Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.     

An Evidence for the Repair of Double-Strand Scissions in DNA of Micrococcus radiodurans after Alpha Bombardment

The conversion of prochaetoglobosins as plausible precursors into mycotoxin chaetoglobosin A (1) in a cell-free system of Chaetomium subaffine was unsuccessful. However, reductase activity of the 20-keto-analogues (1), and prochaetoglobosins II (5) and III (6) were found in a microsomal fraction of this fungi. Two new metabolites of chaetoglobosins, named chaetoglobosin F_ex (2) and 20-dihydro-chaetoglobosin A (3), were also isolated from the same micro-organisms. Their structures were elucidated by spectroscopic data and chemical transformation.     

Calcification process dynamics in coral primary polyps as observed using a calcein incubation method

Calcification processes are largely unknown in scleractinian corals. In this study, live confocal imaging was used to elucidate the spatiotemporal dynamics of the calcification process in aposymbiotic primary polyps of the coral species Acropora digitifera. The fluorophore calcein was used as a calcium deposition marker and a visible indicator of extracellular fluid distribution at the tissue-skeleton interface (subcalicoblastic medium, SCM) in primary polyp tissues. Under continuous incubation in calcein-containing seawater, initial crystallization and skeletal growth were visualized among the calicoblastic cells in live primary polyp tissues. Additionally, the distribution of calcein-stained SCM and contraction movements of the pockets of SCM were captured at intervals of a few minutes. Our experimental system provided several new insights into coral calcification, particularly as a first step in monitoring the relationship between cellular dynamics and calcification in vivo. Our study suggests that coral calcification initiates at intercellular spaces, a finding that may contribute to the general understanding of coral calcification processes.     

Aspergillus niger DLFCC-90 rhamnoside hydrolase, a new type of flavonoid glycoside hydrolase

A novel rutin-α-L-rhamnosidase hydrolyzing α-L-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the α-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13.     

Diversity of Intestinal Bifidobacteria in Patients with Japanese Cedar Pollinosis and Possible Influence of Probiotic Intervention

This study was conducted to evaluate the potential association between intestinal bifidobacteria and Japanese cedar pollinosis (JCPsis) and possible influences of probiotic intervention. In this study, fecal samples were the collected from 29 JCPsis patients. The qualitative and quantitative analyses of fecal bifidobacteria were conducted by quantitative real-time PCR with 16S rRNA-gene-targeted species-specific primers before cedar pollen spread and after a 10-week intervention with fermented milk prepared with Lactobacillus GG and L. gasseri TMC0356 during pollen spread. Each JCPsis patient had a unique diversity of bifidobacteria, which varied qualitatively and quantitatively in an individual-dependent manner during pollen spread. The serum IgE concentration of JCPsis patients with more than 3 detectable Bifidobacterium species was significantly lower than that of patients with less than 2 detected species. The prevalence of B. adolescentis, B. longum, and B. catenulatum increased after probiotic intervention, although the changes were not statistically significant. These results suggest that lower diversity of intestinal Bifidobacterium species might be a pathological aspect of JCPsis. The diversity of intestinal bifidobacteria could be a prospective target for using probiotics in the management of IgE-mediated allergic disorders including JCPsis.     

Functional assembly of the Na+/H+ antiporter of Helicobacter pylori from partial fragments in vivo

Functional assembly of the Helicobacter pylori Na+/H+ antiporter (HPNhaA) from partial fragments was studied. Expression plasmids encoding a series of complementary N- and C-terminal fragment pairs containing the transmembrane domains (TMs) were constructed by inserting a stop or a start codon into each of the loop regions of NhaA. HPNhaA fragments alone or complementary fragment pairs were expressed in DeltanhaA Escherichia coli, and fragment integration into the membrane and antiporter activity were measured. TM1-10, TM1-11, TM2-12, TM6-12, and TM10-12 were found in the membrane fraction, while the other fragments were not. While no single fragment displayed antiporter activity, simultaneous expression of fragments in certain pairs, such as TM1-2 + TM3-12, TM1-8 + TM9-12, or TM1-11 + TM12, reconstituted antiporter activity. With the exception of TM12, all of the fragments in the pairs were detected in the membrane. No single fragments expressed alone for these pairs were found in the membrane, except for TM1-11, suggesting that the interaction between the fragments in these pairs stabilized the fragments and enabled reconstitution of HPNhaA. We also found that the simultaneous expression of three complementary fragments (TM1-2 + TM3-8 + TM9-12) reconstituted HPNhaA activity. Other pairs that were found in the membrane (TM1-5 + TM6-12, TM1-10 + TM11-12, and TM1 + TM2-12) did not reconstitute antiporter activity, suggesting that they may not have the proper conformation. These results revealed that the ability to reconstitute antiporter activity depends on the split position in the loop regions and the interaction between complementary fragment pairs. We propose that formation of the active HPNhaA molecule is initiated by the interaction of short-lived intermediates and maintained by the increased stability of the intermediates within the resulting complex.     

Colonization of vegetation-rich moraines and inference of multiple sources of colonization in the High Arctic for Salix arctica

Vegetation-rich patches in the High Arctic may serve as a significant source for vegetation reconstruction in the climate changes. Diversity and colonization, however, of such potential source populations in the High Arctic has rarely been studied. We examined chloroplast sequence variation in Salix arctica, a key species in the Canadian High Arctic, from four adjacent glacial moraines of differing ages on Ellesmere Island, Canada, as well as two other populations located at the center and southern end of the species’ range. The estimated ages of the moraines varied from 35,000 to 250 years old. The older moraine populations showed higher within-population genetic variation compared with the other moraine populations, which is generally attributed to differences in establishment age associated with plant densities among moraines. The moraines with smaller plant density had lower genetic diversity and had no private haplotypes, indicating the local population size and genetic diversity may not be recovered within a few thousand years. This suggests seed dispersal at a local scale may be limited even in species with high velocity of seed dispersal, and that High Arctic vegetation-rich patches may serve as significant source populations for sustaining local genetic diversity. In addition, the three regions we observed comprised an evolutionarily distinct lineage and significant population differentiation. This implies multiple sources for the colonization during the most recent deglaciation, resulting in the current wide distribution. Local as well as range-wide processes of colonization would be essential to understand vegetation responses in High Arctic to the environmental changes.     

Synthesis of the cyclic and acyclic acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine,a potent antitumor nucleoside. Design of prodrugs to be selectively activated in tumor tissues via the bio-reduction-hydrolysis mechanism

We have designed and synthesized the acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, 1), the 2',3'-O-nitrobenzylidene derivatives 2 and 3 and the 5'-O-(alkoxy)(nitrophenyl)methyl derivatives 6-10 as potential prodrugs of ECyd. These prodrugs can be selectively activated in tumor tissues via a bio-reduction-hydrolysis mechanism owing to the characteristic properties of tumor tissues, such as hypoxia and lower pH. Although the 2',3'-O-(4-nitrobenzylidene) derivatives 2 and 3 were converted bio-reductively into the corresponding 4-aminobenzylidene derivatives by rat S-9 mix, the reduction products, that is, the corresponding amino congeners 4 and 5, proved to be rather stable in an aqueous solution at pH 6.5 used as a pH model for acidic tumor tissues. In contrast, the 5'-O-(alkoxy)(4-nitropheny)methyl derivatives 6-8 were also reduced by rat S-9 mix to the corresponding amino congeners 11-13, which were hydrolyzed to release ECyd more effectively at pH 6.5 than at pH 7.4. Accordingly, the acyclic acetals 6-8 may be efficient prodrugs of ECyd, that are effectively reduced under physiological conditions releasing ECyd in acidic tumor tissues.