Background: Dunaliella salina is a good model organism for studying salt stress. In order to have a global understanding of the expression profiles of Dunaliella salina in response to hypersaline stress, we performed quantitative transcriptomic analysis of Dunaliella salina under hypersaline stress (2.5 M NaCl) of different time duration by the second and third generation sequencing method. Results: Functional enrichment of the up-regulated genes was used to analyze the expression profiles. The enrichment of photosynthesis was observed, accompanied by enrichments of carbon fixation, pigment biosynthetic process and heme biosynthetic process, which also imply the enhancement of photosynthesis. Genes responsible for starch hydrolysis and glycerol synthesis were significantly up-regulated. The enrichment of biosynthesis of unsaturated fatty acids implies the plasma membrane undergoes changes in desaturation pattern. The enrichment of endocytosis implies the degradation of plasma membrane and might help the synthesis of new glycerophospholipid with unsaturated fatty acids. Co-enrichments of protein synthesis and degradation imply a higher protein turnover rate. The enrichments of spliceosome and protein processing in endoplasmic reticulum imply the enhancement of regulations at post-transcriptional and post-translational level. No up-regulation of any Na + or Cl − channels or transporters was detected, which implies that the extra exclusion of the ions by membrane transporters is possibly not needed. Voltage gated Na + and Cl − channels, mechanosensitive ion channel are possible signal receptors of salt stress, and Ca 2+ and MAP kinase pathways might play a role in signal transduction. Conclusion: At global transcriptomic level, the response of Dunaliella salina to hypersaline stress is a systematic work, possibly involving enhancements of photosynthesis, carbon fixation, and heme biosynthetic process, acceleration of protein turnover, spliceosome, protein processing in endoplasmic reticulum, and endocytosis, as well as degradation of starch, synthesis of glycerol, membrane lipid desaturation. Altogether, the changes of these biological processes occurred at trancriptomic level will help understand how a new intracellular balance achieved in Dunaliella salina to adapt to hypersaline environment, which are worth being confirmed at the physiological levels.
Lipase from Aspergillus terreus was purified to homogeneity using ammonium sulfate precipitation and chromatographies with Q-Sepharose and Sephacryl S-200. It showed a single band on SDS-PAGE and IEF-PAGE with a relative molecular mass of 37.2 kDa and pI of 3.2. Its glycoprotein nature was confirmed with the percentage of saccharides of 5.02% and 3.88% determined by the phenol/sulfuric acid and anthrone/ sulfuric acid methods, respectively. The lipase hydrolyzed both plant oils and animal oils, with the K(m) value for substrate p-NPP of 16.42 mM at pH 6.0, 50 °C. The enzyme was tolerant in a wide range of pH (pH 3-12) with optimum activity at pH 4.0. It remained stable under the highest temperature of 65 °C, with maximal activity at 50 °C. Ca²⁺, Co²⁺, Mn²⁺, and Ni²⁺ stimulated enzyme activity, but Hg²⁺ caused inhibition. Detected detergents had no obvious effect on enzyme activity, except SDS, which stimulated the activity at lower concentrations but inhibited the activity at higher concentrations. The inhibitory effect on enzyme activity of phenylmethanesulfonyl fluoride revealed that the Ser was involved in catalysis. Saccharides had no obvious effect on enzyme activity but could enhance its thermostability. Furthermore, the enzyme was resistant to trypsin digestion.
Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disease that may lead to joint damage, deformity, and disability, if not treated effectively. Hedyotis diffusa Willd (HDW) and its main components have been widely used to treat a variety of tumors and inflammatory diseases. The present study utilized a network pharmacology approach, microarray data analysis and molecular docking to predict the key active ingredients and mechanisms of HDW against RA. Eleven active ingredients in HDW and 180 potential anti-RA targets were identified. The ingredients-targets-RA network showed that stigmasterol, beta-sitosterol, quercetin, kaempferol, and 2-methoxy-3-methyl-9,10-anthraquinone were key components for RA treatment. KEGG pathway results revealed that the 180 potential targets were inflammatory-related pathways with predominant enrichment of the AGE-RAGE, TNF, IL17, and PI3K-Akt signaling pathways. Screened through the PPI network and with Cytoscape software, RELA, TNF, IL6, TP53, MAPK1, AKT1, IL10, and ESR1 were identified as the hub targets in the HDW for RA treatment. Molecular docking was used to identify the binding of 5 key components and the 8 related-RA hub targets. Moreover, the results of network pharmacology were verified by vitro experiments. HDW inhibits cell proliferation in MH7A cells in a dose and time-dependent manner. RT-qPCR and WB results suggest that HDW may affect hub targets through PI3K/AKT signaling pathway, thereby exerting anti-RA effect. This study provides evidence for a clinical effect of HDW on RA and a research basis for further investigation into the active ingredients and mechanisms of HDW against RA.
The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.
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