Isolation, structure characterization and in vitro immune-enhancing activity of a glucan from the peels of stem lettuce (Lactuca sativa).

BACKGROUND: Stem lettuce is a medicinal and edible plant. The peels, accounting for 300-400 g kg-1 raw stem lettuce and containing polysaccharides 200 g kg-1 , are discarded as industrial waste, causing environment pollution and resource waste. RESULTS: A polysaccharide named PPSL10-2 was obtained from the peels of stem lettuce after hot water extraction, and gradation with cascade ultrafiltration and purification using DEAE-Sepharose cellulose. The purity and molecular weight of PPSL10-2 is 96.10% and 2.2 × 104 Da respectively, as detected by high-performance gel permeation chromatography. PPSL10-2 was found to be an α-(1→4)-d-glucan that branched at O-6 with a terminal 1-linked α-d-Glcp as side chain, and devoid of helix conformation, which was characterized by monosaccharide composition analysis, Fourier-transform infrared spectroscopy, Congo red test, scanning electron microscopy, methylation analysis and NMR spectroscopy. Furthermore, PPSL10-2 exhibited potent immune-enhancing effect by improving proliferation and phagocytosis, promoting the secretion of nitric oxide and cytokines, as well as the expression of related genes in RAW264.7 macrophages. CONCLUSION: The findings of the present study suggest that peels as an agricultural by-product of stem lettuce are good sources of polysaccharides, which could be developed as immunopotentiator for improving human health. © 2023 Society of Chemical Industry.

Discovery of Novel Prebiotic Carbohydrates and Sugar Mimics of BlMsmE, a Solute-Binding Protein of the ABC Transporter from Bacillus licheniformis.

Stachyose is a typical prebiotic that can be utilized by the probiotic strain Bacillus licheniformis. Pioneering X-ray crystallography has determined the structure of stachyose in complex with the solute-binding protein MsmE in B. licheniformis (BlMsmE). The present work describes a combined strategy for the identification of putative BlMsmE-specific ligands, which can be used for the development of prebiotics. After a ligand-based virtual similarity screening of a large ZINC database containing ∼22 M compounds, we identified 3575 ligands. A total of 600 structures for which the Tanimoto coefficient's value was larger than a cutoff of 0.23 were selected for molecular docking. Based on the docking scores, we identified 100 top-scoring ligands, followed by molecular dynamics (MD) simulations. During simulations, 35 candidates were abandoned because of serious steric clashes in the complexes. Finally, the top 10 ligands with free energies below an energy threshold of -50.84 kcal/mol were selected. The top two ligands were stachyose and raffinose, which have proved their health benefits as prebiotics and their safety. The remaining eight ligands were further analyzed by the in silico ADME tool; only galactinol did not violate any of the criteria required for a lead compound. These three ligands were further analyzed for understanding their binding to BlMsmE. Isothermal titration calorimetry analysis suggested that stachyose, raffinose, and galactinol bound strongly to BlMsmE with Kd values of 299, 170, and 134 nM, respectively. Microsecond MD simulations suggested significant conformational changes of BlMsmE upon ligand binding. Our results provide new insight into the thermodynamics of sugars and MsmE, which would promote the development of novel prebiotics.

Reducing higher alcohols by nitrogen compensation during fermentation of Chinese rice wine.

Excessive amount of higher alcohols in alcoholic beverages causes unwell and side-effect for consumers although adequate consumption offers joy and pleasure. Therefore, reducing higher alcohols in alcoholic beverages is necessary. We used nitrogen compensation to reduce higher alcohols with Chinese rice wine as an experimental model. Higher alcohols including isobutyl alcohols, isoamyl alcohols, and ß-phenethyl alcohols were significantly decreased by 19.27, 23.03 and 19.43%, respectively, when 200 mg/L (NH4)2HPO4, 5% (w/v) yeast, and 11% wheat Koji were added to fermentation broth. Meanwhile, important quality parameters remained unchanged including free amino acids, organic acids, biogenic amines, and esters. The expression of glutamate dehydrogenase 1 gene (GDH1) and glutamine synthetase gene (GLN1) was significantly enhanced, 26.9- and 1.9-folds respectively. These results suggest that ammonium compensation could effectively decrease higher alcohols in Chinese rice wine by activating glutamate dehydrogenase (GDH) pathway in ammonium assimilation.

Proteomic analysis of stachyose contribution to the growth of Lactobacillus acidophilus CICC22162.

Stachyose is a functional oligosaccharide, acting as a potential prebiotic for colonic fermentation. To understand the mechanism of how stachyose promotes the growth of probiotic bacterium, we analyzed the differences of the proteome of Lactobacillus acidophilus grown on stachyose or glucose. By a combination of two-dimensional electrophoresis and mass spectrometry analysis, we observed 16 proteins differentially abundant under these two conditions and identified 9 protein spots. Six of these proteins were highly abundant when stachyose was used as the sole carbon source. They included the phosphotransferase system, the energy coupling factor (ECF) transporter and the mannose-6-phosphate isomerase, involved in the uptake and catabolism of stachyose in Lactobacillus acidophilus CICC22162. Supportively, these observations were validated by quantitative RT-PCR analysis and enzymatic activity determination. Positive correlation was found between the content of the proteins and their mRNA levels. Additionally, we explored the recognition mechanism for stachyose binding to the newly identified ECF transporter by MD simulations and free energy analysis. Taken together, these results provide new insights into the mechanism of stachyose in promoting the growth of probiotic bacterium.

Experimental free-space quantum key distribution with efficient error correction.

We report a 17-km free-space quantum key distribution (QKD) experiment using an engineering model of the space-bound optical transmitter and a ground station for satellite-ground QKD. The final key rate of ~ 0.5 kbps is achieved in this experiment with the quantum bit error rate (QBER) of ~ 3.4%. An efficient error correction algorithm, Turbo Code, is employed. Compared with the current error correction algorithm of Cascade, a high-efficiency error correction is realized by Turbo Code with only one-time data exchange. For a low QBER, with only one-time data exchange, the final key rates based on Turbo code are similar with Cascade. As the QBER increases, Turbo Code gives higher final key rates than Cascade. Our results experimentally demonstrate the feasibility of satellite-ground QKD and show that the efficient error correction based on Turbo Code is potentially useful for the satellite-ground quantum communication.

Proteomic analysis of ginsenoside Re attenuates hydrogen peroxide-induced oxidative stress in human umbilical vein endothelial cells.

Ginsenoside Re is an active component in ginseng that has attracted much attention because of its evident therapeutic effects on the cardiovascular system. However, little basic information is available on the mechanisms and pharmacological effects of ginsenoside Re. The potential mechanisms and protective effects of Re on H2O2-induced oxidative injury in human umbilical vein endothelial cells (HUVECs) were investigated in this study. An oxidative injury model was established using H2O2. The anti-oxidative effects of Re were determined using a series of experiments, such as MTT and anti-oxidative indicator assays. The potential protective mechanisms of Re were explored at the proteomic level, and differentially expressed proteins were validated by quantitative real-time polymerase chain reaction and western blotting. Results indicated that Re could be a potential anti-oxidant to protect HUVECs against oxidative stress damage. Proteomic analysis showed that the expression of 23 protein spots was upregulated in Re and H2O2 groups to resist oxidative stress, 15 of which were identified by their mass spectrum. These upregulated proteins were involved in stress response, anti-oxidative systems, protein synthesis, regulation of transcription and post-translational modifications, and repair of mitochondrial functions. This study may provide new insights into the mechanisms of ginsenoside Re in protecting the cardiovascular system.

The caspase pathway of linoelaidic acid (9t, 12t-c18:2)-induced apoptosis in human umbilical vein endothelial cells.

Trans fatty acids (TFA) are reported to contribute to inflammation and coronary heart disease. The study aim was to investigate the proapoptotic effects of two double bond TFA (TDTFA) on human umbilical vein endothelial cells (HUVEC). The HUVEC were grown in media supplied with linoelaidic acid (9t,12t-C18:2) at 50, 100, 200, 400 µmol/l for 24 or 48 h to examine the effects of TDTFA on the viability and apoptosis of these cells. Flow cytometry analysis and confocal scanning were used to measure apoptosis, cell binding of Annexin V and propidium iodide uptake. Colorimetric assay and RT-PCR were used to analyze enzyme activities and mRNA expression of caspase-3, -8 and -9 in HUVEC. Results showed that 9t,12t-C18:2 inhibited the viability of HUVEC in a dose-dependent and time-dependent manner. The percentages of 9t,12t-C18:2 induced apoptotic and necrotic cells significantly increased compared with that of the control. The activities and mRNA expression of caspase-8, -9 and -3 were significantly increased in 9t,12t-C18:2 treated cells compared to that of the control. Addition of specific inhibitors of caspase-8 (z-IETD-fmk) and caspase-9 (z-LEHD-fmk) to HUVEC was found to completely inhibit 9t,12t-C18:2-induced activation of caspase-3, and z-IETD-fmk inhibited the activation of caspase-9. Meanwhile, it was found that mRNA expression of Bid, Smac/DIABLO and the release of mitochondrial cytochrome c were significantly elevated by 9t,12t-C18:2 treatment. These results suggest that 9t,12t-C18:2 may induce apoptosis of HUVEC through activating caspase-8, -9 and -3. Both the death receptor pathway and the mitochondrial pathway may be involved in the apoptosis induced by 9t,12t-C18:2.

Protective effect of rhein against oxidative stress-related endothelial cell injury.

Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of atherosclerosis. Therefore, phytochemicals or antioxidants that inhibit the production of ROS have clinical value for the treatment of atherosclerosis. Rhein is one of the most important active components of rhubarb (Rheum officinale), a famous traditional Chinese remedy that possesses potent antioxidant properties through undefined mechanism(s). The aim of the present study was to determine whether rhein inhibits hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs). The oxidative injury model was established with H2O2. HUVECs were treated with different concentrations of rhein in the presence/absence of H2O2. The protective effects of rhein against the injury caused by H2O2 were evaluated. HUVECs incubated with 200 µmol/l H2O2 had significantly decreased cell viability, which was accompanied by cell apoptosis and upregulated Bid and caspase-3, -8 and -9 mRNA expression. Meanwhile, H2O2 treatment induced a marked increase in malondialdehyde (MDA) and lactate dehydrogenase (LDH) content and decreased the nitric oxide (NO) content and nitrogen oxide synthase (NOS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity. However, pre-treatment with different rhein concentrations (2, 4, 8 and 16 µmol/l) significantly increased the viability of H2O2-injured HUVECs, decreased the MDA and LDH content, increased the NO content and NOS, SOD and GSH-PX activity in a dose-dependent manner and resulted in significant recovery from H2O2-induced cell apoptosis. In addition, the results of the qRT-PCR indicated that pre­treatment with rhein downregulates the expression of Bid and caspase-3, -8 and -9 mRNA, which plays a key role in H2O2-induced cell apoptosis. The present study shows that rhein protects endothelial cells against oxidative injury induced by H2O2, suggesting that rhein is a potential compound for the prevention and treatment of atherosclerosis.

[Analysis of differential expression proteins and functions in Lactobacillus brevis NCL912 under acid stress].

OBJECT: We investigated the proteomic profile of Lactobacillus brevis NCL912 under optimum pH and acidic pH in the media without the addition of sodium L- glutamate to characterize the differential expression proteins and function by two-dimensional gel electrophoresis. METHODS: The differential expression proteins were separated and analyzed by two-dimensional gel electrophoresis, mass spectrum and bioinformatics. RESULTS: The results showed that the two-dimensional gel electrophoresis profiles of L. brevis NCL912 were uniformity, well-resolution and repeatability. 25 proteins were differently expressed in the two profiles. Among them, 8 proteins were identified and analyzed by the mass spectrum and bioinformatics due to the lack of genome sequence data of L. brevis NCL912. These proteins played the roles of the synthesis of protein and DNA, glycolysis and regulating the cellular energy level. CONCLUSION: The differential expression proteins might play the important role in the acid stress resistance mechanism which may protect cell against acid stress.

[Study on extraction of the total saponins of Chinese yam].

A method for the determination of total saponins of Chinese yam was established. The dioscin was used as a standard compound, the vanillin-perchloric acid as chromogenic agent and glacial acetic acid as solvent. The extraction technique of asponins from Chinese yam was studied by spectrometric method. Extracting temperature, extracting time, ethanol concentration and the ratio of raw material and water were selected as four factors to design the orthogonal test, and the optical condition of extraction was obtained. The results showed that the optical condition of extraction was as following: extracting temperature 60 degrees C, extracting time 6 h, ethanol concetration 80%, and the ratio of raw material and water 1:8.

Antiproliferative effects of conjugated linoleic acid on human colon adenocarcinoma cell line Caco-2.

Conjugated linoleic acid (CLA) reduces body fat reserves, and reduces atherogenesis and type II diabetes in animal experiments. It has been reported that CLA have isomeric-specificity, such as c9, t11 CLA with anticancer activity. The antiproliferative effects of two isomers of CLA (c9, t11-CLA, t9, t11-CLA) and their mixture on the human colon adenocarcinoma cell line Caco-2 were investigated in this paper. Caco-2 were incubated in serum-free medium. The antiproliferative effects of different concentrations (0, 25, 50, 100, 200 micromol/L) of linoleic acid (LA), c9, t11-CLA, t9, t11-CLA (the purity of LA and CLA was 96%) and a mixture of c9, t11-CLA and t9, t11- CLA (1:1 v/v) on caco-2 in various action time (1d, 2d, 3d, 4d) were tested in the present study. The antiproliferative effects of four substances in the same concentration and with the same action time were compared. All substances tested could inhibit Caco-2 cell proliferation. The higher anti-proliferation activity in the four materials is the mixture of CLA, then is t9,t11-CLA, c9,t11-CLA, and linoleic acid respectively. The activity is closely related to treatment time and concentration. The isomer t9, t11-CLA itself was found to have antiproliferative activity.