Functional of tongue sole (Cynoglossus semilaevis) gamma-interferon-inducible lysosomal thiol reductase with implications in innate immune reponse depend on CXXC active site.

The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in promoting the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens. It is also involved in MHC I-restricted antigens catalyzing disulfide bond reduction in fishes' adaptive immunity. The open reading frame of tongue sole (Cynoglossus semilaevis) GILT (tsGILT) gene is 771 bp long, encoding 257 amino acids, with a calculated molecular weight of 28.465 kDa and isoelectric point (pI) of 5.35. After induction with lipopolysaccharide, the expression of tsGILT mRNA was upregulated in spleen and kidney and recombinant tsGILT protein transferred to late endosomes and lysosomes in HeLa cells. The refolded tsGILT was capable of catalyzing the reduction of the interchain disulfide bonds against an IgG substrate depend on the active site CXXC motif at residues 75-78. The process of immune response to bacteria challenge needs GILT to catalyze the reduction of disulfide bond and unfolding native protein antigens, promoting their hydrolysis by proteases. Whether a single mutation or a double mutation of active site CXXC at residues75-78, the 3D structure of tsGILT protein has undergone major changes and lost its activity of catalyzing the reduction of the interchain disulfide bonds.

Optimization of Encapsulation Using Milk Polar Lipid Liposomes with S-Layer Protein and Transport Study of the ACE-Inhibitory Peptide RLSFNP.

The purpose of this study is to develop a new type of nanodrug delivery material by modifying milk polar lipid (MPL) liposomes with the S-layer protein. LIP-RLSFNP (MPL liposomes encapsulating RLSFNP (Arg-Leu-Ser-Phe-Asn-Pro)) and SLP-LIP-RLSFNP (S-layer protein-modified LIP-RLSFNP) were prepared and characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, confocal laser scanning microscopy, surface plasmon resonance, and mastersizer dynamic light scattering measurements. The results showed that the S-layer protein could modify the surface of MPL liposomes, stabilize the shape of the vesicles, and improve the resistance to external interference. Furthermore, SLP-LIP-RLSFNP showed better performance in in vitro and in vivo experiments compared with LIP-RLSFNP in terms of promoting absorption and delayed release. The findings suggested that MPL liposomes modified with the S-layer protein have potential for use as an effective delivery system for therapeutic proteins and peptides.

New Nanocarrier System for Liposomes Coated with Lactobacillus acidophilus S-Layer Protein to Improve Leu-Gln-Pro-Glu Absorption through the Intestinal Epithelium.

The present study describes the development of a novel liposome nanocarrier system. The liposome was coated with Lactobacillus acidophilus CICC 6074 S-layer protein (SLP) to improve the intestinal absorption of the cholesterol-lowering peptide Leu-Gln-Pro-Glu (LQPE). The SLP-coated liposomes were prepared and characterized with morphology, particle size, zeta potential, membrane stability, Fourier transform infrared spectroscopy, and dual-channel surface plasma resonance. The results showed that SLP could successfully self-assemble on liposomes. Then, LQPE liposomes and SLP-coated LQPE liposomes (SLP-L-LQPE) were prepared. SLP-L-LQPE not only showed better sustained release properties and gastrointestinal tolerance in vitro but also increased the retention time in mice intestine. Transepithelial transport experiment indicates that the transshipment of LQPE increased significantly after being embedded by liposomes and coated with SLP. The research provides a theoretical basis for the study of SLP-coated liposomes and a potential drug delivery system for improving the intestinal absorption of peptides.

Prevention of Necrotizing Enterocolitis through Milk Polar Lipids Reducing Intestinal Epithelial Apoptosis.

Neonatal necrotizing enterocolitis (NEC) is a common and devastating disease. The objective of this research was to investigate the protective mechanisms of milk polar lipids (MPLs) on the attenuation of lipopolysaccharides (LPS)-induced intestinal inflammation and apoptosis. MPLs were extracted from buttermilk and analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A neonatal NEC rat model was used to investigate the effects of MPLs on NEC and its underlying mechanisms. Hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay were used to observe intestinal morphological changes and intestinal epithelial cell apoptosis, which showed that MPLs could reduce NEC symptoms and intestinal apoptosis. The expressions of IL-6, IL-8, and TNF-α in the MPL group was significantly downregulated (P < 0.05), and the expression levels of IL-10 were significantly upregulated (P < 0.05). At the same time, MPLs also significantly reduced (P < 0.05) activation of the LPS-induced TLR4/NF-κB signaling pathway. Furthermore, MPLs inhibit apoptosis by reducing the expressions of Bax, caspase-9, and caspase-3 and by increasing the expression of Bcl-2. In conclusion, MPLs could reduce NEC symptoms in mice by inhibiting cell inflammation and protecting against intestinal apoptosis.

iTRAQ-based mitochondrial proteome analysis of the molecular mechanisms underlying postharvest senescence of Zizania latifolia.

To explore the molecular mechanisms underlying postharvest senescence of Zizania latifolia, the changes in the mitochondrial proteome of plants treated with or without (control) 1-methyleyelopropene and ethylene during storage at room temperature for 0, 3 and 6 days were investigated using isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with two-dimensional liquid chromatography-tandem mass spectrometry. A total of 1,390 proteins with two or more peptides were identified, of which 211 showed a significant (p < .05) change (at least twofold) in relative abundance. Monitoring the parallel reaction validated the reliability and accuracy of the iTRAQ results. Bioinformatics and functional analysis of these differentially expressed proteins (DEPs) revealed that postharvest senescence of Z. latifolia could be attributed to (a) strengthened pentose phosphate pathway, (b) imbalanced protein, amino acid, organic acid, and fatty acid metabolism, (c) disordered energy homeostasis, (d) exacerbated oxidative damage, (e) RNA degradation, (f) activation of the Ca2+ , mitogen-activated protein kinase, and jasmonic acid signaling pathways, (g) programed cell death, (h) excessive biosynthesis of secondary metabolites, or (i) degradation of cell structure. Our findings provide integrated insight into the molecular mechanisms of postharvest senescence during storage as well as the DEPs that show promise as targets for controlling senescence-induced quality deterioration of Z. latifolia. PRACTICAL APPLICATIONS: Postharvest senescence is the most important factor that causes fast quality deterioration of Zizania latifolia. The understanding of the processes leading to postharvest senescence of Z. latifolia is essential in enhancing the commercial value and extending the shelf life of the product. It is currently believed that the mitochondrial metabolism is closely related to postharvest senescence. For this, the changes of proteome in Z. latifolia mitochondria treated with or without (control) 1-MCP and ETH during storage at room temperature were investigated. Results showed that a variety of physiobiochemical responses occur during postharvest senescence of Z. latifolia. 1-MCP treatment significantly inhibited the changes of these physiobiochemical processes, finally, retarding the postharvest senescence of Z. latifolia. ETH treatment had opposite effects on proteome changes compared with 1-MCP treatment. Taken together, these results enrich the understanding of the molecular mechanisms of postharvest senescence of Z. latifolia.