Identification and functional analysis of the cathepsin D gene promoter of Bombyx mori.

The gene encoding cathepsin D of silkworm, Bombyx mori (BmCatD) is specifically expressed in the larval fat body and pupal gut, and plays an important role in the programmed cell death during metamorphosis. To identify element involved in this transcription-dependent spatial restriction, truncation and deletion of the 5' terminal from the BmCatD promoter were conducted in vivo. The recombinant AcMNPV vector (Autographa californica multiple nucleopolyhedrovirus) with a dual-luciferase quantitative assay system was used as the transfer. A 289 bp DNA sequence (-1,214 to -925) upstream of the transcriptional start site is found to be responsible for promoting tissue-specific transcription. Further analysis of a series of deletion within the 289 bp region of overlapping deletion showed that a 33 bp region (-1,071 to -1,038) sequence suppresses the ectopic expression of the BmCatD promoter. These results suggest that this 33 bp region could function as a promoter element in the tissue-specificity expression.

Identification of ecdysone response elements (EcREs) in the Bombyx mori cathepsin D promoter.

Bombyx mori Cathepsin D (BmCatD) is specifically expressed in the fat body, and plays a critical role for the programmed cell death of the larval fat body and pupal gut during metamorphosis. To better understand the transcriptional control of BmCatD expression, we conducted this study to identify the ecdysone response elements (EcREs) in the BmCatD promoter and clarify their regulational functions. We inserted EcREs into a recombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector and performed luciferase assay with a dual-luciferase quantitative assay system. Three putative EcREs were located at positions -109 to -99, -836 to -826 and -856 to -846 relative to the transcription start site. Overlapping deletion studies of this EcRE region showed that the three EcREs could suppress the ectopic expression of the BmCatD promoter. EcRE mutations resulted in the loss of the fat body-specific expression of the BmCatD gene. These results suggest that the EcREs are vital for activation of the promoter by 20-hydroxyecdysone (20E) in the larval fat body and further support the crucial role of ecdysone signaling to control cathepsin D gene transcription. It may suggest that the heterodimeric complex EcR/USP mediates the activation of ecdysone-dependent BmCatD transcription in the larval fat body of B. mori.

Antioxidant and Hemolysis Protective Effects of Polyphenol-Rich Extract from Mulberry Fruits.

BACKGROUND: Mulberry fruits are a superior source of polyphenol, especially anthocyanins that contribute potentially to the beneficial effects which include reducing the risk of cardiovascular diseases and cancers with antioxidant, anti-inflammatory, and chemoprotective properties. OBJECTIVES: In this study, purification of the polyphenol-rich extract from mulberry fruit (MPE) was purified and assessed the activities of antioxidant and hemolysis protective in vivo and in vitro. MATERIALS AND METHODS: Antioxidant activities in vitro was measured by quantifying its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, reducing power and Fe2+-chelating ability. MPE was purified by high-pressure liquid chromatography (HPLC) and analyzed individual polyphenols using liquid chromatography-mass spectrometry (LC-MS)/MS. RESULTS: The total polyphenol content was 147.69 ± 0.02 mg gallic acid equivalents (GAE)/g dried weight (DW) in the extract and 403.55 ± 0.02 mg GAE/g DW in the purified extract. Further identification by HPLC-ultraviolet-visible and LC-MS/MS analysis indicated in MPE, an anthocyanin compound, cyanidin-3-O-glucoside. With regard to in vitro assays, MPE possessed antioxidant effect, especially in Fe2+ chelating ability with an IC50 value of 1.016 mg/mL. The protective effects on mouse red blood cell hemolysis and lipid peroxidation ex vivo were dose and time dependent. CONCLUSION: It indicates that MPE could be a good candidate for future biomedical applications to promote human health with limited side effects. SUMMARY: Mulberry fruit is an excellent source of polyphenols, in particular, anthocyanins, which has infinite health benefits. This study determined the predominant anthocyanin, cyanidin-3-glucoside, could possibly be the rationale behind the antioxidant and antihemolytic effect of MPE. Results indicate that MPE could be a good candidate for future biomedical applications to promote human health with limited side effects. Abbreviations used: MPE: Purification of the polyphenol-rich extract from mulberry fruit, LC-MS: Liquid chromatography-mass spectrometry, HPLC: High-pressure liquid chromatography, DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging activity, RBC: Red blood cell, GAE: Gallic acid equivalent, FeCl2: Ferrous chloride, H2O2: Hydrogen peroxide, EDTA-2Na: Ethylenediaminetetraacetic acid disodium salt, PBS: Phosphate-buffered saline, TCA: Trichloroacetic acid, TBA: 2-thiobarbituric acid, FeSO4: Ferrous sulphate, MDA: Malondialdehyde, VC: Vitamin C, DW: Dried weight.

[Responses of ecosystem carbon budget to increasing nitrogen deposition in differently degraded Leymus chinensis steppes in Inner Mongolia, China].

Based on a field manipulative nitrogen (N) addition experiment, the effects of atmospheric N deposition level change on the plant biomass and net primary productivity (NPP), soil respiration (Rs) and net ecosystem exchange (NEE) were investigated respectively in 2009 and 2010 in two differently degraded Leymus chinensis steppes in Inner Mongolia of China, and the difference in the response of NEE to equal amount of N addition [10 g x (M2 x a)(-1), MN] between the two steppes was also discussed. The results indicated that for the light degraded Leymus chinensis steppe (site A) , the average plant aboveground biomass (AGB) in MN treatment were 21.5% and 46.8% higher than those of CK in these two years. But for the moderate degraded Leymus chinensis steppe (site B), the N addition decreased the plant AGB and ANPP in 2009, while showed positive effects in 2010. N addition increased the belowground biomass (BGB) of the both sites and belowground NPP (BNPP) of site B in both years, but decreased the BNPP of site A in 2010. The increase of N input in the two steppes did not change the seasonal variation of Rs. The cumulative annual soil C emissions in MN treatment in site A showed an increase of about 14.6% and 25.7% of those in the CK respectively for these two years, while were decreased by about 10.4% and 11.3%, respectively in site B. The NEE of MN treatments, expressed by C, for the two steppes were 59.22 g x (m2 x a)(1) and 166.68 g x (m2 x a)(-1), as well as 83.27 g x (m2 x a)(-1) and 117.47 g x (m2 x a)(-1), respectively in these two years. The increments in NEE originated from N addition for these two years were 15.79 g x (M2 x a)(-1) and 82.94 g x (M2 x a)(-1) in site A and 74.54 g x (M2 x a)(-1) and 101.23 g x (M2 x a)(-1) in site B. The N input per unit could obtain greater C sink effect in the steppe with lower initial N level.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism.

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

[Microbial response mechanism for drying and rewetting effect on soil respiration in grassland ecosystem: a review].

As one of the most important and wide distribution community type among terrestrial ecosystems, grassland ecosystem plays a critical role in the global carbon cycles and climate regulation. China has extremely rich grassland resources, which have a huge carbon sequestration potential and are an important part of the global carbon cycle. Drying and rewetting is a common natural phenomenon in soil, which might accelerate soil carbon mineralization process, increase soil respiration and exert profound influence on microbial activity and community structure. Under the background of the global change, the changes in rainfall capacity, strength and frequency would inevitably affect soil drying and wetting cycles, and thus change the microbial activity and community structure as well as soil respiration, and then exert important influence on global carbon budget. In this paper, related references in recent ten years were reviewed. The source of soil released, the trend of soil respiration over time and the relationship between soil respiration and microbial biomass, microbial activity and microbial community structure during the processes of dry-rewetting cycle were analyzed and summarized, in order to better understand the microbial response mechanism for drying and rewetting effecting on soil respiration in grassland ecosystem, and provide a certain theoretical basis for more accurate evaluation and prediction of future global carbon balance of terrestrial ecosystems and climate change.

[Reponses of soil total organic carbon and dissolved organic carbon to simulated nitrogen deposition in temperate typical steppe in Inner Mongolia, China].

Based on a field manipulative nitrogen (N) addition experiment, the effects of atmospheric N deposition level change on the contents, inter-annual variation and profile distribution of soil total organic carbon (TOC) and dissolved organic carbon (DOC) were investigated from May, 2008 to October, 2011 in a temperate typical steppe in Inner Mongolia of China, and the relationship between TOC and DOC was also discussed. The treatments in the manipulative experiment included N additions at rates of 0, 5, 10, and 20 g x (m2 x a)(-1), representing the control (CK), low N (LN), medium N (MN), and high N (HN) treatment, respectively. The results indicated that the concentrations of soil TOC and DOC decreased progressively with soil depth in all cases except for the DOC at 10-20 cm depth in individual years. The increase of N input in typical steppe did not change the vertical distribution of soil TOC and DOC, but reduced the vertical variation of TOC and increased the vertical variation of DOC in the surface soil horizon. In addition, the contents of soil TOC and DOC at 0- 10 cm and 10- 20 cm soil layers changed insignificantly after the continuous increase in anthropogenic N input for four years. The soil organic C density of 0-20 cm soil layer for different N treatment levels varied between 3.9 kg x m(-2) and 5.6 kg x m(-2), and the soil organic C densities of fertilized treatments in the first two years were similar to or slightly lower than those of CK, while in the following two years, the increase in N deposition gradually played a positive role in increasing soil organic C density, but the differences in soil TOC and DOC contents between CK and fertilized plots were not significant (P > 0.05). The ratio of soil DOC to TOC (DOC/TOC) varied from 0.32% to 1.09%. The increase in N deposition generally lowered the proportion of DOC in soil TOC, which was conducive to the accumulation of soil organic C. The change of soil DOC was positively correlated with that of TOC (P < 0.01). The temporal variations of soil DOC in different N treatments were all far greater than those of TOC, and the soil DOC was the important sensitive indicator for predicting and evaluating the response of soil C pool to the change in atmospheric N deposition in the temperate grassland ecosystem.

Core promoter regulates the expression of cathepsin B gene in the fat body of Bombyx mori.

Bombyx mori cathepsin B (BmCatB) is involved in the programmed cell death of the fat body during B. mori metamorphosis. For a better understanding of the functional regulatory mechanism, the promoter region of BmCatB in the transcriptional regulation has been identified and analyzed in the present study. BmCatB promoter region performed by the 5' truncation or mutagenesis of EcREs was inserted in the pFA3Luc-A3RL double fluorescence expression vector to activate the fireflies luciferase (FLuc) gene. The results indicated that the dual-luciferase activity of BmCatB gene in the silkworm larval fat body is regulated by the length of promoter. Site-directed mutagenesis of EcRE experiment has shown that the EcREs are up-regulated significantly in the regulation of the BmCatB promoter. A 142bp region (-1165 to -1023) and EcREs are the mainly fat-body tissue-specificity related region and could function as a core promoter element.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.