Recombinant lectin Gg for brain imaging of glycan structure and formation in the CNS node of Ranvier.

The nodes of Ranvier are unmyelinated gaps in the axon, important for the efficient transmission of action potentials. Despite the identification of several glycoproteins involved in node formation and maintenance, glycans' structure and formation in the node remain unclear. Previously, we developed a recombinant lectin from the Clostridium botulinum neurotoxin complex, specific to the galactose and N-acetylgalactosamine terminal epitopes (Gg). Gg stained Neuro2a cells. Here, we show Gg punctuate staining in mouse brain cryosections. Thus, we hypothesized that Gg could help study glycans in the node of Ranvier. Lectin histochemistry on mouse brain cryosections confirmed that Gg binds specifically to the node of Ranvier in the central nervous system (CNS). Using a combination of lectin blotting, glycosidase treatment on tissue sections, and lectin histochemistry, Gg ligands were identified as α-galactose terminal glycoproteins in the perinodal extracellular matrix. Furthermore, we detected the spatiotemporal distribution of galactosylated glycans in the CNS node of Ranvier in mouse brain tissues at different postnatal times. Finally, we observed impaired clustering of galactosylated glycans in the nodes during demyelination and remyelination in cuprizone-induced demyelination and remyelination mouse model. In conclusion, Gg can serve as a novel brain imaging tool in glycobiology and report glycoprotein formation and alterations in the CNS node of Ranvier. Our findings might serve as a first step to establish the role of glycans in the node of Ranvier.

Glycan detecting tools developed from the Clostridium botulinum whole hemagglutinin complex.

Lectins are proteins with the ability to recognize and bind to specific glycan structures. These molecules play important roles in many biological systems and are actively being studied because of their ability to detect glycan biomarkers for many diseases. Hemagglutinin (HA) proteins from Clostridium botulinum type C neurotoxin complex; HA1, HA2, and HA3 are lectins that aid in the internalization of the toxin complex by binding to glycoproteins on the cell surface. HA1 mutants have been previously reported, namely HA1 W176A/D271F and HA1 N278A/Q279A which are specific to galactose (Gal)/N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) sugars, respectively. In this study, we utilized HA1 mutants and expressed them in complex with HA2 WT and HA3 WT to produce glycan detecting tools with high binding affinity. Particularly, two types were made: Gg and Rn. Gg is an Alexa 488 conjugated lectin complex specific to Gal and GalNAc, while Rn is an Alexa 594 conjugated lectin complex specific to Neu5Ac. The specificities of these lectins were identified using a glycan microarray followed by competitive sugar inhibition experiments on cells. In addition, we confirmed that Gg and Rn staining is clearly different depending on cell type, and the staining pattern of these lectins reflects the glycans present on the cell surface as shown in enzyme treatment experiments. The availability of Gg and Rn provide us with new promising tools to study Gal, GalNAc, and Neu5Ac terminal epitopes which can aid in understanding the functional role of glycans in physiological and pathological events.

Two cases of prostheses for flap protection after buccal mucosal incision.

Here we describe two patients in whom prostheses were applied for flap protection after buccal mucosal incision. In the first case, the patient was a 65-year-old man with a diagnosis of buccal mucosa squamous cell carcinoma (T2N0M0). Left buccal mucosa squamous cell tumor resection and dermoplasty were performed, followed by alveolar ridge augmentation and buccal mucosal graft in the scar area. The carcinoma recurred, however, and left buccal mucosa carcinoma resection was performed, followed by reconstruction surgery using a free forearm flap. After a 12-week healing period, a molar support was constructed on the unaffected side and a protective prosthesis placed on the affected side. Training in ingestion and swallowing were given postoperatively. The patient in the second case was a 62-year-old woman with a diagnosis of buccal mucosa squamous cell carcinoma (T2N1M0). Right buccal mucosa carcinoma resection and supraomohyoid neck dissection were performed, followed by reconstruction surgery using a free forearm flap. A molar support was constructed on the unaffected side and a protective prosthesis placed on the affected side at 5 months postoperatively. Training was given in ingestion and swallowing postoperatively. The prostheses prevented bite wounds to the flaps in the affected areas due to jaw movement during swallowing or speaking. The postoperative courses were uneventful, and the average masticatory score was 92.5 (85, 100), not affecting daily life. The prostheses were placed after reconstruction surgery using free flaps after buccal mucosa squamous cell carcinoma resection. Dysphasia recovered to the preoperative level by dysphasia and pronunciation training in both cases. The postoperative prognosis was favorable, with the prosthesis preventing damage to the flap.

Immunohistochemical detection of nestin in the periodontal Ruffini endings of the rat incisor.

Nestin is an intermediate filament which was first identified in neuroepithelial stem cells. This expression has also been reported in restricted locations in adults. Previous studies have suggested that the periodontal Ruffini endings remain immature in nature even in adulthood. The present study reports on a characteristic expression of immunoreaction for nestin in the periodontal Ruffini endings during postnatal development. RT-PCR analysis detected nestin mRNA in a reverse transcripted cDNA sample from both the rat trigeminal ganglion and periodontal ligament. The nestin immunoreaction existed in the periodontal ligament at postnatal day 3 (PO 3 days), when many spindle-shaped Schwann cells were positive for nestin immunoreaction. At PO 1 week, when periodontal nerve fibers displayed a dendritic fashion, the round cells came to show the nestin immunoreaction. These immunopositive cells were also reactive for S-100 protein and non-specific cholinesterase, indicating that these cells could be categorized as terminal Schwann cells associated with the periodontal Ruffini endings. Some ordinary Schwann cells also exhibited nestin immunoreaction. From PO 2 to 3 weeks, nestin positive terminal Schwann cells increased in number in accordance with the postnatal development of the periodontal Ruffini endings, while this immuno-expression pattern remained unchanged. Nestin immunoreaction was also recognizable in the satellite cells - but never in the neurons - in the trigeminal ganglion throughout this observation period. This immuno-expression pattern suggests that nestin serves as an intermediate filament for mechanical stability in the periodontal Ruffini endings against external stimuli.