Inhibition of tyrosinase by gastrodin: An integrated kinetic-computational simulation analysis

We studied the inhibitory effect of gastrodin on tyrosinase using inhibition kinetics and computational simulation. Gastrodin reversibly inhibited tyrosinase in a mixed-type manner with K_i = 123.8 ± 20.2 mM. Time-interval kinetics revealed the inhibition to be a first-order process with mono- and bi-phasic components. Using AutoDock Vina, we calculated a binding energy of ?6.3 kcal/mol for gastrodin and tyrosinase, and we performed a molecular dynamics simulation of the tyrosinase–gastrodin interaction. The simulation results suggested that gastrodin interacts primarily with histidine residues in the active site. A 10-ns molecular dynamics simulation showed that one copper ion in the tyrosinase active site was responsible for the interaction with gastrodin. Our study provides insight into the inhibition of tyrosinase by the hydroxyl groups of gastrodin. A combination of inhibition kinetics and computational calculations may help to confirm the inhibitory action of gastrodin on tyrosinase and define the mechanisms of inhibition.     

Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K^M vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K^M-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5–8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn²⁺, which caused a strong inhibition. The K _m and V _max, towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-β-1,4-d-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.     

Marker-assisted breeding of Thai fragrance rice for semi-dwarf phenotype, submergence tolerance and disease resistance to rice blast and bacterial blight

Rice is a staple food crop for more than half of the world’s population. However, rice production is affected by many types of abiotic and biotic stress. Genetic breeding by utilizing natural resistance or tolerance genes is the most economic and efficient way to combat or adapt to these stresses. Khao Dawk Mali 105 (KDML 105) is an elite cultivar of aromatic rice mainly grown in Thailand. However, the production of KDML 105 is affected by lodging problems due to its tall plant type, regular flash floods or short-term submergence during the monsoon season, and diseases such as blast and bacterial blight. Here we report the pyramiding of semi-dwarf gene sd1, submergence tolerance gene Sub1A, blast resistance gene Pi9 and bacterial blight resistance genes Xa21 and Xa27 in KDML 105 by marker-assisted selection. The improved line, designated T5105, has a semi-dwarf phenotype with improved lodging resistance and a greater harvest index. T5105 survives after 2 weeks of complete submergence without significant loss of viability. T5105 confers high resistance to all five Magnaporthe oryzae isolates tested and provides resistance or moderate resistance to 25 of the 27 Xanthomonas oryzae pv. oryzae strains tested. In addition, T5105 produced higher yield than KDML 105 in two field trials and retains similar good grain quality to KDML 105. The development of T5105 provides a new line to boost the production of high-quality aromatic rice in tropical regions.     

Generation and analysis of expressed sequence tags for microsatellite marker development in Calamus simplicifolius C. F. Wei

Rattans serve as an important source of raw non-wood materials for furniture and handicraft industries worldwide. However, their genomic sequence information in public databases is very limited. In this study, a set of 2,528 good-quality expressed sequence tags (ESTs) were generated from a full-length cDNA library constructed previously with root, stem and male inflorescence tissues of Calamus simplicifolius C. F. Wei, a rattan species native to Hainan Island, China. The ESTs were assembled into 1,588 unigenes, including 1,221 singletons and 367 contigs. BlastX searches against the GenBank non-redundant protein database revealed that 1,248 (78.6 %) unigenes had at least one significant match (E ≤ 10^?5). The gene ontology functional classification assigned 991, 669 and 977 of the unigenes to the cellular component, molecular function and biological process categories, respectively. A total of 71 simple sequence repeat (SSR) loci were developed among these ESTs, including 65 polymorphic across 19 rattan species representing three genera. High levels of cross-species/genus transferability were observed for the EST-SSRs. For the polymorphic EST-SSR markers, the number of alleles per locus and polymorphic information content ranged from 2 to 25 (mean 11.1) and from 0.135 to 0.949 (mean 0.695), respectively. The EST sequences and the EST-SSR primers have been deposited in GenBank databases of EST (IDs JK838364–40891) and Probe (IDs Pr16718978–9048, to be assigned).     

Involvement of FgERG4 in ergosterol biosynthesis,vegetative differentiation and virulence in Fusarium graminearum

The ergosterol biosynthesis pathway is well understood in Saccharomyces cerevisiae, but currently little is known about the pathway in plant‐pathogenic fungi. In this study, we characterized the Fusarium graminearum FgERG4 gene encoding sterol C‐24 reductase, which catalyses the conversion of ergosta‐5,7,22,24‐tetraenol to ergosterol in the final step of ergosterol biosynthesis. The FgERG4 deletion mutant ΔFgErg4‐2 failed to synthesize ergosterol. The mutant exhibited a significant decrease in mycelial growth and conidiation, and produced abnormal conidia. In addition, the mutant showed increased sensitivity to metal cations and to various cell stresses. Surprisingly, mycelia of ΔFgErg4‐2 revealed increased resistance to cell wall‐degrading enzymes. Fungicide sensitivity tests revealed that ΔFgErg4‐2 showed increased resistance to various sterol biosynthesis inhibitors (SBIs), which is consistent with the over‐expression of SBI target genes in the mutant. ΔFgErg4‐2 was impaired dramatically in virulence, although it was able to successfully colonize flowering wheat head and tomato, which is in agreement with the observation that the mutant produces a significantly lower level of trichothecene mycotoxins than does the wild‐type progenitor. All of these phenotypic defects of ΔFgErg4‐2 were complemented by the reintroduction of a full‐length FgERG4 gene. In addition, FgERG4 partially rescued the defect of ergosterol biosynthesis in the Saccharomyces cerevisiae ERG4 deletion mutant. Taken together, the results of this study indicate that FgERG4 plays a crucial role in ergosterol biosynthesis, vegetative differentiation and virulence in the filamentous fungus F. graminearum.     

The Invasive Wetland Plant Alternanthera philoxeroides Shows a Higher Tolerance to Waterlogging than Its Native Congener Alternanthera sessilis

Plant invasion is one of the major threats to natural ecosystems. Phenotypic plasticity is considered to be important for promoting plant invasiveness. High tolerance of stress can also increase survival of invasive plants in adverse habitats. Limited growth and conservation of carbohydrate are considered to increase tolerance of flooding in plants. However, few studies have examined whether invasive species shows a higher phenotypic plasticity in response to waterlogging or a higher tolerance of waterlogging (lower plasticity) than native species. We conducted a greenhouse experiment to compare the growth and morphological and physiological responses to waterlogging of the invasive, clonal, wetland species Alternanthera philoxeroides with those of its co-occurring, native, congeneric, clonal species Alternanthera sessilis. Plants of A. philoxeroides and A. sessilis were subjected to three treatments (control, 0 and 60 cm waterlogging). Both A. philoxeroides and A. sessilis survived all treatments. Overall growth was lower in A. philoxeroides than in A. sessilis, but waterlogging negatively affected the growth of A. philoxeroides less strongly than that of A. sessilis. Alternanthera philoxeroides thus showed less sensitivity of growth traits (lower plasticity) and higher waterlogging tolerance. Moreover, the photosynthetic capacity of A. philoxeroides was higher than that of A. sessilis during waterlogging. Alternanthera philoxeroides also had higher total non-structural and non-soluble carbohydrate concentrations than A. sessilis at the end of treatments. Our results suggest that higher tolerance to waterlogging and higher photosynthetic capacity may partly explain the invasion success of A. philoxeroides in wetlands.     

Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

Background

Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines.

Material and Methods

Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis.

Results

Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression.

Conclusion

Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.     

Correlation of Cytokine Levels and Microglial Cell Infiltration during Retinal Degeneration in RCS Rats

Microglial cells, which are immunocompetent cells, are involved in all diseases of the central nervous system. During their activation in various diseases, a variety of soluble factors are released. In the present study, the correlation between cytokine levels and microglial cell migration in the course of retinal degeneration of Royal College of Surgeons (RCS) rats was evaluated. MFG-E8 and CD11b were used to confirm the microglial cells. In the retina of RCS rats, the mRNA expression of seven genes (MFG-E8 and its integrins αυ and ß5, CD11b and the cytokines TNF-α, IL-1ß, and MCP-1) formed almost similar bimodal peak distributions, which were centred at P7 and P45 to P60. In contrast, in rdy rats, which comprised the control group, a unimodal peak distribution centred at P14 was observed. The gene expression accompanied the activation and migration of microglial cells from the inner to the outer layer of the retina during the process of degeneration. Principal component analysis and discriminant function analysis revealed that the expression of these seven genes, especially TNF-α and CD11b, positively correlated with retinal degeneration and microglial activity during retinal degeneration in RCS rats, but not in the control rats. Furthermore, linear regression analysis demonstrated a significant correlation between the expression of these genes and the activation of microglial cells in the dystrophic retina. Our findings suggest that the suppression of microglial cells and the blockade of their cytotoxic effects may constitute a novel therapeutic strategy for treating photoreceptor death in various retinal disorders.