Effect of Mucine 4 and Fucosyltransferase 1 genetic variants on gut homoeostasis of growing healthy pigs

Putative genetic markers have been associated with ETEC F4 (Mucine 4 [MUC4]; MUC4^GG;CG as susceptible; MUC4^CC as resistant) and F18 (Fucosyltransferase 1 [FUT1]; FUT1^GG;AG as susceptible; FUT1^AA as resistant) resistances respectively. In this study, 71 post‐weaning pigs were followed from d0 (35 days old) to d42 (77 days of age) to investigate the effect of MUC4 or FUT1 genotypes on the mid‐jejunal microbiota composition, pigs expression of genes related to inflammation (IL8, GPX2, REG3G, TFF3, CCL20 and LBPI) and glycomic binding pattern profile (Ulex europaeus agglutinin I [UEA] fucose‐binding lectin and peanut agglutinin [PNA] galactose‐specific), and on blood plasma targeted metabolomics profile, faecal score and performance parameters of growing healthy pigs. The MUC4 and FUT1 resistant genotypes improved the pigs’ growth performance and had firmed faecal score susceptible genotypes in d0–d21 period. Pigs with MUC4^GG genotype had a higher jejunal expression of genes relate to immune function (CCL20 and REG3G) than MUC4^CG and MUC4^CC pigs (p < 0.05). MUC4^CG pigs had higher expression of TFF3 (implicated in mucosal integrity) than MUC4^GG and MUC4^CC (p < 0.05). FUT1 influenced the alpha‐ and beta‐jejunal microbial indices. The FUT1^AA group had a higher number of operational taxonomic units (OTUs) belonging to Lactobacillus genus, while FUT1^GG group had a higher number of OTUs belonging to Veillonella genus. MUC4^CC pigs had lower scores for UEA on brush borders and goblet cells in villi than MUC4^GG (p < 0.05). FUT1^AA pigs had lower UEA positivity and higher PNA positivity on brush borders and goblet cells than FUT1^AG and FUT1^GG (p < 0.05). Both FUT1 and MUC4 influenced the metabolic profile of healthy pigs. Results highlight the role of MUC4 and FUT1 on pig intestinal homoeostasis and improved the knowledge regarding the potential interaction between host genetics, gut microbiota composition and host metabolism in a healthy status.     

Fucose-containing sulfated polysaccharides from brown seaweeds inhibit proliferation of melanoma cells and induce apoptosis by activation of caspase-3 in vitro

Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3.     

羊栖菜和裙带菜中抗凝血活性物质的初步筛选

Components with blood-anticoagulant activity were fractionated from marine algae Sargassumfusiforrne and Undaria pinnatifida and the activity of the components was investigated by using the method of bioassay. It was found that the 80% ethanol soluble fraction of Sargassumfusiforme exhibited no blood-anticoagulant activity, while the components obtained from the 80 % ethanol insoluble fraction (hot water extract) gave obvious bloodanticoagulant activity. A further study suggests that the principal component in the hot water extract from Sargassumfusiforrne with high blood-anticoagulant activity should be sulfated polysaccharides, in which the activity was positively correlated with the content of total sugar and fucose in sulfated polysaccharides. As for Undaria pinnatifida, the n-butanol extract fractionated from the 80% ethanol soluble fraction had blood anticoagulant activity, while the petroleum ether, ether, ethyl acetate, or water extract showed no bloodanticoagulant activity.     

Upregulation of TNF-α Production by IFN-γ and LPS in Cultured Canine Keratinocytes: Application to Monosaccharides Effects

Activated keratinocytes play a key role in the cutaneous immune system by their interactions with other cell types through the production of cytokines with both autocrine and paracrine activity. But there is little knowledge about epidermal cytokines in the dog. In this study, cultured canine keratinocytes were stimulated by human recombinant interferon γ (IFN-γ) and lipopolysaccharide (LPS) and cell supernatants were tested for tumour necrosis factor α (TNF-α) concentration using a cell viability assay on a murine cell line. We show that IFN-γ in combination with LPS significantly increases TNF-α secretion by canine keratinocytes. The best stimulations were obtained using confluent cultures and the association of IFN-γ (400 ng/ml) and LPS (40 μg/ml). The experimental protocol we describe represents a new method for studying keratinocyte activation and its modulation in the dog. We provide an example of application of our method: the study of the effects of different monosaccharides on canine keratinocyte activation.     

Identification of Core Alpha 1,3-Fucosyltransferase Gene From Silkworm: An Insect Popularly Used to Express Mammalian Proteins

Silkworm has great potential as production system of recombinant mammalian proteins. When the protein products are used for medical purpose, it is required to reduce the risk of an allergy, the content of core alpha 1,3-fucosyl residue attached to the N-glycan of proteins, for example. We isolated the gene of an enzyme responsible for the transfer of core alpha 1,3-fucosyl residue, core alpha 1,3-fucosyltransferase (Fuc-T C3), from silkworm. A candidate cDNA for silkworm Fuc-T C3 was isolated as a homolog of the fruit fly enzyme gene fucTA. The gene was located on chromosome 7 of the silkworm genome and was composed of seven exons, which spanned approximately 10 kb on the genome. The coding region of the gene was 1,350 bp and encoded a 450-amino acid protein with a molecular mass of 52.2 kDa. Deduced amino acid sequence of the coding region showed one transmembrane domain in its N-terminal and typical motifs common to fucosyltransferases including Fuc-T C3s of other organisms in its C-terminal. The extract of CHO cells transfected with the cDNA showed Fuc-T C3 activity using GDP-fucose and DABS-GnGn peptide as substrates. These results showed this cDNA clone actually encodes silkworm Fuc-T C3.     

Characterization of the exopolysaccharide produced by Salipiger mucosus A3, a halophilic species belonging to the Alphaproteobacteria, isolated on the Spanish Mediterranean seaboard

We have studied the exopolysaccharide produced by the type strain of Salipiger mucosus, a species of halophilic, EPS-producing (exopolysaccharide-producing) bacterium belonging to the Alphaproteobacteria. The strain, isolated on the Mediterranean seaboard, produced a polysaccharide, mainly during its exponential growth phase but also to a lesser extent during the stationary phase. Culture parameters influenced bacterial growth and EPS production. Yield was always directly related to the quantity of biomass in the culture. The polymer is a heteropolysaccharide with a molecular mass of 250 kDa and its components are glucose (19.7%, w/w), mannose (34%, w/w), galactose (32.9%, w/w) and fucose (13.4%, w/w). Fucose and fucose-rich oligosaccharides have applications in the fields of medicine and cosmetics. The chemical or enzymatic hydrolysis of fucose-rich polysaccharides offers a new efficient way to process fucose. The exopolysaccharide in question produces a solution of very low viscosity that shows pseudoplastic behavior and emulsifying activity on several hydrophobic substrates. It also has a high capacity for binding cations and incorporating considerable quantities of sulfates, this latter feature being very unusual in bacterial polysaccharides.     

黏蛋白侧链岩藻糖在肠道细菌与宿主互作过程中的作用

消化道表面黏液层中含有大量黏蛋白,这些黏蛋白分子侧链往往被糖基化修饰,这些糖基在肠道菌的黏附、定植及免疫调节过程中发挥重要作用。岩藻糖是肠道黏蛋白分子侧链中的一种重要糖基,本文总结了岩藻糖作为肠道菌黏附靶位点和信号调节分子,以及调节肠道功能等方面的作用。     

洋栖菜中褐藻多糖硫酸酯的分离和组分分析

从洋栖菜中提取出水溶性的粗多糖－褐藻糖胶,该多糖为由岩藻糖、半乳糖等糖基组成的杂多糖,并含有硫酸酯．经醇析、氯化钙分级沉淀分离后,用ＤＥＡＥ纤维素阴离子交换柱层析,０．３～１．５ｍｏｌ／ＬＮａＣｌ溶液梯度洗脱得组份Ｆ１、Ｆ２、Ｆ３．分析表明Ｆ１和Ｆ２均为以岩藻糖为主的多糖硫酸酯,岩藻糖、木糖、甘露糖和半乳糖的物质量比分别为１∶ｔｒａｃｅ∶０．０２７∶０．４６和１∶０．０３∶０．１６∶１．３６．氮蓝四唑光化还原试验表明三组份均有一定的抗氧化活性,其中Ｆ１活性最强．     

猪FUT1基因启动子区的确定及活性分析

F18大肠杆菌(Escherichia coli F18,E.coli F18)是养猪(Susscrofa)业中发生最普遍、危害最大的病原菌之一,球系列鞘糖脂生物合成通路及通路中α-(1,2)岩藻糖转移酶1基因(alpha(1,2)fucose transferase 1,FUT1)对断奶仔猪F18抗性具有重要调控作用.本研究运用生物信息学技术挖掘课题组前期获得的断奶仔猪转录组测序结果,确定FUT1基因的转录起始位点和启动子区.同时对启动子区序列进行CpG岛分析;采用双荧光素酶报告基因以及AliBaba 2.0软件,分别分析启动子区活性和CpG岛序列潜在的转录结合位点.通过比对人类(Homo sapiens)和猪的基因序列信息数据库,结果表明,FUT1基因转录起始区域具有5种可变剪接(AS-1,AS-2,AS-3,AS-4和AS-5)和2个启动子区域(启动子1和启动子2);双荧光素酶报告基因检测结果进一步显示,FUT1基因启动子2的转录活性极显著高于启动子1的转录活性(P＜0.01),启动子2的活性是启动子1的2.75倍,根据结果可以推测启动子2在转录过程中起主导作用;CpG岛分析显示,猪FUT1基因启动子1和启动子2分别存在一个CpG岛.FUT1启动子1扩增片转录因子预测分析表明,FUT1基因启动子1存在20个潜在的转录因子结合位点,并且Sp1出现在多个转录结合位点处.本研究结果为猪FUT1基因的甲基化检测和调控机制分析提供一定的基础和依据.     

猪感染猪瘟病毒和猪痢疾密螺旋体后胃肠细胞中荆豆凝集素Ⅰ受体的分布变化

荆豆凝集素 I(UEAI)受体仅存在于健康猪肠吸收细胞胞质中。猪瘟病猪除肠吸收细胞含 UEA_1受体外,其胃肠粘膜上皮细胞、十二指肠腺细胞和部分杯状细胞胞质中出现大量UEA_1受体。猪痢疾密螺旋体病猪的部分肠吸收细胞胞质中的 UEA_1受体完全消失,而极少数胃粘膜上皮细胞、十二指肠腺细胞和杯状细胞胞质中却出现 UEA_1受体。另外,在病理剖检无明显组织病变差别的猪瘟病猪和猪痢疾密螺旋体病猪之间,其 UEA_1受体分布有差异。