardia).     

Synthesis and structure–activity relationships of oxamyl dipeptide caspase inhibitors developed for the treatment of liver disease

The P4 region of a series of oxamyl dipeptide caspase inhibitors was optimized by the combination of anti-apoptotic activity in the Jurkat/Fas (JFas) cellular assay and membrane permeability in the PAMPA assay. Two highly potent anti-apoptotic agents with moderate membrane permeability, 29 and 36, showed strong in vivo efficacy in a murine model of α-Fas-induced liver injury.     

Synthesis of and triplex formation in oligonucleotides containing 2′-deoxy-6-thioxanthosine

This study aimed to synthesize triplex-forming oligonucleotides (TFOs) containing 2′-deoxy-6-thioxanthosine (s⁶X) and 2′-deoxy-6-thioguanosine (s⁶Gs) residues and examined their triplex-forming ability. Consecutive arrangement of s⁶X and s⁶Gs residues increased the triplex-forming ability of the oligonucleotides more than 50 times, compared with the unmodified TFOs. Moreover, the stability of triplex containing a mismatched pair was much lower than that of the full-matched triplex, though s⁶X could form a s⁶X-GC mismatched pair via tautomerization of s⁶X. The present results reveal excellent properties of modified TFOs containing s⁶Xs and s⁶Gs residues, which may be harnessed in gene therapy and DNA nanotechnology.     

Distribution of polymorphic gene duplication at theGpdh locus in natural populations ofDrosophila melanogaster

Summary The glycerol-3-phosphate dehydrogenase (GPDH, E. C. 1.1.1.8) gene ofDrosophila melanogaster contains a tandem duplication of a 4.5-kb-long DNA fragment. Survey of theGpdh gene region by the Southern blot analysis revealed the following features of this gene duplication: (1) The duplication was not observed in chromosome lines that carryIn(2L)t, a cosmopolitan chromosomal inversion in this species. The duplication and the inversion are in linkage disequilibrium. (2) The duplication is polymorphic in the Japan and US natural populations examined. Its frequency is 0.26 on an average inIn(2L)t-free chromosomes. (3) Triplication is absent or has not become frequent in the populations surveyed. Possible evolutionary factors of this duplication polymorphism are discussed.     

On-chip cell culture on a microarray of extracellular matrix with surface modification of poly(dimethylsiloxane)

Microfluidic cell culture chips allow to perform assays of small-volume samples rapidly and reproducibly. Most of these chips are made of poly(dimethylsiloxane) (PDMS), which is a flexible, durable, transparent and inexpensive polymer that can be easily applied to fabrication of microstructures by photolithography and replica molding. However, not many cells are able to grow on unmodified PDMS because the cells need appropriate scaffolds on the surface. Here we report surface modification of a PDMS substrate with a microarray of extracellular matrix (ECM) for on-chip cell culture. The ECM proteins collagen and fibronectin were covalently immobilized on an 8 x 8 microarray format by micropatterned UV-induced graft polymerization through a photomask and dehydration-condensation reaction through a microfabricated stencil. Identical spots of ECMs were successfully formed and the geometry of the spots accurately corresponded to the micropattern of the photomask and stencil. We demonstrate the culture of CHO-K1 cells on the ECM microarray chip. Cells proliferated on the fibronectin spots during the 2-day culture.