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INTRODUCTION Repulsive guidance molecule a (RGMa) is a key protein that negatively regulates neuronal regeneration as its inhibition enhances axonal growth and promotes functional recovery in animal models of spinal cord injury. However, the role of RGMa in traumatic brain injury (TBI) remains elusive. This study aimed to clarify TBI-responsive RGMa expression in a murine model. METHODS Adult male C57BL/6J mice were subjected to controlled cortical impact. Brains were extracted 6 hours and 1, 3, 7, 14 and 21 days after injury (n = 6 in each group). Changes in the messenger RNA (mRNA) expression of RGMa and its receptor, neogenin, were evaluated by quantitative polymerase chain reaction in the damaged area of the cortex and contralateral cortex, along with expression measurement of inflammation-related molecules. Neurological deficit was also assessed by the cylinder test. RESULTS Neurological score was consistently lower in the TBI group compared to the sham group throughout the experimental period. The mRNA expressions of representative inflammatory cytokine TNF-α and chemokine receptor CCR2 were remarkably increased in the injured cortex on day 1 and gradually decreased over time, although remaining at higher values at least until day 14. The mRNA expressions of RGMa and neogenin were significantly suppressed in the damaged cortex until day 3. Interestingly, RGMa expression was suppressed most on day 1 and recovered over time. CONCLUSION In the acute phase of TBI, gene expression of inflammatory cytokines significantly increased, and gene expressions of RGMa and neogenin significantly decreased in the inflammatory milieu of the damaged area. Despite the subsequent remission of inflammation, RGMa gene expression recovered to the normal level 1 week after TBI. Intrinsic regenerative response to acute brain injury might be hampered by the following recovery of RGMa expression, hinting at the possibility of functional RGMa inhibition as a new, effective maneuver against TBI.

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA(+)), ClpB forms a hexameric ring structure, with each protomer containing two AAA(+) modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401–Arg-532 and the inter-subunit pair Asp-404–Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401–Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180–Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large-scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli-based cell-free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation-prone fractions. We translated ∼90 E. coli proteins in the protease-inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large-scale analysis.

要旨 魚骨異物は救急外来で多く遭遇するが，気道緊急を呈することは稀である。今回気道緊急を呈した魚骨異物に対して，1度除去したにも関わらず，残存していた声門下魚骨異物により再度気道緊急を呈した症例を経験した。1歳8ヶ月の男児が夕食後に喘鳴を認め，声門間隙による魚骨異物と診断し全身麻酔下でこれを除去した。除去後に咽喉頭ファイバースコープで確認した際には異物残存を認めなかった。摘出後翌日に退院となったが，退院6日目より再度喘鳴および流涎を認め，退院7日目に再受診した。再度，咽喉頭ファイバースコープを施行したところ，声門下に魚骨異物を認めたため，気管切開後に摘出を行った。声門下異物は稀であるが，気道緊急を呈する。咽喉頭ファイバースコープでは十分に確認できないことがあり，確認のためには気管支ファイバースコープによる観察も考慮する必要がある。 ABSTRACT Fish bone foreign bodies are often seen in the emergency department, but rarely cause airway obstruction. We describe the case of laryngeal fish bone foreign bodies that caused airway obstruction twice. A 20–month–old boy presented stridor after dinner, he was diagnosed with a fish bone foreign body in the glottis by laryngoscopy. We removed it under general anesthesia. After the removal, we confirmed the absence of foreign bodies using a laryngoscope. He was discharged the day after removal, but 6 days after discharge, he presented stridor and salivation appeared again. We performed laryngoscopy and found a subglottic foreign body. We performed tracheotomy and removed the foreign body. Subglottic foreign bodies are rare, but can cause airway obstruction. Laryngoscopy is insufficient to confirm the absence of subglottic foreign bodies, so we should consider bronchoscopy for this purpose.