High-resolution profile of transcriptomes reveals a role of alternative splicing for modulating response to nitrogen in maize.

BACKGROUND: The fluctuation of nitrogen (N) contents profoundly affects the root growth and architecture in maize by altering the expression of thousands of genes. The differentially expressed genes (DEGs) in response to N have been extensively reported. However, information about the effects of N variation on the alternative splicing in genes is limited. RESULTS: To reveal the effects of N on the transcriptome comprehensively, we studied the N-starved roots of B73 in response to nitrate treatment, using a combination of short-read sequencing (RNA-seq) and long-read sequencing (PacBio-sequencing) techniques. Samples were collected before and 30 min after nitrate supply. RNA-seq analysis revealed that the DEGs in response to N treatment were mainly associated with N metabolism and signal transduction. In addition, we developed a workflow that utilizes the RNA-seq data to improve the quality of long reads, increasing the number of high-quality long reads to about 2.5 times. Using this workflow, we identified thousands of novel isoforms; most of them encoded the known functional domains and were supported by the RNA-seq data. Moreover, we found more than 1000 genes that experienced AS events specifically in the N-treated samples, most of them were not differentially expressed after nitrate supply-these genes mainly related to immunity, molecular modification, and transportation. Notably, we found a transcription factor ZmNLP6, a homolog of AtNLP7-a well-known regulator for N-response and root growth-generates several isoforms varied in capacities of activating downstream targets specifically after nitrate supply. We found that one of its isoforms has an increased ability to activate downstream genes. Overlaying DEGs and DAP-seq results revealed that many putative targets of ZmNLP6 are involved in regulating N metabolism, suggesting the involvement of ZmNLP6 in the N-response. CONCLUSIONS: Our study shows that many genes, including the transcription factor ZmNLP6, are involved in modulating early N-responses in maize through the mechanism of AS rather than altering the transcriptional abundance. Thus, AS plays an important role in maize to adapt N fluctuation.

Effects of astaxanthin on blood coagulation, fibrinolysis and platelet aggregation in hyperlipidemic rats.

CONTEXT: Astaxanthin (ASTX) is a xanthophyll carotenoid that reduces hemostasis in hyperlipidemic organisms. Its antihemostatic mechanisms remain unclear. OBJECTIVE: The effects of ASTX on coagulation, the fibrinolytic system and platelet aggregation were investigated in hyperlipidemic rats. MATERIALS AND METHODS: Different doses of ASTX (5, 10 and 30 mg/kg/day, p.o.) were administered for four weeks to high-fat diet-induced hyperlipidemic rats. Serum lipid and lipoprotein levels were measured with an automatic biochemical analyzer. The prothrombin time (PT), activated partial thromboplastin time (APTT) and maximum platelet aggregation rate (MAR) were determined by a coagulation analyzer. The activities of the tissue-type plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1) and endothelial nitric oxide synthase (eNOS), as well as the levels of thromboxane B(2) [TXB(2)], 6-keto prostaglandin F(1α) [6-keto-PGF(1α)] and platelet granule membrane protein (GMP-140), were measured with enzyme-linked immunosorbent assay kits. Gene and protein expression levels were analyzed by reverse transcriptase polymerase chain reaction and Western blot, respectively. RESULTS: ASTX (30 mg/kg) treatment in hyperlipidemic rats reduced serum TG (0.58 ± 0.14 versus 1.12 ± 0.24 mmol/L), serum TC (1.77 ± 0.22 versus 2.24 ± 0.21 mmol/L), serum LDL-C (1.13 ± 0.32 versus 2.04 ± 0.48 mmol/L), serum MDA (69%), plasma MAR (55%), serum TXB2/6-keto-PGF1α (34%) and serum GMP-140 levels (25%), plasma PAI-1 activity (48%) and downregulated the mRNA (33%) and protein (23%) expression of aorta eNOS, the mRNA (79%) and protein (72%) expression levels of aorta PAI-1. However, ASTX (30 mg/kg/d) treatment increased serum SOD activity (2.1 fold), serum GPx activity (1.8 fold), plasma PT (1.3 fold), plasma APTT (1.7 fold), serum NO (1.4-fold), serum 6-keto-PGF1α (1.3 fold). CONCLUSIONS: ASTX reduced blood coagulation and platelet aggregation and promoted fibrinolytic activity in hyperlipidemic rats. These activities were closely correlated with ASTX, maintaining the balance of t-PA/PAI-1, NO/ROS and TXA2/PGI2 in vivo.

Optimization of astilbin extraction from the rhizome of Smilax glabra, and evaluation of its anti-inflammatory effect and probable underlying mechanism in lipopolysaccharide-induced RAW264.7 macrophages.

Astilbin, a dihydroflavonol derivative found in many food and medicine plants, exhibited multiple pharmacological functions. In the present study, the ethanol extraction of astilbin from the rhizome of smilax glabra Roxb was optimized by response surface methodology (RSM) using Box-Behnken design. Results indicated that the obtained experimental data was well fitted to a second-order polynomial equation by using multiple regression analysis, and the optimal extraction conditions were identified as an extraction time of 40 min, ethanol concentration of 60%, temperature of 73.63 °C, and liquid-solid ratio of 29.89 mL/g for the highest predicted yield of astilbin (15.05 mg/g), which was confirmed through validation experiments. In addition, the anti-inflammatory efficiency of astilbin was evaluated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Results showed that astilbin, at non-cytotoxicity concentrations, significantly suppressed the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), as well as the mRNA expression of inducible nitric oxide synthase (iNOS) and TNF-α in LPS-induced RAW 264.7 cells, but did not affect interleukin-6 (IL-6) release or its mRNA expression. These effects may be related to its up-regulation of the phosphorylation of p65, extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK).

Inhibitory Effects of Chemical Compounds Isolated from the Rhizome of Smilax glabra on Nitric Oxide and Tumor Necrosis Factor- α Production in Lipopolysaccharide-Induced RAW264.7 Cell.

The rhizome of Smilax glabra has been used for a long time as both food and folk medicine in many countries. The present study focused on the active constituents from the rhizome of S. glabra, which possess potential anti-inflammatory activities. As a result, nine known compounds were isolated from the rhizome of S. glabra with the bioassay-guiding, and were identified as syringaresinol (1), lasiodiplodin (2), de-O-methyllasiodiplodin (3), syringic acid (4), 1,4-bis(4-hydroxy-3,5-dimethoxyphenyl)-2,3-bis(hydroxymethyl)-1,4-butanediol (5), lyoniresinol (6), trans-resveratrol (7), trans-caffeic acid methyl ester (8), and dihydrokaempferol (9). Among these compounds, 2 and 3 were isolated for the first time from S. glabra. In addition, the potential anti-inflammatory activities of the isolated compounds were evaluated in vitro in lipopolysaccharide- (LPS-) induced RAW264.7 cells. Results indicated that 4 and 7 showed significant inhibitory effects on NO production of RAW264.7 cells, and 1, 2, 3, and 5 showed moderate suppression effects on induced NO production. 1, 7, and 5 exhibited high inhibitory effects on TNF-α production, with the IC50 values less than 2.3, 4.4, and 16.6 µM, respectively. These findings strongly suggest that compounds 1, 2, 3, 4, 5, 7, and 9 were the potential anti-inflammatory active compositions of S. glabra.

Polysaccharides from Smilax glabra inhibit the pro-inflammatory mediators via ERK1/2 and JNK pathways in LPS-induced RAW264.7 cells.

The rhizomes of Smilax glabra have been used as both food and folk medicine in many countries for a long time. However, little research has been reported on polysaccharides of S. glabra. In the present study, two polysaccharide fractions, SGP-1 and SGP-2, were isolated from the rhizomes of S. glabra with the number average molecular weights of 1.72 × 10(2)kDa and 1.31 × 10(2)kDa, and the weight average molecular weights of 1.31 × 10(5)kDa and 1.18 × 10(5)kDa, respectively, and their mainly monosaccharide compositions were both galactose and rhamnose (2.5:1). Both SGP-1 and SGP-2 significantly suppressed the release of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from LPS-induced RAW 264.7 cells, as well as the mRNA expression of inducible nitric oxide synthase (iNOS), TNF-α and IL-6. Additionally, SGP-1 and SGP-2 repressed the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). These findings strongly suggested polysaccharides were also the anti-inflammatory active ingredient for S. glabra, and the potential of SGP-1 and SGP-2 as the anti-inflammatory agents.