Extract Methods, Molecular Characteristics, and Bioactivities of Polysaccharide from Alfalfa (Medicago sativa L.).

The polysaccharide isolated from alfalfa was considered to be a kind of macromolecule with some biological activities; however, its molecular structure and effects on immune cells are still unclear. The objectives of this study were to explore the extraction and purifying methods of alfalfa (Medicago sativa L.) polysaccharide (APS) and decipher its composition and molecular characteristics, as well as its activation to lymphocytes. The crude polysaccharides isolated from alfalfa by water extraction and alcohol precipitation methods were purified by semipermeable membrane dialysis. Five batches of alfalfa samples were obtained from five farms (one composite sample per farm) and three replicates were conducted for each sample in determination. The results from ion chromatography (IC) analysis showed that the APS was composed of fucose, arabinose, galactose, glucose, xylose, mannose, galactose, galacturonic acid (GalA), and glucuronic acid (GlcA) with a molar ratio of 2.6:8.0:4.7:21.3:3.2:1.0:74.2:14.9. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and Z-average molecular weight (Mz) of APS were calculated to be 3.30 × 106, 4.06 × 105, and 1.43 × 108 g/mol, respectively, according to the analysis by gel permeation chromatography-refractive index-multiangle laser light scattering (GPC-RI-MALS). The findings of electron ionization mass spectrometry (EI-MS) suggest that APS consists of seven linkage residues, namely 1,5-Araf, galactose (T-D-Glc), glucose (T-D-Gal), 1,4-Gal-Ac, 1,4-Glc, 1,6-Gal, and 1,3,4-GalA, with molar proportions of 10.30%, 4.02%, 10.28%, 52.29%, 17.02%, 3.52%, and 2.57%, respectively. Additionally, APS markedly increased B-cell proliferation and IgM secretion in a dose- and time-dependent manner but not the proliferation and cytokine (IL-2, -4, and IFN-γ) expression of T cells. Taken together, the present results suggest that APS are macromolecular polymers with a molar mass (indicated by Mw) of 3.3 × 106 g/mol and may be a potential candidate as an immunopotentiating pharmaceutical agent or functional food.

In vitro study of effects of emulsified oil on broiler feed quality.

An experiment was conducted to compare effects of emulsified soybean oil and non-emulsified soybean oil on the quality of broiler feed differing in the feed type and the broiler feeding stage in vitro. A 2 × 2 × 3 factorial arrangement was designed with two fat sources (soybean oil and emulsified oil), two feed types (mash and pellet) and three broiler feeding stages (starter, grower and finisher). Four samples of feeds were collected from each combination of factors at the beginning of the experiment and stored at 20°C. Subsamples were taken at 15-day intervals to determine the moisture content, peroxide value (PV), acid value (AV) and the total fungal count over a 45-day period; weight loss percentage was determined by weighting the samples at days 0, 15, 30 and 45; fines percentage in pellets was only determined at day 0. The emulsified oil reduced (P < 0.05) the fines percentage, increased (P < 0.05) the moisture content, decreased (P < 0.05) the weight loss percentage and PV, did not affect (P > 0.05) the AV and the total fungal count. Results showed that the emulsified oil decreased weight loss, increased pelletability, moisture content and oxidation stability without affecting fungal growth.

Phospholipase A2 group IIA expression correlates with prolonged survival in gastric cancer.

AIMS: The secreted phospholipase A2 type IIA (PLA2G2A) gene has been identified as a modifier of intestinal adenoma multiplicity in Apc(Min/+) mice. The aim of the present study was to analyse the clinical significance of PLA2G2A expression in human gastric cancer. METHODS AND RESULTS: Using immunohistochemistry, cytoplasmic immunoreactivity of PLA2G2A was observed in 27% (40 of 149) of gastric cancer tissues compared with negative staining in normal mucosa. The PLA2G2A expression rate in well-differentiated carcinoma was elevated significantly compared with that in poorly differentiated carcinoma (46% versus 19%, P = 0.001). Statistical analysis also revealed that PLA2G2A expression correlated negatively with depth of mural invasion, lymph node metastasis and tumour-node-metastasis (TNM) stage (P < 0.05). Patients with positive PLA2G2A expression showed higher 5-year overall survival than those with negative expression (P = 0.0004). In intestinal metaplasia, PLA2G2A was found to be abundant in Paneth cells. The coexistence of PLA2G2A and lysozyme was observed in Paneth cell-rich gastric cancer (P < 0.0001). CONCLUSIONS: PLA2G2A may predict survival and might be a potential biomarker for early detection and individualized therapy.

S100A6 overexpression is associated with poor prognosis and is epigenetically up-regulated in gastric cancer.

S100A6 has been implicated in a variety of biological functions as well as tumorigenesis. In this study, we investigated the expression status of S100A6 in relation to the clinicopathological features and prognosis of patients with gastric cancer and further explored a possible association of its expression with epigenetic regulation. S100A6 expression was remarkably increased in 67.5% of gastric cancer tissues as compared with matched noncancerous tissues. Statistical analysis demonstrated a clear correlation between high S100A6 expression and various clinicopathological features, such as depth of wall invasion, positive lymph node involvement, liver metastasis, vascular invasion, and tumor-node metastasis stage (P < 0.05 in all cases), as well as revealed that S100A6 is an independent prognostic predictor (P = 0.026) significantly related to poor prognosis (P = 0.0004). Further exploration found an inverse relationship between S100A6 expression and the methylation status of the seventh and eighth CpG sites in the promoter/first exon and the second to fifth sites in the second exon/second intron. In addition, the level of histone H3 acetylation was found to be significantly higher in S100A6-expressing cancer cells. After 5-azacytidine or trichostatin A treatment, S100A6 expression was clearly increased in S100A6 low-expressing cells. In conclusion, our results suggested that S100A6 plays an important role in the progression of gastric cancer, affecting patient prognosis, and is up-regulated by epigenetic regulation.

Overexpression of endothelial cell specific molecule-1 (ESM-1) in gastric cancer.

BACKGROUND: The endothelial cell-specific molecule-1 (ESM-1) gene is involved in various biological events. This study was designed to clarify its clinical significance and explore its biological behavior in gastric cancer (GC). METHODS: ESM-1 mRNA expression was evaluated by real-time PCR in GC (n = 34) and matched adjacent normal tissues (n = 14). The expression of ESM-1 protein was investigated by immunohistochemistry in GC (n = 159) and matched normal tissues (n = 40), and its correlation with the clinicopathological features and overall survival of patients was analyzed. Microvessel density (MVD) in GC was assessed by anti-CD34 and the pattern of ESM-1 expression in tumor-related vascular was evaluated. The effect of ESM-1 promotion of proliferation in the GC MKN28 cell line and human microvascular endothelial cell line HMEC-1 were tested using the MTT assay. RESULTS: ESM-1 mRNA was significantly overexpressed in GC compared with adjacent noncarcinoma controls (P < 0.01). ESM-1 protein was predominantly expressed in GC. ESM-1 expression was associated with distant metastasis and Borrmann type IV (P < 0.05) and was strongly associated with vascular invasion (P = 0.0057). Patients with ESM-1 expression showed lower 5-year survival rate (P = 0.0339). Multivariate analysis revealed that ESM-1 was an independent prognostic factor. In GC, CD34-MVD of GC vessels positively expressing ESM-1 was higher than that of GC with negative vessels expression of ESM-1 (P < 0.05). Besides, ESM-1 antibody dose-dependently impaired MKN28 and HMEC-1 growth. CONCLUSIONS: ESM-1 is overexpressed in GC and can serve as a tumor biomarker to predict survival of GC patients, and it might promote tumor angiogenesis and growth in GC and, hence, may represent a potential therapeutic target.

Identification of prognosis-related proteins in advanced gastric cancer by mass spectrometry-based comparative proteomics.

PURPOSE: The objective of this study was to identify differentially expressed proteins of advanced gastric cancer from patients with different prognosis using NanoLC-MS/MS (LTQ) (nanoflow liquid chromatography system interfaced with a linear ion trap LTQ mass spectrometer). METHODS: Eight gastric cancer patients with relatively early TNM stage and survival time >34 months were identified as good survival (group G), while the other eight with late stage and survival time <15 months as poor survival (group P). The total protein of the tissue samples from each group was extracted and pooled together respectively. The resulting two protein mixtures were trypsin-digested and analyzed using NanoLC-MS/MS (LTQ). Database searches were done against NCBI non-redundant database and SWISS-PROT database and the identified proteins were classified through an online Web Gene Ontology Annotation Plot tool. Immunohistochemistry was used to verify candidate prognosis-related proteins. RESULTS: There were 284 and 213 proteins identified for group G and group P respectively. And 117 proteins were detected exclusively in group G and 46 proteins exclusively in group P. These protein markers function in calcium ion signaling pathway, cellular metabolism, cytoskeleton formation, stress reaction, etc. Among those, the down-regulated expression of S100P was verified to claim a poor clinical outcome of gastric cancer patients (P = 0.0375). CONCLUSION: The MS-based proteomics approach is efficient in identifying differentially expressed proteins in relation to prognosis of advanced gastric cancer patients. These differentially expressed proteins could be potential prognosis-related cancer markers and deserve further validation and functional study.

[Nuclear expression of S100A4 is associated with lymph node metastasis in gastric carcinoma].

OBJECTIVE: To investigate the intracellular localization of S100A4 in gastric carcinoma cells and the relationship between S100A4 expression status and lymph node metastasis of gastric carcinoma. METHODS: Western blotting analysis was performed to locate the expression of S100A4 protein in sub-fraction components of frozen tissues. S100A4 protein expression was also determined by immunohistochemical method in 131 samples of gastric cancer and 20 samples of matched metastatic lymph nodes. RESULTS: Thirty-two of 131 (24.4%) gastric carcinoma showed positive S100A4 nuclear expression and 50/131 (38.2%) carcinoma showed positive cytoplasmic expression. In 32 samples with positive S100A4 nuclear expression, 30 (93.8%) carcinomas had positive lymph node metastases. S100A4 nuclear expression level was higher in gastric carcinoma with lymph node metastasis (29.1%) than that without lymph node metastasis (7.1%) (P=0.016). CONCLUSION: Nuclear expression of S100A4 is associated with lymph node metastasis of gastric carcinoma.

[Detection of p53 gene mutation in the plasma of gastric cancer patients].

OBJECTIVE: To investigated p53 gene mutation in plasma of gastric cancer patients. METHODS: DNA extracted from plasma and matched tumor and tumor-adjacent normal tissues of 96 gastric cancer patients, and DNA from 20 healthy people were studied. Exons 5, 6, 7, and 8 of p53 were amplified by PCR. The mutation status was analyzed by denaturing high-performance liquid chromatography (DHPLC), followed by direct sequencing of cases with aberrant chromatographic patterns. RESULTS: Heterozygous mutations of p53 gene were detected in 19.9% (19/96) of primary tumor tissues and 5.2% (5/96) of corresponding plasma. All p53 gene mutations detected in plasma DNA consisted with mutations in the matched primary tumor samples. Neither the tumor-adjacent gastric mucosa tissues nor control plasma from healthy volunteers showed p53 gene mutation. No correlation was found between p53 mutation status and clinicopathological features of gastric cancer patients. CONCLUSION: p53 gene mutation in plasma can be detected in tissues and plasma of gastric cancer patients, which could be applied in screening and surveillance of this disease.

[Promoter hypermethylation of the p16 gene in pre- and post-gastrectomy plasma of patients with gastric adenocarcinoma].

OBJECTIVE: To detect promoter hypermethylation of the p16 gene in pre- and post-operative plasma, matched cancer tissues and para-tumor non-cancerous tissues of patients with gastric adenocarcinoma for evaluating the effectiveness of therapeutic intervention. METHODS: Primary tumor tissues and para-tumor tissues and preoperative plasma samples of 84 patients with gastric adenocarcinoma were collected, and 14-21 days' post-operative plasma of 30 of the 84 patients who underwent curative gastrectomy was available. Plasma of 15 healthy people was also collected as control. After sodium-bisulfite treatment, extracted DNA was amplified for p16 promoter hypermethylation by methylation-specific polymerase chain reaction (MSP). The PCR products were detected by both gel-ethidium bromide electrophoresis and high performance liquid chromatogram (HPLC). RESULTS: Among the samples from 84 patients, p16 hypermethylation was detected in 26 (31.0%) cancer tissues and 2 (0.02%) para-tumor non-cancerous tissues and 12 (14.3%) preoperative plasma, while plasma of the healthy people was negative. In all positive plasma, the paired primary tumor tissues were also confirmed to be methylated. As far as samples from 30 patients with post-operative plasma were concerned, 14 cancer tissues and 6 preoperative plasma samples were positive, and only 1 of 6 post-operative plasma remained hypermethylated. The results detected by HPLC exactly matched those by gel-ethidium bromide electrophoresis. CONCLUSION: Promoter hypermethylation of the p16 gene detected in plasma consists with that in primary cancer tissues of patients with gastric adenocarcinoma. The alteration of status of hypermethylation of p16 in post-operative plasma is considered the consequences of surgical intervention. HPLC can be used as an efficient tool in detecting the product of MSP.