Physicochemical properties of silkworm larvae protein isolate and gastrointestinal hydrolysate bioactivities

The objectives of this study were to investigate the amino acid composition and thermal properties of silkworm larvae protein isolate (SLPI) and to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibitory and antioxidant activities of its hydrolysate prepared with gastrointestinal enzymes. The results showed that, SLPI was a high quality protein source with a well-balanced composition of essential amino acids, which was especially rich in glutamic acid (13.79 g/100 g protein), aspartic acid (10.44 g/100 g protein), leucine (8.68 g/100 g protein), lysine (8.01 g/100 g protein) and arginine (6.59 g/100 g protein). In additon, three endothermic denaturation transitions were observed in DSC thermograms of SLPI. The maximum transition peak occurred in the third thermal transition, which denaturation temperature (Td), peak temperature of denaturation (Tp) and enthalpy change (ΔH) were 76.95°C, 80.42°C and 783.75 J/g, respectively. SLPI hydrolysate exhibited strong ACE-inhibitory activity (IC50=8.3 μg/ml) and relatively higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50=57.91 μg/ml) and ferrous ions chelating capacity (IC50=2.03 mg/ml). Moreover, the  hydrolysate showed notable reducing power. It was concluded that, SLPI might be considered as a multifunctional ingredients for functional foods with protein supplements, ACE-inhibitory and antioxidant activity.Key words: Silkworm larvae protein isolates (SLPI), amino acid composition, thermal properties, gastrointestinal enzymes, hydrolysis, ACE inhibition, antioxidant

Construction and Application of an Electronic Spatiotemporal Expression Profile and Gene Ontology Analysis Platform Based on the EST Database of the Silkworm, Bombyx mori

An Expressed Sequence Tag (EST) is a short sub-sequence of a transcribed cDNA sequence. ESTs represent gene expression and give good clues for gene expression analysis. Based on EST data obtained from NCBI, an EST analysis package was developed (apEST). This tool was programmed for electronic expression, protein annotation and Gene Ontology (GO) category analysis in Bombyx mori (L.) (Lepidoptera: Bombycidae). A total of 245,761 ESTs (as of 01 July 2009) were searched and downloaded in FASTA format, from which information for tissue type, development stage, sex and strain were extracted, classified and summed by running apEST. Then, corresponding distribution profiles were formed after redundant parts had been removed. Gene expression profiles for one tissue of different developmental stages and from one development stage of the different tissues were attained. A housekeeping gene and tissue-and-stage-specific genes were selected by running apEST, contrasting with two other online analysis approaches, microarray-based gene expression profile on SilkDB (BmMDB) and EST profile on NCBI. A spatio-temporal expression profile of catalase run by apEST was then presented as a three-dimensional graph for the intuitive visualization of patterns. A total of 37 query genes confirmed from microarray data and RT—PCR experiments were selected as queries to test apEST. The results had great conformity among three approaches. Nevertheless, there were minor differences between apEST and BmMDB because of the unique items investigated. Therefore, complementary analysis was proposed. Application of apEST also led to the acquisition of corresponding protein annotations for EST datasets and eventually for their functions. The results were presented according to statistical information on protein annotation and Gene Ontology (GO) category. These all verified the reliability of apEST and the operability of this platform. The apEST can also be applied in other species by modifying some parameters and serves as a model for gene expression study for Lepidoptera

A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. The life cycle of V. dahliae includes three vegetative phases: parasitic, saprophytic and dormant. The dormant microsclerotia are the primary infectious propagules, which germinate when they are stimulated by root exudates. In this study, we report the first application of Agrobacterium tumefaciens-mediated transformation (ATMT) for construction of insertional mutants from a virulent defoliating isolate of V. dahliae (V592). Changes in morphology, especially a lack of melanized microsclerotia or pigmentation traits, were observed in mutants. Together with the established laboratory unimpaired root dip-inoculation approach, we found insertional mutants to be affected in their pathogenicities in cotton. One of the genes tagged in a pathogenicity mutant encoded a glutamic acid-rich protein (VdGARP1), which shared no significant similarity to any known annotated gene. The vdgarp1 mutant showed vigorous mycelium growth with a significant delay in melanized microsclerotial formation. The expression of VdGARP1 in the wild type V529 was organ-specific and differentially regulated by different stress agencies and conditions, in addition to being stimulated by cotton root extract in liquid culture medium. Under extreme infertile nutrient conditions, VdGARP1 was not necessary for melanized microsclerotial formation. Taken together, our data suggest that VdGARP1 plays an important role in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. Overall, our findings indicate that insertional mutagenesis by ATMT is a valuable tool for the genome-wide analysis of gene function and identification of pathogenicity genes in this important cotton pathogen

A Piezoelectric Immunosensor Using Hybrid Self-Assembled Monolayers for Detection of Schistosoma japonicum

BACKGROUND: The parasite Schistosoma japonicum causes schistosomiasis disease, which threatens human life and hampers economic and social development in some Asian countries. An important lesson learned from efforts to reduce the occurrence of schistosomiasis is that the diagnostic approach must be altered as further progress is made towards the control and ultimate elimination of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Using mixed self-assembled monolayer membrane (mixed SAM) technology, a mixture of mercaptopropionic acid (MPA) and mercaptoethanol (ME) was self-assembled on the surface of quartz crystals by gold-sulphur-bonds. Soluble egg antigens (SEA) of S. japonicum were then cross-linked to the quartz crystal using a special coupling agent. As compared with the traditional single self-assembled monolayer immobilization method, S. japonicum antigen (SjAg) immobilization using mixed self-assembled monolayers exhibits much greater immunoreactivity. Under optimal experimental conditions, the detection range is 1:1500 to 1:60 (infected rabbit serum dilution ratios). We measured several infected rabbit serum samples with varying S. japonicum antibody (SjAb) concentrations using both immunosensor and ELISA techniques and then produced a correlation analysis. The correlation coefficients reached 0.973. CONCLUSIONS/SIGNIFICANCE: We have developed a new, simple, sensitive, and reusable piezoelectric immunosensor that directly detects SjAb in the serum. This method may represent an alternative to the current diagnostic methods for S. japonicum infection in the clinical laboratory or for analysis outside the laboratory

Purification and biochemical characterization of a novel glutathione S-transferase of the silkworm, Bombyx mori

A novel glutathione S-transferase has been purified from Bombyx mori larvae using affinity chromatography on a glutathione agarose column. The purified enzyme appeared as a single band on SDS-PAGE and had a Mr of 28 kDa. Steady state kinetic assays of the enzyme were conducted with 1-chloro-2,4-dinitrobenzene as a substrate. The Km, Vmax, Kcat and Kcat/Km for the purified BmGST were 0.494 mM, 72.07 mmol/min/mg, 65.43 s-1 and 132.45 mM-1·s-1, respectively. The enzyme had a maximum activity at approximately pH 7.1 and 25ºC. BmGST indicated lower inhibitory rate by some inhibitors (albendazol, praziquantel, bile acid and NaCl), suggesting that this novel BmGST could differ structurally or functionally from other animal GSTs

Quantitative risk assessment of the Shilongmen reservoir landslide in the Three Gorges area of China

Many landslides have been triggered by the fluctuation of reservoir water level in the Three Gorges Reservoir area (TGRA) of China. The current research lacks the quantitative risk assessment framework for reservoir landslides. In this study, the risk associated with the Shilongmen reservoir landslide in TGRA is assessed by considering two hazard scenarios corresponding to the deformation and failure phases. The former is modeled via fully coupled finite element analyses, which take into account the reservoir water level and rainfall as triggering factors of landslide displacements; the latter, via the limit equilibrium method and Monte Carlo method, analyzes the failure probability under different conditions induced by the annual reservoir regulation and the return period of rainfall events. The vulnerability is quantitatively modeled as a function of both landslide intensity and the resilience of the elements at risk (EAR). In the deformation scenario, the expected monetary loss is estimated by multiplying the monetary value of the buildings for their vulnerability, and the corresponding risk map displays the potential economic losses to the EAR. In the failure scenario, direct (landslide) and indirect (tsunamis) risks are considered because the rapid decline of reservoir water level and heavy rainfall can cause the landslide rapidly sliding into the Yangtze River. Bearing in mind that there are several unstable slopes in the same geo-environmental context in the TGRA, the proposed research framework could provide a reference for government risk management strategies