The characteristics of protein-glutaminase from an isolated Chryseobacterium cucumeris strain and its deamidation application.

Protein-glutaminase (PG), a deamidation enzyme commercially derived from Chryseobacterium proteolyticum, is used to improve the solubility and other functional properties of food proteins. In this study, a new PG-producing strain, Chryseobacterium cucumeris ZYF120413-7, was isolated from soil, and it had a high PG yield and a short culture time. It gave the maximum PG activity with 0.557 U/ml on Cbz-Gln-Gly after 12 h of culture, indicating that it was more suitable for PG production. The enzyme activity recovery and purification fold were 32.95% and 161.95-fold, respectively, with a specific activity of 27.37 U/mg. The PG was a pre-pro-protein with a 16 amino acids putative signal peptide, a pro-PG of 118 amino acids, and a mature PG of 185 amino acids. The amino acid sequence identity of PG from strain ZYF120413-7 was 74 and 45%, respectively, to that of PG from C. proteolyticum 9670T and BH-PG. The optimum reaction pH and temperature of PG was 6 and 60°C, respectively. Enzyme activity was inhibited by Cu2+. The optimum PG substrate was Cbz-Gln-Gly, and the Km and Vmax values were 1.68 mM and 1.41 µM mg protein-1 min-1, respectively. Degree of deamidation (DD) of soy protein isolate (SPI) treated by purified PG was 40.75% within the first 2 h and 52.35% after 18 h. These results demonstrated that the PG from C. cucumeris ZYF120413-7 was a promising protein-deamidating enzyme for improving the functionality of food proteins.

HMGB1 contributes to the irradiation-induced endothelial barrier injury through receptor for advanced glycation endproducts (RAGE).

This study aimed to investigate whether HMGB1 (high mobility group box-1 protein) and receptor for advanced glycation end products (RAGE) were involved in the irradiation-induced endothelial barrier damage and their mechanism. We constructed the damage model of endothelium barrier model with bEnd.3 cells. The permeability of endothelial barrier was detected by sodium fluorescein (Na-F) permeation test, and the irradiation dose which could induce permeability transition was determined by being exposed to different irradiation doses (5, 10, 15, 20 Gy). MTT assay was applied to detect cell viability under different concentrations of HMGB1, glycyrrhizic acid (GA, a specific inhibitor of HMGB1), and FPS-ZM1 (a blood-brain-barrier permeant blocker of RAGE V domain-mediated ligand binding). The expression of HMGB1, RAGE, and related molecules involved in MAPK signaling pathway, MMP-2, MMP-9, ZO-1, and claudin 5 of differently treated groups were measured by qRT-PCR, western blot, and immunofluorescence. Cells possessed stable endothelial barrier function on 4-7 days after seeded on transwell plates. The permeability of endothelial barrier would change under at least 10 Gy radiation. Both radiation and HMGB1 treatment alone could improve the permeability. After irradiation, the expressions of HMGB1 and RAGE increased and MAPK signal pathway was activated. Meanwhile, MMP-2 and MMP-9 were overexpressed, while the expression of tight junction proteins ZO-1 and claudin 5 was decreased. Radiation could activate MAPK signaling pathway through promoting the expression of HMGB1 and RAGE, which further led to endothelial barrier injury and changed its permeability.

Magnesium Elevation Promotes Neuronal Differentiation While Suppressing Glial Differentiation of Primary Cultured Adult Mouse Neural Progenitor Cells through ERK/CREB Activation.

This study aimed to explore the influence of magnesium elevation on fate determination of adult neural progenitor cells (aNPCs) and the underlying mechanism in vitro. Adult neurogenesis, which is the generation of functional neurons from neural precursors, occurs throughout life in restricted anatomical regions in mammals. Magnesium is the fourth most abundant ion in mammals, and its elevation in the brain has been shown to enhance memory and synaptic plasticity in vivo. However, the effects of magnesium on fate determination of aNPCs, which are vital processes in neurogenesis, remain unknown. NPCs isolated from the dentate gyrus of adult C57/BL6 mice were induced to differentiate in a medium with varying magnesium concentrations (0.6, 0.8, and 1.0 mM) and extracellular signal-regulated kinase (ERK) inhibitor PD0325901. The proportion of cells that differentiated into neurons and glial cells was evaluated using immunofluorescence. Quantitative real-time polymerase chain reaction and Western blot methods were used to determine the expression of ß-III tubulin (Tuj1) and glial fibrillary acidic protein (GFAP). The activation of ERK and cAMP response element-binding protein (CREB) was examined by Western blot to reveal the underlying mechanism. Magnesium elevation increased the proportion of Tju1-positive cells and decreased the proportion of GFAP-positive cells. Also, the expression of Tuj1 was upregulated, whereas the expression of GFAP was downregulated. Moreover, magnesium elevation enhanced the activation of both ERK and CREB. Treatment with PD0325901 reversed these effects in a dose-dependent manner. Magnesium elevation promoted neural differentiation while suppressing glial cell differentiation, possibly via ERK-induced CREB activation.