Possible involvement of Hcn1 ion channel in learning and memory dysfunction in SAMP8 mice

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1). Whole cell patch clamp analysis showed that Hcn1 ion conductivity was significantly lower in SAMP8 compared to that of JF1, which may be associated with learning and memory deficiency.     

Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy

Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.     

Settlement of Planulae of the Moon Jellyfish Aurelia aurita onto Hydrophilic Polycarbonate Plates Modified by Atmospheric Plasma Treatment

It has been reported that planula larvae of some jellyfish prefer artificial substrates for settlement. This research focused on the relationship between the settlement of planulae and the wettability of artificial substrate surfaces. We used atmospheric plasmas to change the wettability of the surfaces of polycarbonate (PC) plates because plasma treatment has no chemical side effects. The treatment made the surfaces hydrophilic, as evidenced by the decrease of contact angle from 85° to 35°. X-ray photoelectron spectroscopy revealed that the change of wettability of the PC plates could be attributed to N₂, which was probably ionized in the air above the plates. Scanning electron microscopy revealed no difference in the surface morphology of the plates before and after plasma treatment. Results of bioassays using treated PC plates showed that planulae tended to preferentially settle on hydrophobic surfaces.     

REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

Fusion of bone marrow-derived cells with renal tubules contributes to renal dysfunction in diabetic nephropathy

Although diabetic nephropathy (DN) is a major cause of end-stage renal disease, the mechanism of dysfunction has not yet been clarified. We previously reported that in diabetes proinsulin-producing bone marrow-derived cells (BMDCs) fuse with hepatocytes and neurons. Fusion cells are polyploidy and produce tumor necrosis factor (TNF)-α, ultimately causing diabetic complications. In this study, we assessed whether the same mechanism is involved in DN. We performed bone marrow transplantation from male GFP-Tg mice to female C57BL/6J mice and produced diabetes by streptozotocin (STZ) or a high-fat diet. In diabetic kidneys, massive infiltration of BMDCs and tubulointerstitial injury were prominent. BMDCs and damaged tubular epithelial cells were positively stained with proinsulin and TNF-α. Cell fusion between BMDCs and renal tubules was confirmed by the presence of Y chromosome. Of tubular epithelial cells, 15.4% contain Y chromosomes in STZ-diabetic mice, 8.6% in HFD-diabetic mice, but only 1.5% in nondiabetic mice. Fusion cells primarily expressed TNF-α and caspase-3 in diabetic kidney. These in vivo findings were confirmed by in vitro coculture experiments between isolated renal tubular cells and BMDCs. It was concluded that cell fusion between BMDCs and renal tubular epithelial cells plays a crucial role in DN.     

Cyclooxygenase-2/prostaglandin E2 accelerates the healing of gastric ulcers via EP4 receptors

We examined the involvement of cyclooxygenase (COX)-1 as well as COX-2 in the healing of gastric ulcers and investigated which prostaglandin (PG) EP receptor subtype is responsible for the healing-promoting action of PGE2. Male SD rats and C57BL/6 mice, including wild-type, COX-1(-/-), and COX-2(-/-), were used. Gastric ulcers were produced by thermocauterization under ether anesthesia. Gastric ulcer healing was significantly delayed in both rats and mice by indomethacin and rofecoxib but not SC-560 given for 14 days after ulceration. The impaired healing was also observed in COX-2(-/-) but not COX-1(-/-) mice. Mucosal PGE2 content increased after ulceration, and this response was significantly suppressed by indomethacin and rofecoxib but not SC-560. The delayed healing in mice caused by indomethacin was significantly reversed by the coadministration of 11-deoxy-PGE1 (EP3/EP4 agonist) but not other prostanoids, including the EP1, EP2, and EP3 agonists. By contrast, CJ-42794 (selective EP(4) antagonist) significantly delayed the ulcer healing in rats and mice. VEGF expression and angiogenesis were both upregulated in the ulcerated mucosa, and these responses were suppressed by indomethacin, rofocoxib, and CJ-42794. The expression of VEGF in primary rat gastric fibroblasts was increased by PGE2 or AE1-329 (EP4 agonist), and these responses were both attenuated by coadministration of CJ-42794. These results confirmed the importance of COX-2/PGE2 in the healing mechanism of gastric ulcers and further suggested that the healing-promoting action of PGE2 is mediated by the activation of EP4 receptors and is associated with VEGF expression.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.