A high-throughput screen for directed evolution of the natural product sulfotransferase LipB

In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.     

Parallel evolution in eight-barbel loaches of the genus Lefua (Balitoridae, Cypriniformes) revealed by mitochondrial and nuclear DNA phylogenies

The evolutionary history of eight-barbel loaches of the genus Lefua contains important phylogenetic information that will aid in resolution of the faunal formations and evolutionary histories of Japanese and East Asian freshwater fishes. Our sequencing of the mitochondrial D-loop region in a large number of samples allowed construction of the most comprehensive phylogeny of these loaches to date; we demonstrated monophyly of five Lefua species and identified populations of Lufua. sp. and Lefua echigonia. Loaches inhabiting the Tokai region in Japan were morphologically and ecologically indistinguishable from Lefua sp. However, they were included in the L. echigonia lineage. We determined a novel phylogeny by sequencing the nuclear ribosomal S7 subunit and showed that nuclear DNA phylogeny essentially matched the mitochondrial DNA phylogeny. Loaches from the Tokai region were part of the L. echigonia lineage, indicating parallel evolution between Tokai loaches and Lefua sp. in western Japan. We presented the most robust phylogeny to date using concatenated mitochondrial and nuclear sequences. The wealth of molecular information allowed us to speculate on evolutionary processes in the genus Lefua.     

Creating localized DNA double-strand breaks with microirradiation

We describe a protocol for creating localized DNA double-strand breaks (DSBs) with minimal requirements that can be applied in cell biology and molecular biology. This protocol is based on the combination of 5-bromo-2'-deoxyuridine (BrdU) labeling and ultraviolet C (UVC) irradiation through porous membranes. Cells are labeled with 10 μM BrdU for 48-72 h, washed with Ca(2+)- and Mg(2+)-free PBS(-), covered by polycarbonate membranes with micropores and exposed to UVC light. With this protocol, localized DSBs are created within subnuclear areas, irrespective of the cell cycle phase. Recruitment of proteins involved in DNA repair, DNA damage response, chromatin remodeling and histone modifications can be visualized without any specialized equipment. The quality is the same as that obtained by laser microirradiation or by any other focal irradiation. DSBs become visible within 30 min of UVC irradiation.     

IGF-1 induction by acylated steryl β-glucosides found in a pre-germinated brown rice diet reduces oxidative stress in streptozotocin-induced diabetes

Background

The pathology of diabetic neuropathy involves oxidative stress on pancreatic β-cells, and is related to decreased levels of Insulin-like growth factor 1 (IGF-1). Acylated steryl β-glucoside (PR-ASG) found in pre-germiated brown rice is a bioactive substance exhibiting properties that enhance activity of homocysteine-thiolactonase (HTase), reducing oxidative stress in diabetic neuropathy. The biological importance of PR-ASG in pancreatic β-cells remains unknown.Here we examined the effects of PR-ASG on IGF-1 and glucose metabolism in β-cells exposed to oxidative stress.

Methodology/Principal Findings

In the present study, a pre-germinated brown rice (PR)-diet was tested in streptozotocin (STZ)-induced diabetic rats. Compared with diabetic rats fed control diets, the PR-diet fed rats showed an improvement of serum metabolic and neurophysiological parameters. In addition, IGF-1 levels were found to be increased in the serum, liver, and pancreas of diabetic rats fed the PR-diet. The increased IGF-1 level in the pancreas led us to hypothesize that PR-ASG is protective for islet β-cells against the extensive injury of advanced or severe diabetes. Thus we examined PR-ASG to determine whether it showed anti-apoptotic, pro-proliferative effects on the insulin-secreting β-cells line, INS-1; and additionally, whether PR-ASG stimulated IGF-1 autocrine secretion/IGF-1-dependent glucose metabolism. We have demonstrated for the first time that PR-ASG increases IGF-1 production and secretion from pancreatic β-cells.

Conclusion/Significance

These findings suggest that PR-ASG may affect pancreatic β-cells through the activation of an IGF-1-dependent mechanism in the diabetic condition. Thus, intake of pre-germinated brown rice may have a beneficial effect in the treatment of diabetes, in particular diabetic neuropathy.     

Predictors of the uptake of A (H1N1) influenza vaccine: findings from a population-based longitudinal study in Tokyo

Background

Overall pandemic A (H1N1) influenza vaccination rates remain low across all nations, including Japan. To increase the rates, it is important to understand the motives and barriers for the acceptance of the vaccine. We conducted this study to determine potential predictors of the uptake of A (H1N1) influenza vaccine in a cohort of Japanese general population.

Methodology/Principal Findings

By using self-administered questionnaires, this population-based longitudinal study was conducted from October 2009 to April 2010 among 428 adults aged 18–65 years randomly selected from each household residing in four wards and one city in Tokyo. Multiple logistic regression analyses were performed. Of total, 38.1% of participants received seasonal influenza vaccine during the preceding season, 57.0% had willingness to accept A (H1N1) influenza vaccine at baseline, and 12.1% had received A (H1N1) influenza vaccine by the time of follow-up. After adjustment for potential confounding variables, people who had been vaccinated were significantly more likely to be living with an underlying disease (p = 0.001), to perceive high susceptibility to influenza (p = 0.03), to have willingness to pay even if the vaccine costs ≥ US$44 (p = 0.04), to have received seasonal influenza vaccine during the preceding season (p<0.001), and to have willingness to accept A (H1N1) influenza vaccine at baseline (p<0.001) compared to those who had not been vaccinated.

Conclusions/Significance

While studies have reported high rates of willingness to receive A (H1N1) influenza vaccine, these rates may not transpire in the actual practices. The uptake of the vaccine may be determined by several potential factors such as perceived susceptibility to influenza and sensitivity to vaccination cost in general population.     

Phylogeography of loaches of the genus lefua (balitoridae, cypriniformes) inferred from mitochondrial DNA sequences

In order to elucidate phylogenetic relationships and intraspecific variations and to infer the evolutionary process of loaches of the genus Lefua, we analyzed nucleotide sequences of the mitochondrial D-loop region of 100 specimens obtained from 97 localities in Japan and Korea. The genus Lefua includes three described species, L. nikkonis, L. echigonia, and L. costata and an undescribed species, Lefua sp. Our results showed that each species of Lefua formed a monophyletic group, indicating clearly that Lefua species can be genetically distinguished from one another. Lefua nikkonis was the most closely related to L. costata, while L. sp. was the most closely related to L. echigonia. Specimens of L. sp. were grouped into two intraspecific populations and specimens of L. echigonia were grouped into six populations. These populations were well separated geographically from one another by mountain ranges and highlands. We estimated the evolutionary time for splitting of the species and intraspecific populations, and speculated on the evolutionary process of the genus Lefua. Species of Lefua are severely threatened. Fundamental genetic information is indispensable for conservation. We presented genetic background in order to protect these threatened loaches.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

Arum- and Paris-type arbuscular mycorrhizas in a mixed pine forest on sand dune soil in Niigata Prefecture, central Honshu, Japan

Arbuscular mycorrhizas (AM) are the most widespread mycorrhiza in nature and form two morphologies, Arum- and Paris-type. The determining factors defining the two different morphologies are not well understood. In this study, the distribution of Arum- and Paris-type AM was determined in a mixed pine forest. A total of 35 plant species belonging to 20 families and 32 genera were identified and examined for AM colonization and morphological types. AM morphological types in 14 families were confirmed as follows: Arum-type in Rosaceae, Oleaceae, Lauraceae, Vitaceae and Compositae, Paris-type in Aquifoliaceae, Ulmaceae, Araliaceae, Theaceae, Magnoliaceae, Rubiaceae and Dioscoraceae, and both and/or intermediate types in Caprifoliaceae and Gramineae. Plant families whose AM morphological status was previously unknown were clarified as follows: Polygonaceae and Commelinaceae showed Arum-type morphology; Celastraceae, Menispermaceae and Elaeagnaceae had typical Paris-type morphology. The proportion of Arum-type to Paris-type species decreased in the following order: annuals > perennials > deciduous species > evergreen species, and pioneer group > early successional group > late successional group. Evergreen plants had a higher tendency to form Paris-type AM than annuals, perennials and deciduous plants. The results indicate that environmental changes, such as shade during plant succession, control the distribution of plant growth forms in mixed pine forest and may also play a part in the distribution of Arum- and Paris-type morphology. The identity of the plant seems to strongly influence AM morphology, though control by the fungal genome cannot be ruled out.     

Transforming growth factor-beta3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during CL/Fr mouse lip fusion

BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. CONCLUSIONS: TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.     

De novo formation of left-right asymmetry by posterior tilt of nodal cilia

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left–right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.