Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.     

Artificial ground shelters for overwintering phytoseiid mites in orchards

Biological control in orchards strongly depends on winter survival of natural enemies, especially in temperate regions. Predacious phytoseiid mites overwinter on trees or on the ground depending on the characteristics of the species. However, the overwintering ecology of phytoseiid mites on the ground is less well known than that of those on trees. We investigated the usefulness of artificial overwintering shelters as a tool for studying the overwintering ecology of phytoseiid mites on the ground. Four kinds of artificial shelter (shading net, felt, cardboard, and urethane foam) were placed on the ground in an apple orchard in Korea. Two dominant phytoseiid species, Neoseiulus makuwa (Ehara) and N. womersleyi (Schicha) (Acari: Phytoseiidae), overwintered in the artificial ground shelters, and numbers were highest in the urethane foam among the four kinds of shelter, and next highest in the shading net. On the other hand, the numbers of phytoseiid mites collected in the ground vegetation plus soil samples under the ground shelters were not significantly different among the five shelter treatments, including the no-shelter control. Our results suggest that artificial overwintering shelters are efficient tools for investigating overwintering ecology of phytoseiid mites on the ground, as well as on trees, in orchards. Furthermore, the artificial shelters would be good sampling units because they are easily formed into identical sizes and can be used almost anywhere in the field with less laborious work. We also discuss some implications about the effects of sheltered structures on the ground on the populations of phytoseiid mites during winter.     

A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.     

Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4 + concentrations in hydroponic conditions

Root system development is an important target for improving yield in cereal crops. Active root systems that can take up nutrients more efficiently are essential for enhancing grain yield. In this study, we attempted to identify quantitative trait loci (QTL) involved in root system development by measuring root length of rice seedlings grown in hydroponic culture. Reliable growth conditions for estimating the root length were first established to renew nutrient solutions daily and supply NH₄ ⁺ as a single nitrogen source. Thirty-eight chromosome segment substitution lines derived from a cross between ‘Koshihikari’, a japonica variety, and ‘Kasalath’, an indica variety, were used to detect QTL for seminal root length of seedlings grown in 5 or 500 μM NH₄ ⁺. Eight chromosomal regions were found to be involved in root elongation. Among them, the most effective QTL was detected on a ‘Kasalath’ segment of SL-218, which was localized to the long-arm of chromosome 6. The ‘Kasalath’ allele at this QTL, qRL6.1, greatly promoted root elongation under all NH₄ ⁺ concentrations tested. The genetic effect of this QTL was confirmed by analysis of the near-isogenic line (NIL) qRL6.1. The seminal root length of the NIL was 13.5–21.1% longer than that of ‘Koshihikari’ under different NH₄ ⁺ concentrations. Toward our goal of applying qRL6.1 in a molecular breeding program to enhance rice yield, a candidate genomic region of qRL6.1 was delimited within a 337 kb region in the ‘Nipponbare’ genome by means of progeny testing of F₂ plants/F₃ lines derived from a cross between SL-218 and ‘Koshihikari’.     

Purification and characterization of fibrinolytic alkaline protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. BLB

Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH₄)₂SO₄ and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus.     

Dual effect of AMD3100, a CXCR4 antagonist, on bleomycin-induced lung inflammation

The chemokine receptor CXCR4, which binds the chemokine stromal cell-derived factor 1, has been reported to be involved in the chemotaxis of inflammatory cells. In addition, AMD3100, an antagonist of CXCR4, has been reported to be an attractive drug candidate for therapeutic intervention in several disorders in which CXCR4 is critically involved. However, little is known about the therapeutic value of AMD3100 in the treatment of pulmonary fibrosis. In this study, we examined the effects of AMD3100 on a murine bleomycin-induced pulmonary fibrosis model. Concurrent administration of AMD3100 and bleomycin apparently attenuated bleomycin-induced pulmonary inflammation. In this process, an inhibition of neutrophil recruitment at early stage followed by the decrease of other inflammatory cell recruitment in the lung were observed. In addition, it also inhibited the expression of cytokines, including MCP-1, MIP-2, MIP-1alpha, and TGF-beta. In contrast, when AMD3100 was administered following bleomycin treatment, the bleomycin-induced lung inflammation progressed and resulted in severe pulmonary fibrosis. In this process, an increase of inflammatory cell recruitment, an up-regulation of lung MCP-1 and TGF-beta, and a remarkable activation of p44/42 MAPK in neutrophils were observed. U0126, an inhibitor of p44/42 MAPK, significantly abolished these effects. Thus, AMD3100 has dual effect on bleomycin-induced pulmonary fibrosis. Difference of inflammatory cell recruitment and activation might be associated with the dual effect of AMD3100 on bleomycin-induced pulmonary fibrosis.     

A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome)

Early infantile epileptic encephalopathy with suppression-burst pattern (EIEE) is one of the most severe and earliest forms of epilepsy, often evolving into West syndrome; however, the pathogenesis of EIEE remains unclear. ARX is a crucial gene for the development of interneurons in the fetal brain, and a polyalanine expansion mutation of ARX causes mental retardation and seizures, including those of West syndrome, in males. We screened the ARX mutation and found a hemizygous, de novo, 33-bp duplication in exon 2, 298_330dupGCGGCA(GCG)9, in two of three unrelated male patients with EIEE. This mutation is thought to expand the original 16 alanine residues to 27 alanine residues (A110_A111insAAAAAAAAAAA) in the first polyalanine tract of the ARX protein. Although EIEE is mainly associated with brain malformations, ARX is the first gene found to be responsible for idiopathic EIEE. Our observation that EIEE had a longer expansion of the polyalanine tract than is seen in West syndrome is consistent with the findings of earlier onset and more-severe phenotypes in EIEE than in West syndrome.     

Single molecule FRET for the study on structural dynamics of biomolecules

Single molecule fluorescence resonance energy transfer (FRET) is the technique that has been developed by combining FRET measurement and single molecule fluorescence imaging. This technique allows us to measure the dynamic changes of the interaction and structures of biomolecules. In this study, the validity of the method was tested using fluorescence dyes on double stranded DNA molecules as a rigid spacer. FRET signals from double stranded DNA molecules were stable and their average FRET values provided the distance between the donor and acceptor in agreement with B-DNA type helix model. Next, the single molecule FRET method was applied to the studies on the dynamic structure of Ras, a signaling protein. The data showed that Ras has multiple conformational states and undergoes transition between them. This study on the dynamic conformation of Ras provided a clue for understanding the molecular mechanism of cell signaling switches.     

Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.