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The venomous phospholipase A2 (AcPLA2) coding reading region of the Chinese honeybee (Apis cerana cerana), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15kD. Detection of western blot using ant-European honeybee (Apis mellifera) phospholipase A2 (AmPLA2) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA2.The result demonstrated that the AcPLA2 peptide had been expressed in E. coli and the AcPLA2 has the similar antigenicity as the AmPLA2.

Royalisin from royal jelly (RJ) is a valuable peptide both for the prevention of honeybee diseases and for RJ preservation. ELISA for fast determination of royalisin content in hemolymphe (RCH) of honeybees with polyclonal antibody against recombinant royalisin from Asian honeybee was established. Assay on RCHs of health samples from Asian honeybee and Western honeybee showed the former (7.06 μg/mL) was significantly higher than that of the latter (5.64 μg/mL, p < 0.01). Moreover, relative to the non infection, the RCHs of Asian honeybees at 24 and 48 h post infection of Eschericha coli were higher than those of Western honeybees by 32.90% and 29.66%, respectively. Evidence revealed that Asian honeybee possesses higher innate immunity and immune response against bacteria in relation to the Western honeybee. The method will be a potential tool for detection of resistant levels to pathogens in honeybees and for quantification of royalisin in RJ products.

Turmeric has been used commonly as a spice, food additive, and an herbal medicine worldwide. Known as a bioactive polyphenolic extract of Turmeric, curcumin has a broad range of health benefit properties for humans. Recently, active research on curcumin with respect to aging and related traits in model organisms has demonstrated that curcumin and its metabolite, tetrahydrocurcumin (THC), increase mean lifespan of at least three model organisms: nematode roundworm, fruit fly Drosophila, and mouse. Nematodes grown on media containing curcumin showed a significantly increased lifespan by reducing the production of reactive oxygen species. Genes osr-1, sek-1, mek-1, skn-1, unc-43, sir-2.1, and age-1 are required for curcumin-mediated lifespan extension. The lifespan extension of Drosophila by curcumin supplementation was associated with increased superoxide dismutase (SOD) activity, and decreased lipofuscin and malondialdehyde levels. Curcumin up-regulated expression of SOD genes and down-regulated expression of several age-related genes, such as dInR, ATTD, Def, CecB, and DptB. In addition, THC extended lifespan in Drosophila and inhibited the oxidative stress response by regulating FOXO and Sir2. Mice fed diets containing THC starting at the age of 13 months had significantly increased mean lifespan. In summary, the positive effects of curcumin on lifespan extension likely arise from beneficial regulation of common oxidative stress responses and age-related genes. Understanding the molecular mechanism(s) of curcumin action has provided base knowledge and rationale for future human clinical trials, and for nutritional intervention in aging and age-associated disorders in humans.

The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST) in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC) of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent.

Drosophila melanogaster has been considered a model organism for investigating human diseases and genetic pathways. Whether Drosophila is an ideal model for nutrigenomics, especially for FA metabolism, however, remains to be illustrated. The aim of this study was to examine the metabolism of C20 and C22 PUFAs in Drosophila. Analysis of FA composition revealed a complete lack of C20 and C22 PUFAs in the body tissue of larvae, pupae, and adult flies fed either a base or supplemented diet abundant in the PUFA precursors linoleic acid and α-linolenic acid. PUFA with >C20 could only be found in flies supplemented with specific FAs. Interestingly, the supplemented C22 PUFAs docosahexaenoic acid (22:6n-3) and docosatetraenoic acid (22:4n-6) were largely converted to the shorter chain C20 PUFAs eicosapentaenoic acid (20:5n-3) and arachidonic acid (20:4n-6), respectively. Furthermore, a genome sequence scan indicated that no gene encoding Δ-6/ Δ-5 desaturases, the key enzymes for the synthesis of C20/C22 PUFA, was present in Drosophila. These findings demonstrate that Drosophila lacks the capability to synthesize the biologically important C20 and C22 PUFAs, and thereby argue that Drosophila is not a valid model for the study of lipid metabolism and related diseases.

Bee venom phospholipase A(2) (BvPLA(2)) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA(2) (AccPLA(2)) gene from the Chinese honeybee (Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa. Products of hexahistidine AccPLA(2) fusion protein accumulated up to 5.32% of the total cellular proteins. The AccPLA(2) fusion protein was cross reactive with the anti-AmPLA(2) (BvPLA(2) of the European honeybee, Apis mellifera) polyclonal serum. The reaction resulted in a double glycosylation band, which agrees with the band generated by the native AmPLA(2) in Western blot analysis. The PLA(2) activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 micromol/(min.mg). In summary, the recombinant AccPLA(2) protein, a native BvPLA(2)-like structure with corresponding biological activities, can be glycosylated in Tn cells. These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA(2) in the pharmaceutical industry.

The anti-yeast activities of a food-grade dilution-stable microemulsion against Candida albicans and Saccharomyces cerevisiae have been studied. The weight ratio of the formulated microemulsion is glycerol monolaurate (GML)/propionic acid/Tween 80/sodium benzoate (SB)/water = 3:9:14:14:24. Results of anti-yeast activity on solid medium by agar diffusion method showed that the anti-yeast activity of the microemulsion at 4.8 mg/ml was comparable to that of natamycin at 0.1 mg/ml as positive control. Results of anti-yeast activity in liquid medium by broth dilution method showed that the growth of both C. albicans and S. cerevisiae was completely inhibited when the liquid medium containing 10(6) cfu/ml was treated with 1.2 mg/ml microemulsion, which was determined as minimum fungicidal concentration. The kinetics of killing results showed that the microemulsion killed over 90% yeast cells rapidly within 15 min and caused a complete loss of viability in 120 min. Among the components, SB and GML had a similar anti-yeast activity, followed by propionic acid, while Tween 80 exhibited no activity and could not enhance the anti-yeast activities of these components, and it was revealed that the anti-yeast activity of the microemulsion was attributed to a combination of propionic acid, GML, and SB. The anti-yeast activity of the microemulsion was in good agreement with the leakage of 260-nm absorbing materials and the observation of transmission electron microscopy, indicating that the microemulsion induced the disruption and dysfunction of the cell membrane.

Apisimin is one of the functional peptides from royal jelly. The aim of this study was to analyze and in vitro express a new gene encoding Acc-apisimin-2 from Chinese honeybee (Apis cerana cerana) in Escherichia coli. Ninety-six clones containing apisimin expressed sequence tag (EST) were identified from 8568 effective ESTs of the cDNA library of Chinese honeybee worker heads. The coding region of the matured peptide from one clone containing Acc-apisimin-2 gene was sub-cloned into the prokaryotic expression vector pGEX-4T-2. The recombinant vector then was transformed into E. coli BL21 (DE3) for expression. The expression product was analyzed with SDS-PAGE and Western blot. The total length of the Acc-apisimin-2 cDNA was 379 bp, containing an open-reading frame (ORF) of 237 nucleotides encoding a 78 amino acid residue precursor. The Acc-apisimin-2 gene shared 100% homologies with Am-apisimin from A. mellifera, but 93% and 91% homologies with Aciapisimin from A. cerana indica and the previously reported Acc-apisimin-1 sequence (AY278991) on a nucleotide level, respectively. The GST-Acc-apisimin-2 fusion protein expressed in the recombinant vector was about 31 kDa in size and accumulated up to about 22.1% of the total bacterial proteins. About 50% of the recombinant protein was soluble. The fusion protein purified through affinity chromatography was cross reactive with GST antibody, which confirmed the successful expression of GST-Acc-apisimin-2.

Classical physiological and biochemical methods, Biolog, and 16S rDNA analysis were used to identify the strain X3 which could control cucumber seedling damping-off. Physiological and biochemical identification showed that it belonged to Pseudomanas aeruginosa, but Biolog identification showed it was P. spinosa. 16S rDNA determination and analysis was used for further identification, which showed that the 16S rDNA sequence of X3 shared 93.7% homologies with published sequence of P. aeruginosa from GenBank, and both sequences constituted a branch in Phylogenetic tree. Based on these results, it is considered that the strain X3 belongs to one strain of P. aeruginosa.