Microsatellite markers from expressed sequence tags (ESTs) of seaweeds in differentiating various Gracilaria species

Gracilaria is a red seaweed that has been cultivated worldwide and is commercially used for food, fertilizers, animal fodder, and phycocolloids. However, the high morphological plasticity of seaweeds often leads to the misidentification in the traditional identification of Gracilaria species. Molecular markers are important especially in the correct identification of Gracilaria species with high economic value. Microsatellite markers were developed from the expressed sequence tags of seaweeds deposited at the National Center for Biotechnology Information database and used for differentiating Gracilaria changii collected at various localities and two other Gracilaria species. Out of 33 primer pairs, only one primer pair gave significant results that can distinguish between three different Gracilaria species as well as G. changii from various localities based on the variation in repeated nucleotides. The unweighted pair group method using arithmetic mean dendrogram analysis grouped Gracilaria species into five main clades: (a) G. changii from Batu Besar (Malacca), Sandakan (Sabah), Bintulu (Sarawak), Batu Tengah (Malacca), Gua Tanah (Malacca), Middle Banks (Penang), Sungai (Sg.) Merbok (Kedah), Teluk Pelandok (Negeri Sembilan), Pantai Dickson (Negeri Sembilan), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore); (b) Gracilaria manilaensis from Cebu, Philippines; (c) G. changii from Morib (Selangor); (d) Gracilaria fisheri from Pattani, Thailand; and (e) G. changii from Pantai Dickson (Negeri Sembilan), Gua Tanah (Malacca), Sg. Merbok (Kedah), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore). This result shows that this primer pair was able to distinguish between three different species, which are G. changii from Morib (Malaysia), G. fisheri from Pattani (Thailand), and G. manilaensis from Cebu (Philippines), and also between different genotypes of G. changii. This suggested that the simple sequence repeat primer we developed was suitable for differentiating between different Gracilaria species due to the polymorphisms caused by the variability in the number of tandem repeats.     

Characterization of saponins and phenolic compounds: antioxidant activity and inhibitory effects on α-glucosidase in different varieties of colored quinoa (Chenopodium quinoa Willd)

ABSTRACT

This study investigated the contents of saponins and phenolic compounds in relation to their antioxidant activity and α-glucosidase inhibition activity of 7 colored quinoa varieties. The total saponin content was significantly different among 7 varieties and ranged from 7.51 to 12.12 mg OAE/g DW. Darker quinoa had a higher content of phenolic compounds, as well as higher flavonoids and antioxidant activity than that of light varieties. Nine individual phenolic compounds were detected in free and bound form, with gallic acid and ferulic acid representing the major compounds. The free and bound phenolic compounds (gallic acid and ferulic acid in particular) exhibited high linear correlation with their corresponding antioxidant values. In addition, the free phenolic extracts from colored quinoa exhibited higher inhibitory activity against α-glucosidase than the bound phenolic extracts. These findings imply that colored quinoa with abundant bioactive phytochemicals could be an important natural source for preparing functional food.     

Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant human melanocortin-4 receptors

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor that plays an essential role in regulating energy homeostasis. Defects in MC4R are the most common monogenic form of obesity, with about 170 distinct mutations identified in human. In addition to the conventional G_s-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2). Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the G_s-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2-fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the G_s-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.     

Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods

Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16–20 h is an effective and simple modified enzyme digestion method.     

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H₂S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H₂S in kidney fibrosis remains to be defined. Here, we investigated the role of H₂S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H₂S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H₂S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H₂S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H₂S in UO-induced kidney fibrosis, suggesting that H₂S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.     

3,5-Diiodo-l-thyronine ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats

3, 5-Diiodothyronine (T2), a natural metabolite of triiodothyronine (T3) from deiodination pathway, can mimic biologic effects of T3 without inducing thyrotoxic effects. Recent studies revealed T3 acted as a protective factor against diabetic nephropathy (DN). Nevertheless, little is known about the effect of T2 on DN. This study was designed to investigate whether and how T2 affects experimental models of DN in vivo and in vitro. Administration of T2 was found to prevent significant decrease in SIRT1 protein expression and activity as well as increases in blood glucose, urine albumin excretion, matrix expansion, transforming growth factor-β1 expression, fibronectin and type IV collagen deposition in the diabetic kidney. Concordantly, similar effects of T2 were exhibited in the cultured rat mesangial cells (RMC) exposed to high glucose and that could be abolished by a known SIRT1 inhibitor, sirtinol. Moreover, enhanced NF-κB acetylation and JNK phosphorylation present in both diabetic rats and high glucose-treated RMC were distinctly dampened by T2. Collectively, these results suggested that T2 was a protective agent against renal damage in diabetic nephropathy, whose action involved regulation of SIRT1.     

Genome-wide analysis of Nilaparvata lugens nymphal responses to high-density and low-quality rice hosts

The brown planthopper （BPH） Nilaparvata lugens is an economically impor- tant pest on rice plants. In this study, the higher population density and yellow-ripe stage of rice plants were used to construct adverse survival conditions （ASC） against BPH nymphs. Simultaneously, the low population density and tillering stage of rice plants were used to establish a suitable survival condition （SSC） as a control. Solexa/Illumina sequencing was used to identify genes of BPH nymphs responding to ASC. Significantly longer duration development of BPH nymphs and significantly lower brachypterous ratio of BPH adults were observed by ASC compared with SSC. A total of 2 544 differentially expressed genes （DEGs） were obtained and analyzed by BLASTx, Gene Ontology and KEGG Orthology. Gene ontology analysis revealed that the DEGs were mainly involved in categories of cell, cell part, cellular process, binding, catalytic, organelle and metabolic processes. 1138 DEGs having enzyme commission numbers were assigned to different metabolic pathways. The largest clusters were neurodegenerative diseases （137, 12.0%）, followed by carbohy- drate metabolism （113, 9.9%）, amino acid metabolism （94, 8.3%）, nucleotide metabolism （76, 6.7%）, energy metabolism （64, 5.6%）, translation （60, 5.3%）, lipid metabolism （58, 5.1%）, and folding, sorting and degradation （52, 4.6%）. Expressing profile of 11 DEGs during eight nymphal developmental stages of BPH were analyzed by quantitative real- time polymerase chain reaction. The 11 genes exhibited differential expression between ASC and SSC during at least one developmental stage. The DEGs identified in this study provide molecular proof of how BPH reconfigures its gene expression profile to adapt to overcrowding and low-quality hosts.     

双歧杆菌预防早产儿坏死性小肠结肠炎的疗效观察

目的 探讨双歧杆菌预防早产儿坏死性小肠结肠炎(NEC)的疗效.方法 随机分成试验组和对照组,对照组给予常规治疗,试验组在对照组基础上给予双歧杆菌治疗.观察两组不同胎龄和不同出生体重早产儿NEC患病率、治疗前后肠道各菌群变化的差异.结果 (1)试验组NEC总发生率显著低于对照组,差异有统计学意义(P＜0.01);(2)试验组出生体重＜1500g早产儿NEC发生率显著低于对照组,差异有统计学意义(P＜0.05);(3)治疗后试验组细菌总数、球菌总数及杆菌总数上升幅度显著大于对照组,差异均有统计学意义(P＜0.01);治疗后试验组杆球菌比值较对照组显著下降,差异有统计学意义(P＜0.01).结论 双歧杆菌可有效预防早产儿坏死性小肠结肠炎.     

Micro-CT检测中等强度跑台运动对去卵巢大鼠腰椎微结构的影响

目的用micro-CT方法,评估中等强度跑台运动对去卵巢大鼠腰椎微结构的影响。方法将30只3月龄雌性SD大鼠按体重分层后随机分为假手术、去卵巢静止和去卵巢运动三个组。运动组每周进行4次45min、速度18 m/min、坡度5°的跑台训练。正式运动处理14周时,取第2腰椎检测骨密度,取第4腰椎行micro-CT分析及三维结构重建;取第3腰椎椎体进行椎体压缩实验。结果去卵巢运动组第2腰椎骨密度、第3腰椎最大载荷、最大应力和弹性模量以及第4腰椎骨小梁体积和骨小梁数目显著高于去卵巢静止组,骨小梁分离度显著低于去卵巢静止组,而骨小梁厚度无显著变化。结论中等强度跑台运动能改善去卵巢大鼠腰椎的微结构。