Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion

Autophagy involves de novo formation of double membrane-bound structures called autophagosomes, which engulf material to be degraded in lytic compartments. Atg8 is a ubiquitin-like protein required for this process in Saccharomyces cerevisiae that can be conjugated to the lipid phosphatidylethanolamine by a ubiquitin-like system. Here, we show using an in vitro system that Atg8 mediates the tethering and hemifusion of membranes, which are evoked by the lipidation of the protein and reversibly modulated by the deconjugation enzyme Atg4. Mutational analyses suggest that membrane tethering and hemifusion observed in vitro represent an authentic function of Atg8 in autophagosome formation in vivo. In addition, electron microscopic analyses indicate that these functions of Atg8 are involved in the expansion of autophagosomal membranes. Our results provide further insights into the mechanisms underlying the unique membrane dynamics of autophagy and also indicate the functional versatility of ubiquitin-like proteins.     

Bezafibrate regulates the expression and enzyme activity of 11beta-hydroxysteroid dehydrogenase type 1 in murine adipose tissue and 3T3-L1 adipocytes

A clinically employed antihyperlipidemic drug, bezafibrate, has been characterized as a PPAR(alpha, -gamma, and -delta) pan-agonist in vitro. Recent extended trials have highlighted its antidiabetic properties in humans. However, the underlying molecular mechanism is not fully elucidated. The present study was designed to explore potential regulatory mechanisms of intracellular glucocorticoid reactivating enzyme, 11beta-HSD1 and anti-diabetic hormone, adiponectin by bezafibrate in murine adipose tissue, and cultured adipocytes. Treatment of db/db mice with bezafibrate significantly ameliorated hyperglycemia and insulin resistance, accompanied by a marked reduction of triglyceride and nonesterified fatty acids. Despite equipotent in lipid-lowering effects, another fibrate, fenofibrate, did not show such beneficial effects on glycemic control. Treatment of bezafibrate caused a marked decrease in the mRNA level of 11beta-HSD1 preferentially in adipose tissue of db/db mice (-47%, P<0.05), concomitant with a significant increase in plasma adiponectin level (+37%, P<0.01). Notably, treatment of bezafibrate caused a marked decrease in the mRNA level (-34%, P<0.01) and enzyme activity (-32%, P<0.01) of 11beta-HSD1, whereas the treatment substantially augmented the expression (+71%, P<0.01) and secretion (+27%, P<0.01) of adiponectin in 3T3-L1 adipocytes. Knockdown of 11beta-HSD1 by siRNA confirmed that 11beta-HSD1 acts as a distinct oxoreductase in adipocytes and validated the enzyme activity assays in the present study. Effects of bezafibrate on regulation of 11beta-HSD1 and adiponectin in murine adipocytes were comparable with those in thiazolidinediones. This is the first demonstration that bezafibrate directly regulates 11beta-HSD1 and adiponectin in murine adipocytes, both of which may contribute to metabolically-beneficial effects by bezafibrate.     

Genomic regions involved in yield potential detected by genome-wide association analysis in Japanese high-yielding rice cultivars

Background

High-yielding cultivars of rice (Oryza sativa L.) have been developed in Japan from crosses between overseas indica and domestic japonica cultivars. Recently, next-generation sequencing technology and high-throughput genotyping systems have shown many single-nucleotide polymorphisms (SNPs) that are proving useful for detailed analysis of genome composition. These SNPs can be used in genome-wide association studies to detect candidate genome regions associated with economically important traits. In this study, we used a custom SNP set to identify introgressed chromosomal regions in a set of high-yielding Japanese rice cultivars, and we performed an association study to identify genome regions associated with yield.

Results

An informative set of 1152 SNPs was established by screening 14 high-yielding or primary ancestral cultivars for 5760 validated SNPs. Analysis of the population structure of high-yielding cultivars showed three genome types: japonica-type, indica-type and a mixture of the two. SNP allele frequencies showed several regions derived predominantly from one of the two parental genome types. Distinct regions skewed for the presence of parental alleles were observed on chromosomes 1, 2, 7, 8, 11 and 12 (indica) and on chromosomes 1, 2 and 6 (japonica). A possible relationship between these introgressed regions and six yield traits (blast susceptibility, heading date, length of unhusked seeds, number of panicles, surface area of unhusked seeds and 1000-grain weight) was detected in eight genome regions dominated by alleles of one parental origin. Two of these regions were near Ghd7, a heading date locus, and Pi-ta, a blast resistance locus. The allele types (i.e., japonica or indica) of significant SNPs coincided with those previously reported for candidate genes Ghd7 and Pi-ta.

Conclusions

Introgression breeding is an established strategy for the accumulation of QTLs and genes controlling high yield. Our custom SNP set is an effective tool for the identification of introgressed genome regions from a particular genetic background. This study demonstrates that changes in genome structure occurred during artificial selection for high yield, and provides information on several genomic regions associated with yield performance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-346) contains supplementary material, which is available to authorized users.     

Disturbance by large herbivores alters the relative importance of the ecological processes that influence the assembly pattern in heterogeneous meta‐communities

Disturbance caused by large herbivores can affect the relative importance of ecological processes in determining community assembly and may cause a systematic loss of biodiversity across scales. To examine changes in the community assembly pattern caused by an overabundance of large herbivores in Japan, we analyzed community composition data from before and after the overabundance occurred. The community assembly pattern becomes more random after the deer overabundance. In addition, result of variation partitioning revealed decrease in importance of environmental processes and increase in importance of spatial processes. However, response of turnover rate, niche breadth, and niche overlap was heterogeneous, according to scale of each environmental gradient. Our results emphasize the importance of conserving habitat specialists that represent the local environment (habitat type and topography) at various altitudinal ranges to maintain biodiversity at regional scales under the increasing pressure of large herbivores.     

Aqueous microwave-assisted solid-phase peptide synthesis using Fmoc strategy. III: Racemization studies and water-based synthesis of histidine-containing peptides

In this study, we describe the first aqueous microwave-assisted synthesis of histidine-containing peptides in high purity and with low racemization. We have previously shown the effectiveness of our synthesis methodology for peptides including difficult sequences using water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles. It is an organic solvent-free, environmentally friendly method for chemical peptide synthesis. Here, we studied the racemization of histidine during an aqueous-based coupling reaction with microwave irradiation. Under our microwave-assisted protocol using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, the coupling reaction can be efficiently performed with low levels of racemization of histidine. Application of this water-based microwave-assisted protocol with water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles led to the successful synthesis of the histidine-containing hexapeptide neuropeptide W-30 (10–15), Tyr-His-Thr-Val-Gly-Arg-NH₂, in high yield and with greatly reduced histidine racemization.     

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.     

EFA6A,a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin‐1 and regulates neurite outgrowth

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6‐specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin‐1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two‐hybrid screening. The interaction was mediated by the C‐terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull‐down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post‐synaptic density and endosomes in dendritic spines. In Neuro‐2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co‐expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6‐mediated neurite formation through the interaction with SNX1.

     

A novel murine gene, Sickle tail, linked to the Danforth's short tail locus, is required for normal development of the intervertebral disc

We established the mutant mouse line, B6;CB-SktGtAyu8021IMEG (SktGt), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called Sickle tail (Skt), is composed of 19 exons and encodes a protein of 1352 amino acids. Expression of a reporter gene was detected in the notochord during embryogenesis and in the nucleus pulposus of mice. Compression of some of the nuclei pulposi in the intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and annulus fibrosus of IVDs in SktGt/Gt mice. These phenotypes were different from those in Danforth's short tail (Sd) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to those seen in the annulus fibrosus in all IVDs. The Skt gene maps to the proximal part of mouse chromosome 2, near the Sd locus. The genetic distance between them was 0.95 cM. The number of vertebrae in both [Sd +/+ SktGt] and [Sd SktGt/+ +] compound heterozygotes was less than that of Sd heterozygotes. Furthermore, the enhancer trap locus Etl4lacZ, which was previously reported to be an allele of Sd, was located in the third intron of the Skt gene.