Lys-63-linked Ubiquitination by E3 Ubiquitin Ligase Nedd4-1 Facilitates Endosomal Sequestration of Internalized α-Synuclein

α-Synuclein (aS) is a major constituent of Lewy bodies, which are not only a pathological marker for Parkinson disease but also a trigger for neurodegeneration. Cumulative evidence suggests that aS spreads from cell to cell and thereby propagates neurodegeneration to neighboring cells. Recently, Nedd4-1 (neural precursor cell expressed developmentally down-regulated protein 4-1), an E3 ubiquitin ligase, was shown to catalyze the Lys-63-linked polyubiquitination of intracellular aS and thereby facilitate aS degradation by the endolysosomal pathway. Because Nedd4-1 exerts its activity in close proximity to the inner leaflet of the plasma membrane, we speculate that after the internalization of aS the membrane resident aS is preferentially ubiquitinated by Nedd4-1. To clarify the role of Nedd4-1 in aS internalization and endolysosomal sequestration, we generated aS mutants, including ΔPR1(1–119 and 129–140), ΔC(1–119), and ΔPR2(1–119 and 134–140), that lack the proline-rich sequence, a putative Nedd4-1 recognition site. We show that wild type aS, but not ΔPR1, ΔPR2, or ΔC aS, is modified by Nedd4-1 in vitro, acquiring a Lys-63-linked ubiquitin chain. Compared with the mutants lacking the proline-rich sequence, wild type-aS is preferentially internalized and translocated to endosomes. The overexpression of Nedd4-1 increased aS in endosomes, whereas RNAi-mediated silencing of Nedd4-1 decreased endosomal aS. Although aS freely passes through plasma membranes within minutes, a pulse-chase experiment revealed that the overexpression of Nedd4-1 markedly decreased the re-secretion of internalized aS. Together, these findings demonstrate that Nedd4-1-linked Lys-63 ubiquitination specifies the fate of extrinsic and de novo synthesized aS by facilitating their targeting to endosomes.     

Newly identified type of beta actin reduces invasiveness of mouse B16-melanoma

Low metastatic parent B16 melanoma and isolated B16-F1 cell lines have a third actin designated as beta m(Ax:previously). beta m actin is scantily or not at all detected in highly metastatic cell lines, such as B16-F10 and BL6. To directly assess the physiological role of beta m in phenotypic changes of B16 melanoma, we transfected expression plasmids of beta m into B16-F10 cells. The actin expressed in the transfectants is located largely in cytoskeletal fractions. The transfectants exhibited a larger number of stress fibers and a lower invasiveness than did the recipient cells. Thus, beta m actin plays an important role in the organization of actin stress fibers, the result being a decrease in invasiveness of B16 melanoma.     

REVISION OF THE MASTOPHOROIDEAE (CORALLINALES,RHODOPHYTA) AND POLYPHYLY IN NONGENICULATE SPECIES WIDELY DISTRIBUTED ON PACIFIC CORAL REEFS1

The subfamily Mastophoroideae (Corallinaceae, Rhodophyta) is characterized by species possessing nongeniculate, uniporate tetrasporangial conceptacles without apical plugs, the presence of cell fusions, and the absence of secondary pit connections. However, molecular phylogenetic studies not including the type genus Mastophora indicated that the Mastophoroideae was polyphyletic. Our molecular phylogenetic analysis of the subfamily including the type genus using DNA sequences of SSU rDNA and plastid‐encoded gene of PSII reaction center protein D1 (psbA) revealed that Mastophora formed a robust clade only with Metamastophora. The other mastophoroid genera were divided into six lineages within the family Corallinaceae. Five supported lineages—(i) Pneophyllum; (ii) Hydrolithon gardineri (Foslie) Verheij et Prud’homme, Hydrolithon onkodes (Heydr.) Penrose et Woelk., and Hydrolithon pachydermum (Foslie) J. C. Bailey, J. E. Gabel et Freshwater; (iii) Hydrolithon reinboldii (Weber Bosse et Foslie) Foslie; (iv) Spongites; and (v) Neogoniolithon—were clearly distinguished by the combination of characters including the presence or absence of palisade cells and trichocytes in large, tightly packed horizontal fields and features of tetrasporangial and spermatangial conceptacles. Therefore, we amend the Mastophoroideae to be limited to Mastophora and Metamastophora with a thin thallus with basal filaments comprised of palisade cells, tetrasporangial conceptacles formed by filaments peripheral to fertile areas, and spermatangia derived only from the floor of male conceptacles. This emendation supports Setchell’s (1943) original definition of the Mastophoroideae as having thin thalli. We also propose the establishment of three new subfamilies, Hydrolithoideae subfam. nov. including Hydrolithon, Porolithoideae subfam. nov. including the resurrected genus Porolithon, and Neogoniolithoideae subfam. nov. including Neogoniolithon. Taxonomic revisions of Pneophyllum and Spongites were not made because we did not examine their type species.     

Vaccination with CD133+ melanoma induces specific Th17 and Th1 cell�Cmediated antitumor reactivity against parental tumor

Accumulating evidence suggests that cancer cells possess a small subpopulation that survives during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular-targeting therapy. CD133 is a putative marker that distinguishes a minor subpopulation from normal differentiated tumor cells in many cancers. Although it is necessary to eradicate all cancer cells to obtain a cure, effective treatment to eliminate the CD133(+) treatment-tolerant cells has not been elucidated. In this study, we demonstrated that a CD133(+) subpopulation in murine melanoma is immunogenic and that effector T cells specific for the CD133(+) melanoma cells mediated potent antitumor reactivity, curing the mice of the parental melanoma. CD133(+) melanoma antigens preferentially induced type 17 T helper (Th17) cells and Th1 cells but not Th2 cells. CD133(+) melanoma cell-specific CD4(+) T-cell treatment eradicated not only CD133(+) tumor cells but also CD133(-) tumor cells while inducing long-lasting accumulation of lymphocytes and dendritic cells with upregulated MHC class II in tumor tissues. Further, the treatment prevented regulatory T-cell induction. These results indicate that T-cell immunotherapy is a promising treatment option to eradicate CD133(+) drug-tolerant cells to obtain a cure for cancer.     

Asymmetric synthesis and sensory evaluation of sedanenolide

The synthesis and sensory evaluation of enantiomeric sets of sedanenolide (1) and 3-butylphthalide (3) are described. The asymmetric synthesis was achieved via the intramolecular Diels-Alder reaction of chiral propargylester (5) which was prepared from optically active propargyl alcohol (4) and 2,4-pentadienoic acid. The sensory evaluation of these enantiomers revealed that there were distinct differences between their aroma character and odor threshold.     

Assessment of willow (Salix sp.) as a woody heavy metal accumulator: field survey and in vivo X-ray analyses

Trees that accumulate metals are important plants for restoring contaminated soil because of their high biomass. In our previous study, we discovered that Salix miyabeana has the capability to take up high levels of Cd, and identified the several accumulation sites of the endogenous metals in the leaf parts of plants. To analyze the detailed localization of Cd in apoplastic and symplastic compartments in S. miyabeana, synchrotron radiation-based micro X-ray fluorescence (SR-μ-XRF) analysis and micro X-ray absorption near edge structure (μ-XANES) measurements were performed on beam line 37XU of the SPring-8 (Hyogo, Japan). The two-dimensional metal distribution of segments of young stems was obtained by μ-XRF with approximately 2 μm(2) X-ray beams and showed the predominant localization of Cd in the apoplastic region. μ-XANES analyses suggest that the apoplastic detoxification of Cd in willow depends on Cd-oxygen, but not on Cd-sulfur, interaction. S. miyabeana growing near an old mining site in Japan was then examined to evaluate the metal accumulating ability of this plant in the field. The metal concentration in the leaves of the plants was compared to that in the soil and enrichment factors (EFs) were calculated for Cu, Zn, Cd and Pb. Results showed efficient removal of Cd and Zn from the contaminated sites by the willow plants. In order to discuss the combined and long-term effect of multiple heavy metals on S. miyabeana grown in soil, variable-pressure scanning electron microscopy fitted with energy dispersive X-ray analysis (VPSEM-EDX) and SR-μ-XRF was used to characterize the serration of leaves. The combination of 2D elemental images revealed metal accumulation in the tip cells in serrations without any exudation of heavy metals from the hydathodes.     

B vitamins, methionine and alcohol intake and risk of colon cancer in relation to BRAF mutation and CpG island methylator phenotype (CIMP)

Background

One-carbon metabolism appears to play an important role in DNA methylation reaction. Evidence suggests that a low intake of B vitamins or high alcohol consumption increases colorectal cancer risk. How one-carbon nutrients affect the CpG island methylator phenotype (CIMP) or BRAF mutation status in colon cancer remains uncertain.

Methods

Utilizing incident colon cancers in a large prospective cohort of women (the Nurses'' Health Study), we determined BRAF status (N = 386) and CIMP status (N = 375) by 8 CIMP-specific markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and 8 other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT-1, MINT-31, p14, and WRN). We examined the relationship between intake of one-carbon nutrients and alcohol and colon cancer risk, by BRAF mutation or CIMP status.

Results

Higher folate intake was associated with a trend towards low risk of CIMP-low/0 tumors [total folate intake ≥400 µg/day vs. <200 µg/day; the multivariate relative risk = 0.73; 95% CI = 0.53–1.02], whereas total folate intake had no influence on CIMP-high tumor risks (P_{heterogeneity} = 0.73). Neither vitamin B₆, methionine or alcohol intake appeared to differentially influence risks for CIMP-high and CIMP-low/0 tumors. Using the 16-marker CIMP panel did not substantially alter our results. B vitamins, methionine or alcohol intake did not affect colon cancer risk differentially by BRAF status.

Conclusions

This molecular pathological epidemiology study suggests that low level intake of folate may be associated with an increased risk of CIMP-low/0 colon tumors, but not that of CIMP-high tumors. However, the difference between CIMP-high and CIMP-low/0 cancer risks was not statistically significant, and additional studies are necessary to confirm these observations.     

A macro-scale perspective on within-farm management: how climate and topography alter the effect of farming practices

Organic farming has the potential to reverse biodiversity loss in farmland and benefit agriculture by enhancing ecosystem services. Although the mixed success of organic farming in enhancing biodiversity has been attributed to differences in taxa and landscape context, no studies have focused on the effect of macro-scale factors such as climate and topography. This study provides the first assessment of the impact of macro-scale factors on the effectiveness of within-farm management on biodiversity, using spiders in Japan as an example. A multilevel modelling approach revealed that reducing pesticide applications increases spider abundance, particularly in areas with high precipitation, which were also associated with high potential spider abundance. Using the model we identified areas throughout Japan that can potentially benefit from organic farming. The alteration of local habitat-abundance relations by macro-scale factors could explain the reported low spatial generality in the effects of organic farming and patterns of habitat association.