Establishment and characterization of primary canine hepatocellular carcinoma cell lines producing alpha-fetoprotein

A primary cultured cell line named CHKS was established from a hepatocellular carcinoma (HCC) of a dog showing a high level of serum alpha-fetoprotein (AFP). CHKS secreted a 66 KDD AFP into the growth medium regardless of the presence or absence of fetal bovine serum (FBS). Cloning CHKS with limiting dilution produced 4 clones, CHKS-1, -2, -3, and -4, which secreted 826, 471, 70, and less than 10 ng/ml, respectively, of AFP into the culture medium. In culture, these cell lines were similar in morphology and proliferation pattern to epithelial cells and positive to periodic acid-Schiff (PAS) staining. The presence of mRNA for canine albumin was demonstrated by nested PCR. The doubling times of the clone cell lines were 21, 45, 36, and 35 h, saturation densities 34, 18, 22, and 24 x 10(4)/cm(2), and plating efficiencies 18, 45, 46, and 45%, respectively. Chromosome analysis of these cell lines showed near triploidy. These results show that CHKS and its clones have hepatic cell functions and are useful for carcinogenetic and clinical studies of canine HCC.     

Analysis of urbanization characteristics causing farmland loss in a rapid growth area using GIS and RS

This study aims to analyze the spatial characteristics of urbanization in a rapid growth area. Remote sensing (RS) and geographic information system (GIS) techniques were used to classify land uses in 1986 and 1996, the most rapid growth period. The two land-use maps were classified with accuracies of 90% and 86% in verification areas for 1986 and 1996, respectively, by a supervised maximum likelihood method. Urbanization in the greenbelt within the study area was restricted, while outside the greenbelt it occurred rapidly. Fifty seven percent of the total increase in built-up area in the study area was concentrated in a 10-km GIS-defined buffer zone adjacent to the greenbelt. The priority for new residential development was put on areas with slopes of less than 15 degrees and potential farmland in the areas surrounding Seoul. In the priority area, farmland loss was very significant. These results show that although the environmental protection of ecologically valuable land was well enforced by the greenbelt from urbanization, farmland was not conserved in comparison to new residential development in the rapid growth area.     

Study on structure and function of an earthen bund irrigation system in Northeast Thailand

In Northeast Thailand, which overlaps with an erosional plain that is not suitable for irrigation because of poor water resources and terrains that prevent efficient water distribution, farmers have long supplied water to surrounding paddy fields by blocking rivers completely with earthen bunds. Although such a traditional irrigation system fits well with the characteristics of Northeast Thailand, those who are concerned with modern irrigation development projects in Northeast Thailand seem to have been paying little attention to it. The present study was performed to facilitate development of more appropriate irrigation systems in Northeast Thailand by providing information regarding traditional irrigation methods. We investigated the traditional irrigation methods with earthen bunds at three study sites, and our results indicated that styles of irrigation vary with riverbed slope. The traditional irrigation system that is used in most of Northeast Thailand is different from ordinal weir irrigation, in that paddy fields are not irrigated by gravity flow but by backwater of earthen bunds. Our results suggest that the development of more successful irrigation systems would be possible through improvement of the design ideas of traditional irrigation methods.

DNA methylation-dependent modulator of Gsg2/Haspin gene expression

The Gsg2 (Haspin) gene encodes a serine/threonine protein kinase and is predominantly expressed in haploid germ cells. In proliferating somatic cells, Gsg2 is shown to be expressed weakly but plays an essential role in mitosis. Although the Gsg2 minimal promoter recognized by the spermatogenic cell-specific nuclear factor(s) has been found, to date, the molecular mechanism that differentially controls Gsg2 expression levels in germ and somatic cells remains to be sufficiently clarified. In this study, we analyzed the DNA methylation status of the upstream region containing the Gsg2 promoter. We found a tissue-dependent and differentially methylated region (T-DMR) upstream (-641 to -517) of the authentic promoter that is hypomethylated in germ cells but hypermethylated in other somatic tissues. Profiling of Gsg2 expression and DNA methylation status at the T-DMR in spermatogenic cells indicated that the hypomethylation of the T-DMR is maintained during spermatogenesis. Using the reporter assay, we also demonstrated that DNA methylation at the T-DMR of Gsg2 reduced the promoter activity by 60-80%, but did not fully suppress it. Therefore, the T-DMR functions as a modulator in a DNA methylation-dependent manner. In conclusion, Gsg2 is under epigenetic control.     

Disease-dependent differently methylated regions (D-DMRs) of DNA are enriched on the X chromosome in uterine leiomyoma

Uterine leiomyoma is the most common benign tumor in women. Although responsible gene mutations have not been found in leiomyomas, they represent a progressive disease with irreversible symptoms. To characterize epigenetic features of uterine leiomyomas, the DNA methylation status of a paired sample of leiomyoma and normal myometrium was subjected to a microarray-based DNA methylation analysis with restriction tag-mediated amplification (D-REAM). In the leiomyoma, we identified an aberrant DNA methylation status for 463 hypomethylated and 318 hypermethylated genes. Although these changes occurred on all chromosomes, aberrantly hypomethylated genes were preferentially located on the X chromosome. Using paired samples of normal myometrium and leiomyoma from 6 hysterectomy patients, methylation-sensitive quantitative real-time PCR revealed 14 shared X chromosome genes with an abnormal DNA hypomethylation status (FAM9A, CPXCR1, CXORF45, TAF1, NXF5, VBP1, GABRE, DDX53, FHL1, BRCC3, DMD, GJB1, AP1S2 and PCDH11X) and one hypermethylated locus (HDAC8). Expression of XIST, which is involved in X chromosome inactivation, was equivalent in the normal myometrium and leiomyoma, indicating that the epigenetic abnormality on the X chromosome did not result from aberration of XIST gene expression. Based on these data, a unique epigenetic signature for uterine leiomyomas has emerged. The 14 hypomethylated and one hypermethylated loci provide valuable biomarkers for understanding the molecular pathogenesis of leiomyoma.     

Optical and acoustic camera observations of the behavior of the Kuril harbor seal <Emphasis Type="Italic">Phoca vitulina stejnegeri</Emphasis> after invading a salmon setnet

It has previously been confirmed that Kuril harbor seals Phoca vitulina stejnegeri cause damage to the chum salmon Oncorhynchus keta setnet fishery along the east coast of Hokkaido, Japan, but the level of damage has increased markedly with the recovery of their population in recent years. In this study, we attached an optical camera (Trawl Camera) and a dual-frequency identification sonar (DIDSON) acoustic camera to a setnet to observe the behavior of seals as they invaded the setnet, and to determine the number of salmon inside the net, to help inform the development of modified fishing gear. Salmon were observed at all times during daytime with the Trawl Camera, while seals were only observed once. Observations using the DIDSON in its low-frequency mode confirmed that the behavior of seals became vigorous from around sunset to nighttime within the recording time (1530–2100 hours). Observations using the DIDSON high-frequency mode showed that the overall lengths and body widths of seals ranged from 1.0 to 1.6 m and 0.15 to 0.35 m, respectively, while their swimming speeds ranged from 0.4 to 2.6 m/s, increasing around sunset and declining into the night. These results imply that seals mainly invade the setnet from evening to nighttime to predate on salmon.     

Identification and Characterization of Yeasts with Tolerance to High Acidity and Resistance to Aluminum Isolated from Tea Soils

From acidic tea soils in Kagoshima Prefecture in Japan, 38 strains of acid-tolerant microorganisms were isolated and some soil properties were determined. Different Al concentrations were applied to YG media to estimate the Al resistance. Selected microbial strains were able to grow actively in liquid media in the presence of 100 mM Al and to survive even in 300 mM Al at pH 3.0. The base sequences of 28S rDNA-D1/D2 were determined and sequence data were searched using the Basic Local Alignment Search Tool (BLAST) system. The results of sequencing revealed that the isolates belonged to two different species, Cryptococcus sp. and Candida palmioleophila . Subsequently, Neighbor-Joining molecular phylogenetic trees were constructed. When the yeasts were cultured at various Al concentrations, the growth was inhibited at a concentration of 200 mM. Pre-culture of these strains with 0–30 mM Al did not promote the growth response induced by Al. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to assess the elimination of Al from YG media. The amount of Al remaining in the culture media decreased considerably after culture. Due to a capacity for resistance to significant Al concentrations as well as high Al elimination, these acid-tolerant and Al-resistant yeasts may offer potential applications for the bio- and phyto-remediation of acid soils with a high level of Al.     

Nutritional values and antioxidative activities of whole peanuts and peanut skins for ruminant feeds

This study was conducted to investigate the nutritive values of two peanut by‐products, nonstandardized whole peanuts and peanut skins, along with their effects on microbial growth and fermentation in the rumen, their roughage values, and their antioxidative activities by a digestion trial using four goats. The experimental rations were alfalfa haycube (basal ration), 85% alfalfa with 15% whole peanuts, and 70% alfalfa with 15% whole peanuts and 15% peanut skins. The ether extracts and crude protein in whole peanuts were 47% and 27% on a dry matter basis (DM) both with over 90% of digestibilities, resulting in total digestive nutrients (TDN) of 140%. Peanut skins also had a high energy value with 91% of TDN. Ruminal concentrations of total volatile fatty acids (VFA) and acetic acid decreased in the rations containing the peanut by‐products, but the NDF digestibility and ruminal microbial protein estimated from urinary purines was not altered by feeding the peanut by‐products. Plasma oxidative stress maker, malondialdehyde, tended to be lower when peanut skins were supplemented. Whole peanuts and peanut skins could be used as high‐energy and high‐protein diets for ruminants, and peanut skins would be expected as a feed having antioxidant functions.