Monitoring Autophagy Flux and Activity: Principles and Applications

Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi-step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome-dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been developed. In these assays, autophagosome marker proteins, microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) and gamma-aminobutyric acid receptor-associated protein-II (GABARAP-II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3-II and/or GABARAP-II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure.     

Chemical Characterization and Phytotoxic Effects of the Aerial Parts of Ruzigrass (Urochloa ruziziensis)

Studies of the phytotoxic effects between plants can be a crucial tool in the discovery of innovative compounds with herbicide potential. In this sense, we can highlight ruzigrass (Urochloa ruziziensis), which is traditionally used in the crop rotation system in order to reduce weed emergence. The aim of this work was to characterize the secondary metabolites of ruzigrass and to evaluate its phytotoxic effects. In total, eight compounds were isolated: friedelin, oleanolic acid, α‐amyrin, 1‐dehydrodiosgenone, sitosterol and stigmasterol glycosides, tricin and p‐coumaric acid. Phytotoxic effects of the crude methanolic extract and fractions of ruzigrass were assessed using germination rate, initial seedling growth, and biomass of Bidens pilosa, Euphorbia heterophylla and Ipomoea grandifolia. Chemometric analysis discriminated the weed species into three groups, and B. pilosa was the most affected by fractions of ruzigrass. The phytotoxic activities of 1‐dehydrodiosgenone, tricin, and p‐coumaric acid are also reported, and p‐coumaric acid and 1‐dehydrodiosgenone were active against B. pilosa.     

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus

ABSTRACT

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2–7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1–7) and the chemically prepared compounds (8–10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3–10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1–10 showed significant inhibition activity against Axl and/or immune checkpoint.     

Plant stem cell research is uncovering the secrets of longevity and persistent growth

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project ‘Principles of pluripotent stem cells underlying plant vitality’ was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.     

Kip-related protein 3 is required for control of endoreduplication in the shoot apical meristem and leaves of Arabidopsis

The cell cycle plays an important role in the development and adaptation of multicellular organisms; specifically, it allows them to optimally adjust their architecture in response to environmental changes. Kip-related proteins (KRPs) are important negative regulators of cyclin-dependent kinases (CDKs), which positively control the cell cycle during plant development. The Arabidopsis genome possesses seven KRP genes with low sequence similarity and distinct expression patterns; however, why Arabidopsis needs seven KRP genes and how these genes function in cell cycle regulation are unknown. Here, we focused on the characterization of KRP3, which was found to have unique functions in the shoot apical meristem (SAM) and leaves. KRP3 protein was localized to the SAM, including the ground meristem and vascular tissues in the ground part of the SAM and cotyledons. In addition, KRP3 protein was stabilized when treated with MG132, an inhibitor of the 26S proteasome, indicating that the protein may be regulated by 26S proteasome-mediated protein degradation. KRP3-overexpressing (KRP3 OE) transgenic plants showed reduced organ size, serrated leaves, and reduced fertility. Interestingly, the KRP3 OE transgenic plants showed a significant reduction in the size of the SAM with alterations in cell arrangement. In addition, compared to the wild type, the KRP3 OE transgenic plants had a higher DNA ploidy level in the SAM and leaves. Taken together, our data suggest that KRP3 plays important regulatory roles in the cell cycle and endoreduplication in the SAM and leaves.     

The biomechanical effect of the collar of a femoral stem on total hip arthroplasty

To investigate the biomechanical effect of collars, finite element analyses are carried out through two hip joints that are implanted using collared and collarless stems, respectively, and an intact hip joint model. For the analyses, the sacrum, coxal bone, and the cancellous and cortical bones of a femur are modelled using finite elements based on X-ray computed tomographic images taken from a 27-year-old woman. From the results, it is found that a collar with perfect calcar contact prevents stem subsidence and decreases the proximal–lateral gap and the lateral stem tilting. Therefore, it can impart reasonable biomechanical stability for total hip arthroplasty. However, its low load transmission ability and increased stem tilting effect due to the imperfect contact between the collar and the calcar are found to be serious problems that need to be solved. Results of clinical follow-up are presented for supporting the computational results.     

Production of Hydroxyl Radicals and α-dicarbonyl Compounds Associated with Amadori Compound-Cu 2+ Complex Degradation

The chemical properties of Amadori compounds in the presence of transition metal ions were studied, using the analogs 1-deoxy-1- n -butylamino- d -fructose (DBF) and N &#102 -formyl-fructoselysine (fFL). The following characteristics were revealed: (a) DBF combined easily with Cu 2+ (but no other transition metal ions) to form a DBF-Cu 2+ complex in phosphate buffer, pH 7.4; (b) the complex was unstable, and degraded with the release of Cu + during incubation at 37°C; (c) degradation of the complex was associated with the production of hydroxyl radicals by the Fenton reaction and &#102 -dicarbonyl compounds by non-autoxidative degradation; and (d) properties of DBF were similar to those of fFL. The above properties were additionally observed in glycated poly-Lys (GPL). Our findings indicate a novel mechanism for the generation of hydroxyl radicals and &#102 -dicarbonyl compounds from Amadori adducts in the presence of Cu 2+ .     

No ATG is an island—the connection of autophagy with diverse pathways and functions

The sixth International Symposium on Autophagy took place in October 2012 in Okinawa, Japan. It brought together scientists from all over the world to cultivate a better understanding of cutting‐edge autophagy research from molecular mechanisms to disease states.