Yizhen Wang

Preparation, characterization and immunomodulatory activity of selenium-enriched exopolysaccharide produced by bacterium Enterobacter cloacae Z0206

The tolerant-selenium exopolysaccharide-producing bacterial strain Enterobacter cloacae Z0206 was batch cultured in PDA medium containing optimal concentration of sodium selenite. Selenium was accumulated efficiently in Enterobacter cloacae Z0206 during cultivation with selenium. Inorganic selenite could be transformed into organic forms. Selenium-enriched exopolysaccharide (Se-ECZ-EPS-1) was purified from the fermentation liquid. Selenium content of Se-ECZ-EPS-1 was 12.962microg/g. Se-ECZ-EPS-1 with Mw of 29,300Ka was composed of Glc, Gal and Mann with molar ratio of 8.530:0.061:0.706. Administration of Se-ECZ-EPS-1 to cyclophosphamide (CP)-exposed animals resulted in improvement of cellular and humoral immune responses. These findings indicated that Se-ECZ-EPS-1 may act as potent immunomodulatory agents.

Design of hybrid β-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity.

Antimicrobial peptides (AMPs) have attracted considerable attention for their broad-spectrum antimicrobial activity and reduced tendency to cause bacterial resistance. Emerging concerns over the host cytotoxicity of AMPs, however, may ultimately compromise their development as pharmaceuticals. In order to optimize AMPs with potent cell specificity and anti-inflammatory activity, we designed β-hairpin hybrid peptides based upon progetrin-1, bovine lactoferricin and cecropin A. The synthetic hybrid peptides LB-PG and CA-PG demonstrated high selectivity over a wide range of microbes from Gram-positive and Gram-negative bacteria in porcine red blood cells. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) show that these peptides kill microbial cells by penetrating the cell membrane and damaging the membrane envelope. Gel retardation demonstrates that the peptides have a high affinity for DNA, indicating an additional possible intracellular bactericidal mechanism. Moreover, the hybrid peptides inhibit the expression of LPS-induced proinflammatory cytokines and chemokines, such as tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein 1(MCP-1), following LPS stimulation in RAW264.7 cells. Our results indicate that these hybrid peptides have considerable potential for future development as antimicrobial and anti-inflammatory agents.

Distinctive Genes Determine Different Intramuscular Fat and Muscle Fiber Ratios of the longissimus dorsi Muscles in Jinhua and Landrace Pigs

Meat quality is determined by properties such as carcass color, tenderness and drip loss. These properties are closely associated with meat composition, which includes the types of muscle fiber and content of intramuscular fat (IMF). Muscle fibers are the main contributors to meat mass, while IMF not only contributes to the sensory properties but also to the plethora of physical, chemical and technological properties of meat. However, little is known about the molecular mechanisms that determine meat composition in different pig breeds. In this report we show that Jinhua pigs, a Chinese breed, contains much higher levels of IMF than do Landrace pigs, a Danish breed. We analyzed global gene expression profiles in the longissimus dorsi muscles in Jinhua and Landrace breeds at the ages of 30, 90 and 150 days. Cross-comparison analysis revealed that genes that regulate fatty acid biosynthesis (e.g., fatty acid synthase and stearoyl-CoA desaturase) are expressed at higher levels in Jinhua pigs whereas those that regulate myogenesis (e.g., myogenic factor 6 and forkhead box O1) are expressed at higher levels in Landrace pigs. Among those genes which are highly expressed in Jinhua pigs at 90 days (d90), we identified a novel gene porcine FLJ36031 (pFLJ), which functions as a positive regulator of fat deposition in cultured intramuscular adipocytes. In summary, our data showed that the up-regulation of fatty acid biosynthesis regulatory genes such as pFLJ and myogenesis inhibitory genes such as myostatin in the longissimus dorsi muscles of Jinhua pigs could explain why this local breed produces meat with high levels of IMF.

Comparisons of different muscle metabolic enzymes and muscle fiber types in Jinhua and Landrace pigs.

Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality, however, the underlying molecular mechanism remains unclear. The main objective of this study was to evaluate the breed-specific mechanisms controlling meat quality and postmortem muscle metabolism. The specific purpose was to investigate the variations in meat quality, muscle fiber type, and enzyme activity between local Jinhua and exotic Landrace pigs at the same age (180 d of age), as well as the same BW of 64 kg, respectively. We compared differentially expressed muscle fiber types such as types I and IIa (oxidative), type IIb (glycolytic), as well as type IIx (intermediate) fibers in LM and soleus muscles of Jinhua and Landrace pigs using real-time reverse-transcription PCR. Furthermore, the metabolic enzyme activities of lactate dehydrogenase, as well as succinic dehydrogenase and malate dehydrogenase, were used as markers of glycolytic and oxidative capacities, respectively. Results showed that Jinhua pigs exhibited greater intramuscular fat content and less drip loss compared with the Landrace (P < 0.01). Meanwhile, the mRNA abundance of oxidative and intermediate fibers was increased in Jinhua pigs, whereas the glycolytic fibers were more highly expressed in the Landrace (P < 0.01). In addition, Jinhua pigs possessed greater oxidative capacity than that of the Landrace (P < 0.05). These results suggested that the increased expression of the oxidative and intermediate fibers and greater activities of oxidative enzymes in Jinhua pigs were related to meat quality as indicated by a greater intramuscular fat and reduced drip loss. Based on these results, we conclude that muscle fiber composition and postmortem muscle metabolism can explain, in part, the variation of meat quality in Jinhua and Landrace pigs. These results may provide valuable information for understanding the molecular mechanism responsible for breed specific differences in growth performance and meat quality.

Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig.

DNA N6-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1–0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome.

Effects of Se-enriched polysaccharides produced by Enterobacter cloacae Z0206 on alloxan-induced diabetic mice

In this study, the water-soluble selenium-enriched exopolysaccharides (Se-ECZ-EPS) were isolated from submerged culture broth of Enterobacter cloacae Z0206 through fermentation, ethanol precipitation and deproteinization. The protective effects of Se-ECZ-EPS on alloxan-induced diabetic mice were investigated. Diabetes was induced in ICR (Institute of Cancer Research) mice by administration of single doses of alloxan intraperitoneally (190 mg/kg body weight). Se-ECZ-EPS at a dose of 200 mg/kg body weight were administered per os (p.o.) as single dose per day to diabetes-induced mice for a period of 42 days. The decrease in body weight, serum insulin level, and the increase in blood glucose level, glycosylated serum protein (GSP), total cholesterol (TC) and triglycerides (TG) in liver were observed in diabetic mice. On the other hand, oral administration of Se-ECZ-EPS resulted in a significant reduction in fasting blood glucose levels, GSP, TC and TG contents in liver coupled with improvement of body weight and serum insulin level in comparison with diabetic control group. These results suggest that Se-ECZ-EPS possess significant protective and anti-diabetic effects in alloxan-induced diabetic mice.

The Effect of Insulin, TNFα and DHA on the Proliferation, Differentiation and Lipolysis of Preadipocytes Isolated from Large Yellow Croaker (Pseudosciaena Crocea R.)

Fish final product can be affected by excessive lipid accumulation. Therefore, it is important to develop strategies to control obesity in cultivated fish to strengthen the sustainability of the aquaculture industry. As in mammals, the development of adiposity in fish depends on hormonal, cytokine and dietary factors. In this study, we investigated the proliferation and differentiation of preadipocytes isolated from the large yellow croaker and examined the effects of critical factors such as insulin, TNFα and DHA on the proliferation, differentiation and lipolysis of adipocytes. Preadipocytes were isolated by collagenase digestion, after which their proliferation was evaluated. The differentiation process was optimized by assaying glycerol-3-phosphate dehydrogenase (GPDH) activity. Oil red O staining and electron microscopy were performed to visualize the accumulated triacylglycerol. Gene transcript levels were measured using SYBR green quantitative real-time PCR. Insulin promoted preadipocytes proliferation, stimulated cell differentiation and decreased lipolysis of mature adipocytes. TNFα and DHA inhibited cell proliferation and differentiation. While TNFα stimulated mature adipocyte lipolysis, DHA showed no lipolytic effect on adipocytes. The expressions of adipose triglyceride lipase (ATGL), fatty acid synthase (FAS), lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor α, γ (PPARα, PPARγ) were quantified during preadipocytes differentiation and adipocytes lipolysis to partly explain the regulation mechanisms. In summary, the results of this study indicated that although preadipocytes proliferation and the differentiation process in large yellow croaker are similar to these processes in mammals, the effects of critical factors such as insulin, TNFα and DHA on fish adipocytes development are not exactly the same. Our findings fill in the gaps in the basic data regarding the effects of critical factors on adiposity development in fish and will facilitate the further study of molecular mechanism by which these factors act in fish and the application of this knowledge to eventually control obesity in cultured species.

Lkb1 controls brown adipose tissue growth and thermogenesis by regulating the intracellular localization of CRTC3

Brown adipose tissue (BAT) dissipates energy through Ucp1-mediated uncoupled respiration and its activation may represent a therapeutic strategy to combat obesity. Here we show that Lkb1 controls BAT expansion and UCP1 expression in mice. We generate adipocyte-specific Lkb1 knockout mice and show that, compared with wild-type littermates, these mice exhibit elevated UCP1 expression in BAT and subcutaneous white adipose tissue, have increased BAT mass and higher energy expenditure. Consequently, KO mice have improved glucose tolerance and insulin sensitivity, and are more resistant to high-fat diet (HFD)-induced obesity. Deletion of Lkb1 results in a cytoplasm to nuclear translocation of CRTC3 in brown adipocytes, where it recruits C/EBPβ to enhance Ucp1 transcription. In parallel, the absence of Lkb1 also suppresses AMPK activity, leading to activation of the mTOR signalling pathway and subsequent BAT expansion. These data suggest that inhibition of Lkb1 or its downstream signalling in adipocytes could be a novel strategy to increase energy expenditure in the context of obesity, diabetes and other metabolic diseases.

Hypoglycemic and hypolipidemic properties of polysaccharides from Enterobacter cloacae Z0206 in KKAy mice.

The water-soluble polysaccharides (EPS) were isolated from culture broth of Enterobacter cloacae Z0206, and the hypoglycemic and hypolipidemic effects of EPS were investigated. In this study, KKAy mice were gavaged once daily with either distilled water or EPS (200 mg/kg body weight) for 6 weeks. Results showed that EPS possessed significant hypoglycemic effects. Improved oral glucose tolerance, reduced serum insulin levels as well as decreased serum triglycerides (TG), cholesterol (TC) and low density lipoprotein cholesterol (LDL-c) were observed after treatment with EPS. Furthermore, EPS upregulated the expression of glucokinase (GK), HSL (Hormone Sensitive Lipase), adipose triglyceride lipase (ATGL), carnitine palmitoyl transferase 1-α (CPT1-α), glucose transporter 2 (Glut2), adenosine monophosphate activated protein kinase (AMPK) and silent information regulator 1 (Sirt1), but downregulated the gene expression of glucose-6-phosphatase (G6P) and fatty acid synthase (FAS) in the liver. These results suggest that EPS exhibits hypoglycemic and hypolipidemic effects possibly through regulating AMPK- and SirT1-mediated effects on carbohydrate and lipid metabolism.

Sulfated modification and antioxidant activity of exopolysaccahrides produced by Enterobacter cloacae Z0206

Nine modification conditions were designed to sulfate exopolysaccharides (EPS) produced by Enterobacter cloacae Z0206 by chlorosulfonic acid-pyridine (CSA-Pyr) method according to the orthogonal test and focusing on three affecting factors such as the ratio of CSA to Pyr, reaction temperature and reaction time. And nine sulfated derivatives with various degrees of substitution (DS) were obtained. Their antioxidant activities were evaluated in vitro, by scavenging abilities on superoxide radical and hydroxyl radical. The results indicated that sulfated derivatives of EPS showed noticeable effects on scavenging superoxide radical and hydroxyl radical compared with native one, and sulfated derivative with moderate DS of 0.60 showed highest antioxidant activities. The optimum sulfated conditions of EPS were the ratio of CSA to Pyr of 1:2, the reaction time of 2h and reaction temperature of 70 °C.