Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling.

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11-12 (E11-12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12-13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140-144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

[Improvement of Nitrite Analysis Method in Food Products].

The conventional analysis method has problems with extraction efficiency, operability, and reproducibility. In this study, we attempted to solve these problems and improve the analytical method to obtain sufficient extraction efficiency and good operability and accuracy. The conventional method was able to get sufficient extraction in dried meat products, where the extraction efficiency of the conventional method was low, by increasing the concentration of sodium hydroxide solution at the time of homogenization. Suction filtration after adding the defoaming agent was added allowed for accurate volume adjustment. The turbidity of the extract caused by insufficient addition of zinc acetate solution was removed by increasing the amount of zinc acetate solution that was added. Turbidity caused by starch was removed by adding pancreatin. The RSD of the quantitative values was improved by adding sodium hydroxide solution and 80-90℃ water and immediately homogenizing. Furthermore, by changing the dilution factor of the extract solution in the colorimetric method, the inhibition of coloration by reducing substances was suppressed, and more accurate quantitative values could be obtained than with the conventional method. The recovery rate was 78.5-105% (RSD 0.7-5.8%), which was a good result. This method was considered to be a useful analytical method that can contribute to improving the inspection accuracy of nitrite ion analysis.

Recipient colon preoperative treatment with type I collagenase and fibronectin promotes the growth of transplanted enteric neural crest cells into Auerbach's plexus.

PURPOSE: Cell-based therapy is a potential treatment option for neurointestinal diseases by serving as a source of neural progenitor cells to replace missing or abnormal enteric neurons. Using an ex vivo transplantation model, we recently demonstrated that treatment with collagenase and fibronectin promotes infiltration of transplanted enteric neural crest cells (ENCCs) toward the colon lumen. The aim of this study was to determine whether this new method also promotes colonization of transplanted ENCCs in vivo. METHODS: Collagenase was applied locally on the anti-mesenteric area of the recipient colon using filter paper, followed by fibronectin. Neurospheres were generated from ENCCs isolated from fetal mouse intestines and transplanted into the collagenase and fibronectin-treated colon. Engraftment of neurospheres was confirmed by immunofluorescence. RESULTS: Neurospheres transplanted onto PBS- or fibronectin-treated colons were not observed to infiltrate to the muscle layer. However, when used in combination with type I collagenase and fibronectin in the recipient colon, transplanted neurospheres reached Auerbach's plexus. CONCLUSION: We demonstrated that transplanted neurospheres grow into Auerbach's plexus in the recipient colon pretreated with collagenase and fibronectin.

[Determination of Nitrate Ion in Cheese and Sake].

The Japanese official analysis method for determination of nitrate ions in food products used as food additives is associated with various challenges. In some kinds of cheese, the extract becomes suspended. The volume of extracted solution is often not accurate owing to the presence of residues in the solution. Moreover, the determination with liquid chromatography-ultraviolet detection (HPLC-UV) is difficult owing to the influence of impurities. Sake usually does not contain lipids or proteins ; therefore, its analysis can be simplified by omitting the co-precipitation steps to remove them. In the present study, for cheese, the amount of sodium hydroxide solution that causes suspension was reduced, and the influence of residues was removed by adjusting the volume after suction filtration. Whereas, sake was diluted with water and centrifuged. Furthermore, solid-phase extraction (SPE) method using cartridge containing carbon molecular sieve to remove the influence of impurities on the chromatogram was successfully established. The recoveries of the nitrate ions were good outcomes of 91.3-99.6% (CV 0.9-4.5%) (n=5). The analysis range was 0.010-0.20 g/kg for cheese, 0.010-0.20 g/L for milk, and 0.010-0.10 g/kg for sake. The developed analysis methods are considered useful, because various challenges of the official analysis method can be solved and the operation are efficient.

[Quantitative Analysis of L-Ascorbic Acid and Erythorbic Acid in Foods by HPLC, and Confirmation Method by LC-MS/MS].

Analysis of L-ascorbic acid (AsA) and erythorbic acid (ErA) in foods is generally performed by HPLC measurement after extraction with metaphosphoric acid solution. But this method can not always measure the concentrations of AsA and ErA precisely due to the presence of interfering compounds, and the reproducibility of retention time is poor. We considered that quantitative analysis by HPLC and confirmation by LC-MS/MS using an identical extraction solvent might be an effective approach for AsA and ErA analysis. Chelate fiber was added to the sample, followed by extraction with acetic acid solution containing ethylenediaminetetraacetic acid disodium salt, purification with Oasis MCX, and 2-fold dilution with methanol. The resulting solution was used for quantification by HPLC using a ZIC-HILIC column and identification by LC-MS/MS. In recovery tests with 8 kinds of foods, the recovery of AsA was over 91%, and that of ErA was over 88%. The RSD was 5.1% or less for both analytes. Analysis of 8 kinds of foods by both methods showed that this method gave better RSD values than the conventional method. AsA and ErA in all samples were confirmed by product ion scanning and selected reaction monitoring of LC-MS/MS.

Vitamins K1 and K2 potentiate hyperthermia by down-regulating Hsp72 expression in vitro and in vivo.

Hyperthermia is used to treat various malignancies, including esophageal, stomach and rectal cancer. Since hyperthermia alone has produced limited results, much attention has been focused on combining hyperthermia with chemotherapy and on searching for substances able to sensitize tumor cells to hyperthermia-induced damage. Here, we show that vitamins K1 and K2 (VK1, VK2) inhibited the expression of heat-shock protein 72 (Hsp72) but did not affect the constitutive expression of Hsc70 or calnexin in vitro and in vivo. VK1 and VK2 sensitized A549 cells to heat-shock induced cell death, while the compounds alone had no effect on cell viability. The suppression of Hsp72 was apparently at the protein level because the mRNA expression of Hsp72 was unchanged. Moreover, the chaperone activity of Hsp72 was compromised after heat-shock when cells were pre-treated with VK2. The effect of VK2 on Hsp72 suppression, however, was also observed in normal mouse tissue after the mice were subjected to whole-body hyperthermia. To eliminate this side effect, local hyperthermia was performed on tumors in mice. The pre-treatment with VK2 potentiated the effect of local hyperthermia on tumor growth suppression. The findings here that VK1 and VK2 inhibit heat-shock-induced Hsp72 suggest their possible use as an adjuvant for hyperthermia in cancer therapy.