Regulatory DNA sequence conserved in the course of BK virus evolution

Summary Within the genome of human polyomavirus BK (BKV), there exists a noncoding regulatory region toward the late region side of the origin of DNA replication. In most BKV strains isolated by viral culture, this regulatory region contains tandem repeats varying in size. Recently. however, several laboratories isolated new BKV strains (designated as archetypal strains) lacking such repeat sequences. To examine the genetic relationship between archetypal strains, a phylogenetic tree was constructed for seven BKV strains, including three archetypal strains, from DNA sequence data on the late genes, those for leader protein (agnoprotein), and those for structural proteins (VP1, VP2, and VP3). For three strains data previously reported were used, whereas for the others sequences were determined in this study. From total numbers of nucleotide substitutions in each pair of strains, a phylogenetic tree was constructed by the unweighted pair-group method. The phylogenetic tree obtained reveals that BKV strains containing the archetypal regulatory region do not constitute a cluster of closely related strains and that these strains, together with those carrying the major part of the archetypal regulatory region, are widespread in the BKV population. This finding suggests that the basic structure of the archetypal regulatory region has been conserved in the course of BKV evolution.     

Viral delivery of miR-196a ameliorates the SBMA phenotype via the silencing of CELF2

Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) tract of the androgen receptor (AR-polyQ). Characteristics of SBMA include proximal muscular atrophy, weakness, contraction fasciculation and bulbar involvement. MicroRNAs (miRNAs) are a diverse class of highly conserved small RNA molecules that function as crucial regulators of gene expression in animals and plants. Recent functional studies have shown the potent activity of specific miRNAs as disease modifiers both in vitro and in vivo. Thus, potential therapeutic approaches that target the miRNA processing pathway have recently attracted attention. Here we describe a novel therapeutic approach using the adeno-associated virus (AAV) vector–mediated delivery of a specific miRNA for SBMA. We found that miR-196a enhanced the decay of the AR mRNA by silencing CUGBP, Elav-like family member 2 (CELF2). CELF2 directly acted on AR mRNA and enhanced the stability of AR mRNA. Furthermore, we found that the early intervention of miR-196a delivered by an AAV vector ameliorated the SBMA phenotypes in a mouse model. Our results establish the proof of principle that disease-specific miRNA delivery could be useful in neurodegenerative diseases.     

An attempt of in vitro portal invasion model of hepatocellular carcinoma utilizing permeable collagen membrane

Hepatocellular carcinoma (HCC) possessed the ability of vascular invasiveness toward hepatic portal vein on the process of progression. This biological character of HCC can influence the patients survival on clinically. In this paper, we tried to establish the in vitro portal invasion model with human materials. The hepatic portal vein endothelial cell (HPVEC) derived from intrahepatic portal veins by surgically, have been propagated, as outgrowth cultures in RPMI-1640 medium with 10% fetal bovine serum, on permeable collagen membranes (KOKEN, Tokyo) containing mainly type I collagen, covered with a solubilized tissue basement membrane (MATRIGEL, Collaborate Res., Inc., Bedford MA) involving type IV collagen, laminin and proteoglycan. The primary cultured HPVEC with polygonal shaped cells forming a pavement stone sheet, were positively stained with Factor VIII related antigen and synthesized both prostacyclin and collagenase inhibitor. Co-culture of primary human HPVEC and HuH-7 (human HCC cell line obtained from Prof. Satoh, Okayama Univ.,) cells were inoculated onto reverse side between collagen membrane and gell formed basement membrane. Morphological alterations on the side of HPVEC can be obtained such as polylayered cells and different cytoplasmic cells among HPVEC. These results indicate that this experimental model can provide an useful in vitro model for the study of HCC portal invasion.     

Complex Interrelationships Among Aboveground Biomass,Soil Chemical Properties,and Events Caused by Feral Goats and Their Eradication in a Grassland Ecosystem of an Island

This study examined the recovery, via biotic and abiotic pathways, of a grassland ecosystem after eradication of introduced exotic goats. We used path analyses to evaluate the relative strength of relationships among aboveground biomass, soil chemical properties (carbon, nitrogen, and phosphorus content; soil acidity), presence of nesting seabirds after goat eradication, extent of vegetation degraded by goats before their eradication, plant species composition after removal of goats, and topography. Models including the same variables with different paths were constructed using the Bayesian estimation method, and the best-fit models were constructed by comparing deviance information criterion values. Results of the path analyses demonstrated that vegetation degradation and soil erosion prior to goat eradication increased soil exchangeable acidity, which resulted in limitation of aboveground biomass. Seabird nesting after goat eradication increased the quantity of soil nutrients, possibly through inputs of feces, eggshells, and dead chicks or adults. The increase in nutrients was affected indirectly, via seabird nesting, by topography and vegetation type after goat eradication. The direct and indirect relationships demonstrated by our results suggest the existence of complex interrelationships during recovery of ecosystem function after eradication of exotic mammals.     

Molecular Epidemiology of Cases of Mycoplasma californicum Infection in Japan

Bovine mastitis due to Mycoplasmacalifornicum is often accompanied by huge economic losses, and the disease spreads very quickly. An appropriate molecular epidemiological analysis is needed to prevent and control infectious disease, but molecular epidemiological analysis methods for M. californicum have not yet been reported. Here we developed a combination of multiple-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) methods, which are common genotyping methods for various bacteria, for M. californicum. The MLVA is based on four interspersed repeat units that were found in the M. californicum genome data. The MLVA using these repeat units showed sufficient discriminatory power for a molecular epidemiological analysis; i.e., a Hunter-Gaston diversity index (HGDI) of 0.949, against M. californicum strains in Japan and M. californicum strain ATCC 33461. The PFGE for M. californicum also showed sufficient discriminatory power, with an HGDI of 0.985. Strain ATCC 33461 showed MLVA profiles and pulsotypes that differed greatly from those of strains from Japan. These results indicate that MLVA and PFGE are good tools for identifying M. californicum transmission events more accurately. Our combined MLVA and PFGE analysis suggests the persistence of M. californicum infection among herds in a specific area for a long period of time, as well as the movement of cows and heifers accompanying the expansion of M. californicum infection. Failure to identify asymptomatic infected cows is suspected as one of the central causes of the present M. californicum infection scenario in Japan.     

Variable maternal methylation overlapping the nc886/vtRNA2-1 locus is locked between hypermethylated repeats and is frequently altered in cancer

Cancer is as much an epigenetic disease as a genetic one; however, the interplay between these two processes is unclear. Recently, it has been shown that a large proportion of DNA methylation variability can be explained by allele-specific methylation (ASM), either at classical imprinted loci or those regulated by underlying genetic variants. During a recent screen for imprinted differentially methylated regions, we identified the genomic interval overlapping the non-coding nc886 RNA (previously known as vtRNA2-1) as an atypical ASM that shows variable levels of methylation, predominantly on the maternal allele in many tissues. Here we show that the nc886 interval is the first example of a polymorphic imprinted DMR in humans. Further analysis of the region suggests that the interval subjected to ASM is approximately 2 kb in size and somatically acquired. An in depth analysis of this region in primary cancer samples with matching normal adjacent tissue from the Cancer Genome Atlas revealed that aberrant methylation in bladder, breast, colon and lung tumors occurred in approximately 27% of cases. Hypermethylation occurred more frequently than hypomethylation. Using additional normal-tumor paired samples we show that on rare occasions the aberrant methylation profile is due to loss-of-heterozygosity. This work therefore suggests that the nc886 locus is subject to variable allelic methylation that undergoes cancer-associated epigenetic changes in solid tumors.