Enzymatic basis for stepwise C-glycosylation in the formation of flavonoid di-C-glycosides in sacred lotus (Nelumbo nucifera Gaertn.).

Lotus plumule, the embryo of the seed of the sacred lotus (Nelumbo nucifera), contains a high accumulation of secondary metabolites including flavonoids and possesses important pharmaceutical value. Flavonoid C-glycosides, which accumulate exclusively in lotus plumule, have attracted considerable attention in recent decades due to their unique chemical structure and special bioactivities. As well as mono-C-glycosides, lotus plumule also accumulates various kinds of di-C-glycosides by mechanisms which are as yet unclear. In this study we identified two C-glycosyltransferase (CGT) genes by mining sacred lotus genome data and provide in vitro and in planta evidence that these two enzymes (NnCGT1 and NnCGT2, also designated as UGT708N1 and UGT708N2, respectively) exhibit CGT activity. Recombinant UGT708N1 and UGT708N2 can C-glycosylate 2-hydroxyflavanones and 2-hydroxynaringenin C-glucoside, forming flavone mono-C-glycosides and di-C-glycosides, respectively, after dehydration. In addition, the above reactions were successfully catalysed by cell-free extracts from tobacco leaves transiently expressing NnCGT1 or NnCGT2. Finally, enzyme assays using cell-free extracts of lotus plumule suggested that flavone di-C-glycosides (vicenin-1, vicenin-3, schaftoside and isoschaftoside) are biosynthesized through sequentially C-glucosylating and C-arabinosylating/C-xylosylating 2-hydroxynaringenin. Taken together, our results provide novel insights into the biosynthesis of flavonoid di-C-glycosides by proposing a new biosynthetic pathway for flavone C-glycosides in N. nucifera and identifying a novel uridine diphosphate-glycosyltransferase (UGT708N2) that specifically catalyses the second glycsosylation, C-arabinosylating and C-xylosylating 2-hydroxynaringenin C-glucoside.

Clinical effect of Changweishu on gastrointestinal dysfunction in patients with sepsis.

OBJECTIVE: To investigate Changweishu's clinical effect on gastrointestinal dysfunction in patients with sepsis. METHODS: Fifty patients with gastrointestinal dysfunction and sepsis were randomly divided into treatment and control groups. The control group patients received routine Western medicine treatments (meropenem, noradrenaline, glutamine glue, Bifidobacterium lactis triple-strain tablet), and the treatment group patients received routine Western medicine treatment combined with Changweishu. Treatments in both groups lasted 7 days. Changes in APACHE II score, gastrointestinal dysfunction score, serum levels of diamine oxidase (DAO), D-lactic acid, inflammatory factors (tumor necrosis factor (TNF)-α, interleukin (IL)-6, and high-mobility group box 1 (HMGB-1)), and the incidence of multiple organ dysfunction syndrome (MODS) and mortality were observed. RESULTS: After treatment, APACHE II score, gastrointestinal dysfunction score, and DAO, D-lactic acid, TNF-α, IL-6, and HMGB-1 levels decreased significantly in both groups, but the decrease was more significant in the treatment group than in the control group. The incidence of MODS and mortality were significantly lower in the treatment group than in the control group. CONCLUSION: The addition of Changweishu to routine Western treatments can improve gastrointestinal function in patients with sepsis and gastrointestinal dysfunction, as well as decreasing the incidence of MODS and mortality and improving patient prognosis.

Fatty acid desaturase 3 (PsFAD3) from Paeonia suffruticosa reveals high α-linolenic acid accumulation.

α-linolenic acid (ALA) deficiency and a skewed ω6: ω3 fatty acid ratio in the diet are thought to be a major cause for the high incidence of cardiovascular, inflammatory, and autoimmune diseases. Recent years, tree peony (Paeonia suffruticosa Andr.) with the high proportion of ALA (more than 45% in seed oil) is widely concerned. However, the underlying accumulation mechanism of the ALA in tree peony seeds remains unknown. In this study, comparative transcriptome analysis was performed between two cultivars ('Saiguifei' and 'Jingshenhuanfa') with different ALA contents. The analysis of the metabolic enzymes associated with ALA biosynthesis and temporal accumulation patterns of unsaturated fatty acids demonstrated the importance of microsomal ω-3 fatty acid desaturase 3 (FAD3). Moreover, PsFAD3 gene was identified from tree peony seeds, which was located in endoplasmic reticulum and the expression levels of PsFAD3 were consistent with ALA accumulation patterns in seeds. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated PsFAD3 protein could catalyze ALA synthesis. These results indicated that PsFAD3 was involved in the synthesis of ALA in seeds and could be exploited by the genetic breeding of new cultivars with high ALA content in tree peony as well as other potential crops.

Identification of microRNAs and long non-coding RNAs involved in fatty acid biosynthesis in tree peony seeds.

MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) act as important molecular regulators in a wide range of biological processes during plant development and seed formation, including oil production. Tree peony seeds contain >90% unsaturated fatty acids (UFAs) and high proportions of α-linolenic acid (ALA, > 40%). To dissect the non-coding RNAs (ncRNAs) pathway involved in fatty acids synthesis in tree peony seeds, we construct six small RNA libraries and six transcriptome libraries from developing seeds of two cultivars (J and S) containing different content of fatty acid compositions. After deep sequencing the RNA libraries, the ncRNA expression profiles of tree peony seeds in two cultivars were systematically and comparatively analyzed. A total of 318 known and 153 new miRNAs and 22,430 lncRNAs were identified, among which 106 conserved and 9 novel miRNAs and 2785 lncRNAs were differentially expressed between the two cultivars. In addition, potential target genes of the microRNA and lncRNAs were also predicted and annotated. Among them, 9 miRNAs and 39 lncRNAs were predicted to target lipid related genes. Results showed that all of miR414, miR156b, miR2673b, miR7826, novel-m0027-5p, TR24651|c0_g1, TR24544|c0_g15, and TR27305|c0_g1 were up-regulated and expressed at a higher level in high-ALA cultivar J when compared to low-ALA cultivar S, suggesting that these ncRNAs and target genes are possibly involved in different fatty acid synthesis and lipid metabolism through post-transcriptional regulation. These results provide a better understanding of the roles of ncRNAs during fatty acid biosynthesis and metabolism in tree peony seeds.

Ebselen protects mitochondrial function and oxidative stress while inhibiting the mitochondrial apoptosis pathway after acute spinal cord injury.

Ebselen is a fat-soluble small molecule and organic selenium compound that regulates the activity of glutathione peroxidase to alleviate mitochondrial oxidative stress and improve mitochondrial function. In the present study, we aimed to investigate the effects of ebselen on mitochondrial oxidative stress response, mitochondrial apotosis, and motor behaviors after spinal cord injury (SCI). We found that ebselen significantly increased the BBB score in motor behavior, thus suggesting a rescue effect of ebselen on motor function after SCI in rats. Meanwhile, we revealed that ebselen can increase glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities after SCI-this suggests ebselen has an antioxidant effect. Furthermore, the ATP content and Na+-K+-ATPase activity in mitochondria were increased by ebselen after SCI, while the mitochondrial membrane potential (MMP) was decreased by ebselen. The Cytochrome C and Smac release from mitochondria were reduced by ebselen after SCI, thus indicating improved membrane permeability by ebselen. Moreover, the alterations in caspase-3, Bax and Bcl-2 protein expression, as well as the proportion of cell apoptosis were improved by ebselen treatment, which together suggested that ebselen has an inhibitory effect on mitochondrial apotosis pathways after SCI. Taken together, our results suggest that ebselen can inhibit secondary damage caused by spinal cord injury. Indeed it plays a neuroprotective role in spinal cord injury perhaps by improving mitochondrial function and inhibiting the mitochondrial apoptosis pathway.

An Arabidopsis mutant of inositol pentakisphosphate 2-kinase AtIPK1 displays reduced arsenate tolerance.

Arsenate [As(V)] toxicity is considered to be derived from similarities in the chemical properties of As(V) and phosphate (Pi). An Arabidopsis thaliana mutant of inositol pentakisphosphate 2-kinase (AtIPK1), atipk1-1, has previously exhibited lower level of phytate and higher level of Pi, relative to wild-type (WT). Here, atipk1-1 displayed hypersensitivity to As(V) stress and less As(V) uptake when compared to WT. Overexpression of AtIPK1 controlled by the CaMV 35S promoter partially rescued the As(V)-sensitive phenotype of atipk1-1. When compared to control Pi status, addition of Pi enhanced As(V) tolerance of both WT and atipk1-1 plants, while the arsenic concentration was less reduced in the latter genotype. Despite the higher Pi level in atipk1-1 than did WT plants, the mutant suffered more severe Pi starvation under Pi limitation stress, indicating that Pi homeostasis was altered in the mutant. Gene expression analysis of WT and atipk1-1 plants showed the diverse effect of As(V) stress on Pi starvation-dependent regulation of Pi-responsive genes. Our study suggested that a particular mechanism of As(V) toxicity existed in atipk1-1 mutant, and may offer new insights into the interactions between Pi homeostasis and As(V) detoxification in plants.

[Expression of neuropeptides ghrelin and Nesfatin-1 in kainic acid kindling rats].

OBJECTIVE: To observed the post-seizure expression level of neuropeptides ghrelin and Nesfatin-1 between the untreated and treated groups in kainic acid-kindling rats and understand the significance of their basic expressions. METHODS: The male S-D rats were divided randomly into NS and VPA groups after seizure. Each group was divided into 7 subgroups of 3 h, 6 h, 12 h, 24 h, 3 d, 7 d and 14 d. And the blank group (without NS or VPA) and sham group (normal saline instead of kainic acid) were established. RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay) and immunohistochemistry were used to detect the expression levels of ghrelin and Nesfatin-1 in the serum and hypothalamus of kainic acid-kindling rats. RESULTS: Compared with the blank and sham groups, the seizure rats had a decreased expression of ghrelin while there was an elevated expression of Nesfatin-1 in hypothalamus. This trend was consistent with the serum expressions of deacylated ghrelin and Nesfatin-1. The expression of ghrelin became elevated at all stages in the VPA group. Especially the subgroups of 24 h (0.320 ± 0.007) and 7 d (0.284 ± 0.004) had a significant increase (P < 0.05) while their expression levels remained lowed than that of the blank group (0.342 ± 0.039). The expressions of Nesfatin-1 had a significant decrease (P < 0.05) in all VPA subgroups while they were higher than those in the blank group (0.124 ± 0.007). CONCLUSION: An accurate detection of serum expression level of deacylated ghrelin and Nesfatin-1 may reflect the fluctuation trend of neuropeptides in hypothalamus. It may offer a simple, sensitive and noninvasive method of diagnosing and treating epilepsy.

[mRNA expression of activity-regulated cytoskeletal-associated protein in hippocampus induced by insular-kindled rats].

OBJECTIVE: To investigate the expression of Arc mRNA in insular electrical kindled and single electrical stimulated rats and its significance. METHODS: Male SD rats were divided randomly into kindled group, single electrical stimulated group, sham-operated group and control group. Each group was divided into 2 sub-groups at different time points. Kindled group: establishing chronic insular electrical kindled model, decapitation to perform RT-PCR of Arc mRNA on hippocampus and to applying in situ hybridization of Arc mRNA on dentate gyrus; Single electrical stimulated group: using the same method as the kindled group with only electrical stimulation twice; Sham-operated group: using the same method to the kindled group without electrical stimulation; CONTROL GROUP: no surgery. RESULTS: Expression of Arc mRNA in the hippocampus of single electrical stimulated, sham-operated and control groups were 0.72 ± 0.14, 0.75 ± 0.16 and 0.71 ± 0.14 respectively. They were significantly less than the kindled group of 1.78 ± 0.43(P < 0.01) at 3 h. The expression of Arc mRNA had no significant difference (P > 0.05) among 4 groups at 6 h; in situ hybridization (cell count) of Arc mRNA showed the expression of Arc mRNA in the kindled group was 112.8 ± 6.0. And it was significantly higher than the other three groups of 46.25 ± 4.35, 45.25 ± 6.23, 44.75 ± 6.49 (P < 0.01) after 3 h, there were no significant difference among 4 groups at 6 h (P > 0.05). CONCLUSION: Insular epilepsy increase the expression of Arc mRNA in hippocampus. Arc may play an important role in the synaptic plasticity of insular epilepsy.

Transcriptional regulation of wheat VER2 promoter in rice in response to abscisic acid, jasmonate, and light.

VER2 is a key gene associated with vernalization process in winter wheat. The expression of VER2 can be induced by low temperature treatment. To further understand how the expression of this gene is mediated by various external and internal factors, different lengths of the VER2 promoter region have been transcriptionally fused with a reporter gene, green fluorescence protein (GFP), and transformed into the model plant, rice (Oryza sativa L.). Using confocal and Western blot analyses, we determined several possible response elements in the promoter region, which could sense ABA, JA, and other environmental cues.

[Functional analysis of vernalization-related gene VER17 in flower development using antisense RNA strategy in winter wheat].

To understand the function of vernalization-related gene VER17 in winter wheat (Triticum aestivum L. cv. Jingdong No.1), an antisense RNA strategy was used. The antisense VER17 with a vector pBI121 was constructed and transformed into winter wheat by using the pollen-tube-pathway method. Fourteen independent transgenic plants transformed with antisense VER17 and five control transformants transformed with pBI121 blank vector were obtained and confirmed by GUS histochemical assay and PCR-Southern blot analysis. Phenotypes of T(0) and T(1) transgenic plants showed that the plants of the antisense VER17 transgenic lines degenerated top or basal spikelets and had delayed flowering time, which suggested that the VER17 gene functions in accelerating flowering and the development of the top or basal spikelets and the stamen which were impacted by vernalization treatment.

Vernalization-induced flowering in wheat is mediated by a lectin-like gene VER2.

A vernalization-related gene VER2 was isolated from winter wheat ( Triticum aestivum L.) using a differential screening approach. The deduced VER2 is a lectin-like protein of 300 amino acids, which contains the presence of a jacalin-like GWG domain. RNA in situ hybridization results demonstrated that VER2 gene expression is restricted to the marginal meristems of immature leaves in vernalized wheat seedlings. No hybridization signal was detected in the epidermal tissue and vascular bundles. However, "devernalization" resulted in the silencing of VER2 gene activity. The gene expression pattern of VER2 induced by jasmonate was similar to that induced by vernalization. Antisense inhibition of VER2 in transgenic wheat showed that heading and maturation time were delayed up to 6 weeks compared with non-transformed wheat and the pBI121empty-vector-transformed wheat. Tissue degeneration at the top of the spike was also noticed in the antisense inhibited transgenic wheat. These results suggest that VER2 plays an important role in vernalization signaling and spike development in winter wheat.