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 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.     

Blockade of sphingosine 1-phosphate receptor 2 signaling attenuates streptozotocin-induced apoptosis of pancreatic β-cells

Sphingosine 1-phosphate (S1P) is a potent sphingolipid mediator that acts through five cognate G protein-coupled receptors (S1P₁-S1P₅) and regulates many critical biological processes. Recent studies indicated that S1P at nanomolar concentrations significantly reduces cytokine-induced apoptosis of pancreatic β-cells in which genes for S1P₁-S1P₄ are co-expressed. However, the S1P receptor subtype(s) involved in this effect remains to be clarified. In this study, we investigated the potential role of S1P₂ in streptozotocin (STZ)-induced apoptosis of pancreatic β-cells and progression of diabetes. S1P₂-deficient (S1P₂^-/-) mice displayed a greater survive ability, lower blood glucose levels, and smaller numbers of TUNEL-positive apoptotic β-cells to administration of a high dose of STZ than wild-type (WT) mice. S1P₂^-/- mice showed higher insulin/glucose ratios (an index of relative insulin deficiency) and larger insulin-positive islet areas to administration of a low dose of STZ than WT mice. Moreover, administration of JTE-013, a S1P₂-specific antagonist, to WT mice ameliorated STZ-induced blood glucose elevation and reduced the incidence of diabetes. Our findings indicate that blockade of S1P₂ signaling attenuates STZ-induced apoptosis of pancreatic β-cells and decreases the incidence of diabetes.     

Mechanisms of heart development in the Japanese lamprey, Lethenteron japonicum

SUMMARY Vertebrate hearts have evolved from undivided tubular hearts of chordate ancestors. One of the most intriguing issues in heart evolution is the abrupt appearance of multichambered hearts in the agnathan vertebrates. To explore the developmental mechanisms behind the drastic morphological changes that led to complex vertebrate hearts, we examined the developmental patterning of the agnathan lamprey Lethenteron japonicum . We isolated lamprey orthologs of genes thought to be essential for heart development in chicken and mouse embryos, including genes responsible for differentiation and proliferation of the myocardium ( LjTbx20, LjTbx4/5 , and LjIsl1/2A ), establishment of left–right heart asymmetry ( LjPitxA ), and partitioning of the heart tube ( LjTbx2/3A ), and studied their expression patterns during lamprey cardiogenesis. We confirmed the presence of the cardiac progenitors expressing LjIsl1/2A in the pharyngeal and splanchnic mesoderm and the heart tube of the lamprey. The presence of LjIsl1/2A -positive cardiac progenitor cells in cardiogenesis may have permitted an increase of myocardial size in vertebrates. We also observed LjPitxA expression in the left side of lamprey cardiac mesoderm, suggesting that asymmetric expression of Pitx in the heart has been acquired in the vertebrate lineage. Additionally, we observed LjTbx2/3A expression in the nonchambered myocardium, supporting the view that acquisition of Tbx2/3 expression may have allowed primitive tubular hearts to partition, giving rise to multichambered hearts.     

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

SUMMARY Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA‐expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU‐incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell‐lineage‐specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell‐lineage‐specific markers during archeocyte differentiation into other cell types. Choanocytes are food‐entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell‐like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.     

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

A Mathematical Modelling for the Cheliped Regeneration with Handedness in Fiddler Crab

An enormously developed giant cheliped with the other small one characterizes the adult male fiddler crab. Some experiments with artificial severances of cheliped indicate that such a handedness in the cheliped size is maintained even after the regeneration of severed cheliped. Other experimental researches give some results about an unknown physiological system which controls the emergence and the regeneration of the handedness in the cheliped size. In this paper, with two hypothesized factors relevant to the regeneration of a severed cheliped, we propose a simple mathematical model to describe the experimental result about the cheliped regeneration with a handedness after the cheliped severance for the fiddler crab. Our model gives a suggestion about an underlying system for the cheliped regeneration in the fiddler crab or some other crustacean species.     

Sequence variation in the Tbx4 gene in marine mammals

The amino-acid sequences of the T-domain region of the Tbx4 gene, which is required for hindlimb development, are 100% identical in humans and mice. Cetaceans have lost most of their hindlimb structure, although hindlimb buds are present in very early cetacean embryos. To examine whether the Tbx4 gene has the same function in cetaceans as in other mammals, we analyzed Tbx4 sequences from cetaceans, dugong, artiodactyls and marine carnivores. A total of 39 primers were designed using human and dog Tbx4 nucleotide sequences. Exons 3, 4, 5, 6, 7, and 8 of the Tbx4 genes from cetaceans, artiodactyls, and marine carnivores were sequenced. Non-synonymous substitution sites were detected in the T-domain regions from some cetacean species, but were not detected in those from artiodactyls, the dugong, or the carnivores. The C-terminal regions contained a number of non-synonymous substitutions. Although some indels were present, they were in groups of three nucleotides and therefore did not cause frame shifts. The dN/dS values for the T-domain and C-terminal regions of the cetacean and artiodactylous Tbx4 genes were much lower than 1, indicating that the Tbx4 gene maintains it function in cetaceans, although full expression leading to hindlimb development is suppressed.