A potential therapeutic agent for the treatment of hyperuricemia and gout: 3,4-Dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazide.

Uric acid, the metabolic product of purines, relies on xanthine oxidase (XOD) for production. XOD is a target for the development of drugs for hyperuricemia (HUA) and gout. Currently, treatment options remain limited for gout patients. 3, 4-Dihydroxy-5-nitrobenzaldehyde (DHNB) is a derivative of the natural product protocatechualdehyde with good biological activity. In this work, we identify a DHNB thiosemicarbazide class of compounds that targets XOD. 3,4-Dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazone can effectively inhibit XOD activity (IC50 value: 0.0437 µM) and exhibits a mixed inhibitory effect. In a mouse model of acute hyperuricemia, a moderate dose (10 mg/kg.w) of 3,4-dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazide effectively controlled the serum uric acid content and significantly inhibited serum XOD activity. In addition, 3,4-Dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazide showed favorable safety profiles, and mice treated with the target compound did not show any symptoms of general toxicity following a single dose of 500 mg/kg. In the allopurinol group, 50 % of the mice died. These results provide a structural framework and mechanism of XOD inhibition that may facilitate the design of hyperuricemia and gout treatments.

Carvacrol protects mice against LPS-induced sepsis and attenuates inflammatory response in macrophages by modulating the ERK1/2 pathway.

Macrophages play an important role in the development of life-threatening sepsis, which is characterized by multiorgan dysfunction, through their ability to produce inflammatory cytokines. Carvacrol is a phenolic compound that has been confirmed to possess strong anti­inflammatory activity. In this study, we mainly investigated the effect of carvacrol on lipopolysaccharide (LPS)-induced macrophage proinflammatory responses and endotoxic shock. The results showed that carvacrol significantly reduced mouse body weight loss and ameliorated pathological damage to the liver, lung, and heart under LPS-induced sepsis. Carvacrol attenuated inflammatory responses by inhibiting the LPS-induced production of inflammatory cytokine interleukin-6 (IL-6) in vivo and in vitro. Mechanistically, carvacrol inhibited IL-6 production mainly through the ERK1/2 signalling pathway in macrophages. Furthermore, carvacrol improved the survival of septic mice. This study sheds light on the role of carvacrol in the pathogenesis of LPS-induced sepsis, and thus, its potential in treating sepsis patients may be considered.

Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg.

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.

Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea.

Brassica species, including crops such as cabbage, turnip and oilseed, display enormous phenotypic variation. Brassica genomes have all undergone a whole-genome triplication (WGT) event with unknown effects on phenotype diversification. We resequenced 199 Brassica rapa and 119 Brassica oleracea accessions representing various morphotypes and identified signals of selection at the mesohexaploid subgenome level. For cabbage morphotypes with their typical leaf-heading trait, we identified four subgenome loci that show signs of parallel selection among subgenomes within B. rapa, as well as four such loci within B. oleracea. Fifteen subgenome loci are under selection and are shared by these two species. We also detected strong subgenome parallel selection linked to the domestication of the tuberous morphotypes, turnip (B. rapa) and kohlrabi (B. oleracea). Overall, we demonstrated that the mesohexaploidization of the two Brassica genomes contributed to their diversification into heading and tuber-forming morphotypes through convergent subgenome parallel selection of paralogous genes.

Jasmonic acid is a crucial signal transducer in heat shock induced sesquiterpene formation in Aquilaria sinensis.

Agarwood, a highly valuable resinous and fragrant heartwood of Aquilaria plants, is widely used in traditional medicines, incense and perfume. Only when Aquilaria trees are wounded by external stimuli do they form agarwood sesquiterpene defensive compounds. Therefore, understanding the signaling pathway of wound-induced agarwood formation is important. Jasmonic acid (JA) is a well-characterized molecule that mediates a plant's defense response and secondary metabolism. However, little is known about the function of endogenous JA in agarwood sesquiterpene biosynthesis. Here, we report that heat shock can up-regulate the expression of genes in JA signaling pathway, induce JA production and the accumulation of agarwood sesquiterpene in A. sinensis cell suspension cultures. A specific inhibitor of JA, nordihydroguaiaretic acid (NDGA), could block the JA signaling pathway and reduce the accumulation of sesquiterpene compounds. Additionally, compared to SA and H2O2, exogenously supplied methyl jasmonate has the strongest stimulation effect on the production of sesquiterpene compounds. These results clearly demonstrate the central induction role of JA in heat-shock-induced sesquiterpene production in A. sinensis.

[Prokaryotic expression and purification of Aquilaria sinensis(Lour.) Gilg AsMYC2 protein].

The MYC2 transcription factor is a member of the important plant b HLH transcription factor families, and it is also the core regulatory elements in jasmonate（JA） signaling pathway. However, there is a little information about AsMYC2 gene in Aquilaria sinensis. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length coding sequence (CDS) of AsMYC2 gene was amplified using RT-PCR method and subcloned into pGEX-4T-1 vector by the gene recombination technique. The recombinant vector pGEX-4T-1-AsMYC2 was verified by restriction enzyme digestion and nucleotide sequencing, and was transformed into E. coli BL21（DE3） to express the protein. A maximum expression of soluble protein was observed with induction by 0.1 mmol·L(-1) IPTG at 37 ℃ for 4 hours. The fusion protein was purified through a Sepharose-Glutathione column, and verified by SDS-PAGE and Western blotting using an anti-GST polyclonal antibody. We successfully constructed the GST-AsMYC2 plasmid, produced and purified the GST-AsMYC2 fusion protein, which would provide the basic material for polyclonal antibody preparation, interactive factors screening and gene function research. According to the tissue-specific expression pattern analysis by q RT-PCR method, the AsMYC2 gene in A. sinensis tissues is mainly expressed in roots and stems, the main agarwood formation parts, and lowest expressed in leaves. These results indicate that AsMYC2 gene likely play some roles in agarwood formation in A. sinensis.

[Construction and expression of prokaryotic vector of AsJAZ1 gene from Aquilaria sinensis].

The full-length coding sequence (cds) of jasmonate-zim-domain protein (AsJAZ1) gene was cloned from Aquilaria sinensis, the prokaryotic vector was constructed and the recombinant proteins expression was induced to provide the basic material for interactive proteins screen and gene function research. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length cds of AsJAZ1 gene was amplified using RT-PCR method and subcloned into pET-28a vector. The recombinant plasmid identified by restriction enzyme digestion and nucleotide sequencing was transformed into E. coli BL21(DE3). Inducing with 0.5 mmol•L⁻¹ IPTG at 37 ℃ for 4 hours, a fusion protein about 39 kDa was maximumly obtained. AsJAZ1 fusion protein had been expressed successfully mainly in the form of inclusion bodies and only a very small amount was secreted into the cytoplasm in the supernatant.

Cloning, expression and characterization of COI1 gene (AsCOI1) from Aquilaria sinensis (Lour.) Gilg.

Aquilaria sinensis, a kind of typically wounding-induced medicinal plant with a great economical value, is widely used in the production of traditional Chinese medicine, perfume and incense. Coronatine-insensitive protein 1 (COI1) acts as a receptor in jasmonate (JA) signaling pathway, and regulates the expression of JA-responsive genes in plant defense. However, little is known about the COI1 gene in A. sinensis. Here, based on the transcriptome data, a full-length cDNA sequence of COI1 (termed as AsCOI1) was firstly cloned by RT-PCR and rapid-amplification of cDNA ends (RACE) strategies. AsCOI1 is 2330 bp in length (GenBank accession No. KM189194), and contains a complete open frame (ORF) of 1839 bp. The deduced protein was composed of 612 amino acids, with a predicted molecular weight of 68.93 kDa and an isoelectric point of 6.56, and was predicted to possess F-box and LRRs domains. Combining bioinformatics prediction with subcellular localization experiment analysis, AsCOI1 was appeared to locate in nucleus. AsCOI1 gene was highly expressed in roots and stems, the major organs of agarwood formation. Methyl jasmonate (MeJA), mechanical wounding and heat stress could significantly induce the expression level of AsCOI1 gene. AsCOI1 is an early wound-responsive gene, and it likely plays some role in agarwood formation.

[Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis].

The study aimed to clone the open reading frame of cinnamate 4-hydroxylase (C4H) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. One unique sequence containing C4H domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of C4H was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. C4H expression profiles in responds to MeJA (methyl jasmonate) application were analyzed by real-time PCR. The length of C4H open reading frame (ORF) was 1 515 bp, encoding 514 amino acids. The GenBank accession number is KF134783. Inducible-experiments showed that the genes were induced by mechanical wound as well as MeJA induction, and reached the highest expression level at 8 h and 20 h, respectively. The full-length cDNA of C4H and its expression patterns will provide a foundation for further research on its function in the molecular mechanisms of aromatic compounds and flavonoids biosynthesis.

A sequence-based genetic linkage map as a reference for Brassica rapa pseudochromosome assembly.

BACKGROUND: Brassica rapa is an economically important crop and a model plant for studies concerning polyploidization and the evolution of extreme morphology. The multinational B. rapa Genome Sequencing Project (BrGSP) was launched in 2003. In 2008, next generation sequencing technology was used to sequence the B. rapa genome. Several maps concerning B. rapa pseudochromosome assembly have been published but their coverage of the genome is incomplete, anchoring approximately 73.6% of the scaffolds on to chromosomes. Therefore, a new genetic map to aid pseudochromosome assembly is required. RESULTS: This study concerns the construction of a reference genetic linkage map for Brassica rapa, forming the backbone for anchoring sequence scaffolds of the B. rapa genome resulting from recent sequencing efforts. One hundred and nineteen doubled haploid (DH) lines derived from microspore cultures of an F1 cross between a Chinese cabbage (B. rapa ssp. pekinensis) DH line (Z16) and a rapid cycling inbred line (L144) were used to construct the linkage map. PCR-based insertion/deletion (InDel) markers were developed by re-sequencing the two parental lines. The map comprises a total of 507 markers including 415 InDels and 92 SSRs. Alignment and orientation using SSR markers in common with existing B. rapa linkage maps allowed ten linkage groups to be identified, designated A01-A10. The total length of the linkage map was 1234.2 cM, with an average distance of 2.43 cM between adjacent marker loci. The lengths of linkage groups ranged from 71.5 cM to 188.5 cM for A08 and A09, respectively. Using the developed linkage map, 152 scaffolds were anchored on to the chromosomes, encompassing more than 82.9% of the B. rapa genome. Taken together with the previously available linkage maps, 183 scaffolds were anchored on to the chromosomes and the total coverage of the genome was 88.9%. CONCLUSIONS: The development of this linkage map is vital for the integration of genome sequences and genetic information, and provides a useful resource for the international Brassica research community.