Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

Molecular Monitoring and Isolation of Previously Uncultured Bacterial Strains from the Sheep Rumen

To estimate the contribution of uncultured bacterial groups to fiber degradation, we attempted to retrieve both ecological and functional information on uncultured groups in the rumen. Among previously reported uncultured bacteria, fiber-associated groups U2 and U3, belonging to the low-GC Gram-positive bacterial group, were targeted. PCR primers and fluorescence in situ hybridization (FISH) probe targeting 16S rRNA genes or rRNA were designed and used to monitor the distribution of targets. The population size of group U2 in the rumen was as high as 1.87%, while that of group U3 was only 0.03%. Strong fluorescence signals were observed from group U2 cells attached to plant fibers in the rumen. These findings indicate the ecological significance of group U2 in the rumen. We succeeded in enriching group U2 using rumen-incubated rice straw as the inoculum followed by incubation in an appropriate medium with an agent inhibitory for Gram-negative bacteria. Consequently, we successfully isolated two strains, designated B76 and R-25, belonging to group U2. Both strains were Gram-positive short rods or cocci that were 0.5 to 0.8 μm in size. Strain B76 possessed xylanase and α-l-arabinofuranosidase activity. In particular, the xylanase activity of strain B76 was higher than that of xylanolytic Butyrivibrio fibrisolvens H17c grown on cellobiose. Strain R-25 showed an α-l-arabinofuranosidase activity higher than that of strain B76. These results suggest that strains B76 and R-25 contribute to hemicellulose degradation in the rumen.Ruminants can utilize plant fiber as an energy source with the aid of a symbiotic relationship with microbes in the rumen. The rumen is a complex microbial ecosystem comprised of bacteria (10¹⁰ to 10¹¹ per ml), protozoa (10⁴ to 10⁶ per ml), and fungi (10³ to 10⁶ per ml) (8, 23, 39). Of the rumen microbes, bacteria are considered to be primarily responsible for the biological degradation of plant fiber, due to their high fibrolytic activity and large biomass in the rumen. In order to determine the mechanism of plant fiber degradation in the rumen, numerous studies have been performed on both the physiological and ecological characteristics of rumen bacteria (16, 27, 36). In particular, various aspects of bacterial attachment to feed particles have been investigated (19, 21, 25), because attachment to plant fiber is a critical step in initiating fiber degradation (20).Recent advances in molecular techniques have allowed recognition of a predominance of uncultured bacteria in the rumen (6, 24, 33). The majority (77%) of fiber-associated community members are uncultured bacteria, although 17% of cloned bacterial 16S rRNA gene sequences were classified as known fibrolytic species, such as Fibrobacter succinogenes and Butyrivibrio fibrisolvens (12). These findings clearly indicate the possibility for involvement of uncultured bacteria in ruminal fiber degradation. Through the phylogenetic analysis of fiber-associated community members, the unidentified bacterial groups were detected and designated uncultured group 2 (U2) and uncultured group 3 (U3). However, their roles in plant fiber digestion have yet to be determined.The predominance of uncultured bacteria has also been pointed out in other environments (26). Recently, new strategies for cultivation have been introduced to resolve the problem of the bacteria being unculturable. Sait et al. (28) reported that culturing with a polymeric growth substrate and longer incubation time was effective for the isolation of previously uncultured bacteria from soil. Cultivation on low-nutrient medium, using increased incubation times, with simulated natural environments or using a membrane as a solid support for growth has apparently led to improvements in bacterial cultivation (7, 31). On the other hand, the majority of rumen bacteria have yet to be isolated (10) despite great efforts toward the isolation of rumen bacterial strains over the past 50 years. Considering the ecological significance of uncultured rumen bacteria, it is important to cultivate and characterize these bacteria to fully understand the ecology of fiber digestion.In the present study, molecular monitoring tools were developed to obtain ecological information on target uncultured bacterial groups in the rumen. Previously uncultured rumen bacteria were then isolated and characterized to retrieve functional information.     

Long-term trends in food habits of Asiatic black bears in the Misaka Mountains on the Pacific coast of central Japan

I did the food habits of the Asiatic black bear Ursus thibetanus from 1013 fecal samples collected between 1999 and 2005 in the Misaka Mountains on the Pacific coast of central Japan. The food habits of the bears showed clear seasonal changes, and I classified the food resources of the bears into three types. Staple foods were green vegetation in spring, soft mast (Prunus spp.) and insects in summer, and hard mast (Quercus spp.) in autumn. Alternative foods were green vegetation and other soft mast (Rubus spp.) in summer and Japanese chestnuts Castanea crenata and vine fruits in autumn. Foods of opportunity were hard mast (Quercus spp.) that had been shed in the previous autumn and were found in spring and other fruits in autumn. Seasonal food habits showed yearly variations: bears used alternative foods and foods of opportunity in response to the yearly variation in staple food amount, but the magnitude of variability of food habits differed among seasons, with large variability in autumn and small variability in summer and spring. The primary influence on the yearly variation in food habit is presumably the fluctuation in fruit production among years. Summer is probably the most difficult season in terms of the bear's food supply, because the number of fruiting species is limited and staple foods such as new green vegetation and fruits are less available. Long-term studies of the availability of the main food items and food habits of bears will be critical for further understanding these animals’ feeding ecology and for determining the factors that influence their behavior.     

Molecular cloning and characterization of FRAT2, encoding a positive regulator of the WNT signaling pathway

FRAT1 positively regulates the WNT signaling pathway by stabilizing beta-catenin through the association with glycogen synthase kinase-3beta. Here, we have cloned FRAT2 cDNAs, spanning the complete coding sequence, from a human fetal lung cDNA library. FRAT2 encoded 233 amino-acid protein, which showed 77.3% total amino-acid identity with FRAT1. FRAT2 and FRAT1 were more homologous in the acidic domain (96% identity), the proline-rich domain (92% identity), and the GSK-3beta binding domain (100% identity). The FRAT2 gene was mapped to human chromosome 10q24.1. The FRAT2 mRNA of 2.4-kb in size was relatively highly expressed in MKN45 (gastric cancer), HeLa S3 (cervical cancer), and K-562 (chronic myelogenous leukemia). Xenopus axis duplication assay revealed that the wild-type FRAT2 mRNA, but not the mutant FRAT2 mRNA lacking the acidic domain and the proline-rich domain, has the capacity to induce the secondary axis. These results indicate that FRAT2, just like FRAT1, functions as a positive regulator of the WNT signaling pathway. Thus, up-regulation of FRAT2 in human cancer might be implicated in carcinogenesis through activation of the WNT signaling pathway.     

Chemical and physical defence in early and late leaves in three heterophyllous birch species native to northern Japan

BACKGROUND AND AIMS: Betula ermanii, B. maximowicziana and B. platyphylla var. japonica have heterophyllous leaves (i.e. early leaves and late leaves) and are typical pioneer species in northern Japan. Chemical and physical defences against herbivores in early and late leaves of these species were studied. METHODS: Two-year-old seedlings were grown under full sunlight in a single growing season. Three-week-old leaves of each seedling were harvested three times (May, July and October). Total phenolics and condensed tannin content were determined for chemical defence and leaf toughness and trichome density were assessed for physical defence. Defoliation of early leaves in May was also performed to study the contribution of early leaves to subsequent growth. KEY RESULTS: Chemical and physical defences were greater in early than late leaves in B. platyphylla and B. ermanii, whereas the reverse was true in B. maximowicziana. In contrast to its weak chemical defences, the trichome density in B. maximowicziana was very high. In B. platyphylla and B. ermanii, the relative growth rates (RGR) were greater early in the growing season. Negative effects on growth of removal of early leaves were significant only in B. platyphylla. CONCLUSIONS: B. platyphylla and B. ermanii invest in defence in early rather than late leaves, since early leaves are crucial to subsequent growth. In contrast, B. maximowicziana more strongly defends its late leaves, since its RGR is maintained at the same level throughout the growing season.     

Recovery of photosynthetic systems during rewetting is quite rapid in a terrestrial cyanobacterium, Nostoc commune

Recovery processes of photosynthetic systems during rewetting were studied in detail in a terrestrial, highly drought-tolerant cyanobacterium, Nostoc commune. With absorption of water, the weight of N. commune colony increased in three phases with half-increase times of about 1 min, 2 h and 9 h. Fluorescence intensities of phycobiliproteins and photosystem (PS) I complexes were recovered almost completely within 1 min, suggesting that their functional forms were restored very quickly. Energy transfer from allophycocyanin to the core-membrane linker peptide (L(CM)) was recovered within 1 min, but not that from L(CM) to PSII. PSI activity and cyclic electron flow around PSI recovered within 2 min, while the PSII activity recovered in two phases after a time lag of about 5 min, with half times of about 20 min and 2 h. Photosynthetic CO(2) fixation was restored almost in parallel with the first recovery phase of the PSII reaction center activity. Although the amount of absorbed water became more than 20 times the initial dry weight of the N. commune colony in the presence of sufficient water, about twice the initial dry weight was enough for recovery and maintenance of the PSII activity.     

Proinflammatory cytokines in bovine colostrum potentiate the mitogenic response of peripheral blood mononuclear cells from newborn calves through IL-2 and CD25 expression

Abstract: Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist, IL-1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL-2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL-1beta, TNF-alpha or IFN-gamma significantly enhanced the ConA response, whereas IL-1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL-2 mRNA expression in ConA-stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA-stimulated controls, the expression levels became comparable after pretreatment with IL-1beta, TNF-alpha or IFN-gamma. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL-1beta, TNF-beta and IFN-gamma. These results suggest that pretreatment of neonatal PBMC with IL-1beta, TNF-alpha or IFN-gamma promotes mitogenic response to ConA through up-regulating the production of IL-2 and the expression of the mature IL-2 receptor.     

Horticultural characterization of<Emphasis Type="Italic"> Angelonia salicariifolia</Emphasis> plants transformed with wild-type strains of<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis>

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.