Lirong Shen

Drosophila lacks C20 and C22 PUFAs

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.

Curcumin and aging.

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.

Mechanism of action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive bacteria.

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.

Expression of Acc-royalisin gene from Royal Jelly of Chinese honeybee in Escherichia coli and its antibacterial activity.

Royalisin is an antibacterial peptide found in Royal Jelly. Two gene fragments of Chinese honeybee (Apis cerana cerana) head, 280 bp cDNA encoding pre-pro-Acc-royalisin (PPAR) of 95 amino acid residues, and 165 bp cDNA encoding mature Acc-royalisin (MAR) of 51 amino acid residues were cloned into the pGEX-4T-2 vector. They were then transformed individually into Escherichia coli for expression. Two expressed fusion proteins, glutathione S-transferase (GST)-PPAR of 36 kDa and GST-MAR of 32 kDa were obtained, which were cross reacted with GST antibody accounting for up to 16.3% and 15.4% of bacterial protein, respectively. In addition, 41% of GST-PPAR and nearly 100% of GST-MAR were soluble proteins. Both lysates of the two purified fusion proteins displayed antibacterial activities, similar to that of nisin against Gram-positive bacteria strains, Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus. MAR peptide released from the thrombin-cleaved GST-MAR fusion protein has a stronger antibacterial activity than that of GST-MAR fusion protein.

Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line.

Major royal jelly protein 1 (MRJP1) is the most abundant member of the major royal jelly protein (MRJP) family of honeybee. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in molecular weight to the glycosylated AmMRJP1 from the Western honeybee (Apis mellifera). Western blots probed with anti-AccMRJP1 antibody demonstrated that recombinant AccMRJP1 and soluble protein of the Western honeybee RJ (AmSPRJ) contained immunoreactive MRJP1. The 57 kDa protein in AmSPRJ contained an N-terminal amino sequence of N-I-L-R-G-E, which is identical to that previously characterized in AmMRJP1. The molecular weight of recombinant AccMRJP1 was decreased from 57 to 48 kDa after deglycosylation, indicating that AccMRJP1 was glycosylated. The recombinant AccMRJP1 significantly stimulated Tn-5B-4 cell growth, similar to AmSPRJ and fetal bovine serum, and affected cell shape and adhesion to the substrate.

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody.

Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.摘要目的为蜂王浆主蛋白1 (MRJP1)的快速检测和鉴别提供科学依据, 为蜂王浆的质量控制提供技术支持。创新点首次比较了MRJP1特异性多克隆抗体与MRJP1重组表达蛋白多克隆抗体对王浆主蛋白家族的免疫反应差异, 验证了蜂王浆中MRJP1蛋白降解与保温时间的相关性, 建立了以MRJP1作为蜂王浆新鲜度生物标志物的快速检测方法。方法通过蜂王浆主蛋白家族蛋白的氨基酸序列同源性分析, 筛选出MRJP1的特异性多肽区域, 进行人工合成, 免疫兔子后取血清制备成特异性多克隆抗体。 用蛋白质印迹法(Western blot)检测了MRJP1特异性多克隆抗体与MRJP1重组表达蛋白多克隆抗体对王浆主蛋白家族的免疫反应。 以新鲜蜂王浆为对照品, 用MRJP1特异性抗体酶联接免疫吸附剂测定(ELISA)法和变性电泳胶灰度扫描法分别测定保温(40 °C) 7∼49天的蜂王浆中MRJP1含量的变化, 并进行了相关性分析。结论MRJP1的特异性抗体对MRJP1蛋白具有专一的免疫识别特性, 可特异性地检测代表蜂王浆新鲜度的MRJP1含量变化, 并鉴别蜂王浆的真伪。

Effect of Major Royal Jelly Proteins on Spatial Memory in Aged Rats: Metabolomics Analysis in Urine

Royal jelly (RJ) produced by worker honeybees is the sole food for the queen bee throughout her life as well as the larvae of worker bees for the first 3 days after hatching. Supplementation of RJ in the diet has been shown to increase spatial memory in rodents. However, the key constituents in RJ responsible for improvement of cognitive function are unknown. Our objective was to determine if the major royal jelly proteins (MRJPs) extracted from RJ can improve the spatial memory of aged rats. The spatial memory assay using the Morris water maze test was administered once to rats after a 14-week feeding. Metabolomics analysis based on quadrupole time-of-flight mass spectrometry was conducted to examine the differences in compounds from urine. Aged male rats fed MRJPs showed improved spatial memory up to 48.5% when compared to the control male aged rats fed distilled water. The metabolite pattern of the MRJPs-fed aged rats was regressed to that of the young rats. Compounds altered by MRJPs were mapped to nicotinate and nicotinamide metabolism, cysteine taurine metabolism, and energy metabolism pathways. In summary, MRJPs may improve spatial memory and possess the potential for prevention of cognitive impairment via the cysteine and taurine metabolism and energy metabolism pathways in aged rats.

Drosophila Fed ARA and EPA Yields Eicosanoids, 15S-Hydroxy-5Z,8Z, 11Z, 13E-Eicosatetraenoic Acid, and 15S-Hydroxy-5Z,8Z,11Z,13E,17Z-Eicosapentaenoic Acid

Drosophila melanogaster has been a widely used as a model system for its powerful genetic tools. However, it remains to be illustrated if Drosophila can be used to examine the biochemical and physiological metabolism of eicosanoids. Thus, the analysis on the metabolism of C20 polyunsaturated fatty acids (PUFA) in Drosophila was implemented with high performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Fatty acid (FA) analysis of the whole body, head, and thorax-abdomen in Drosophila showed C20 PUFA could only be found in Drosophila fed diets supplemented with eicosapentaenoic acid (EPA) and arachidonic acid (ARA), but not in Drosophila fed base diets. The C20 PUFA were found in abundance in the head. Drosophila fed ARA- and EPA-supplemented diets yielded 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid [15(S)-HETE] and 15S-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid [15(S)-HEPE], respectively, while other sampled eicosanoids could not be detected. Similar results were obtained by incubating fly tissue supplemented with ARA or EPA. Furthermore, a genome sequence scan indicated that no gene encoding the key enzymes synthesizing eicosanoids were found in Drosophila. These findings demonstrate that Drosophila may possess a special lipid metabolic system, which is different from mammals.

Supplementation with Major Royal-Jelly Proteins Increases Lifespan, Feeding, and Fecundity in Drosophila.

The major royal-jelly proteins (MRJPs) are the main constituents responsible for the specific physiological role of royal jelly (RJ) in honeybees. Male and female Drosophila flies were fed diets containing either no MRJPs (A) or casein (B) at 1.25% (w/w) of diet or MRJPs at 1.25% (C), 2.50% (D), or 5.00% (E). Diets B, C, D, and E increased mean lifespan by 4.3%, 9.0%, 12.4%, and 13.9% in males and by 5.8%, 9.7%, 20.0%, and 11.8% in females in comparison to results from diet A, respectively. The diet supplemented with 2.50% MRJPs seems to have the optimal dose to improve both physiological and biochemical measures related to aging in both sexes. Interestingly, lifespan extension by MRJPs in Drosophila was positively associated with feeding and fecundity and up-regulation of copper and zinc-superoxide dismutase (CuZn-SOD) and the Egfr-mediated signaling pathway. This study provides strong evidence that MRJPs are important components of RJ for prolonging lifespan in Drosophila.

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines

Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%‒56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.中文概要目 的为MRJPs 应用于人体细胞培养提供科学依据, 为发展蜂王浆在细胞工程的新用途提供技术支撑。创新点首次验证MRJPs 对多种人体细胞的促分裂效果, 证实MRJPs 可部分替代FBS 培养人体细胞, 并取得培养人体细胞的MRJPs 与FBS 和细胞因子的适合比例。方 法鲜蜂王浆中分离得到的MRJPs 溶液与FBS 按不同比例(0/10、3/7、6/4 和9/1)复配成复合血清, 分别添加于无血清培养基中用于培养293T、HFL-I、231、HCT116 和Changliver 等5 种人体细胞。以添加体积分数为10%的FBS 的无血清培养基为对照, 根据MTT 法测定的吸光度和细胞形态比较分析结果, 得到促进细胞分裂的MRJPs溶液与FBS 最佳配合比例。然后在筛选出的最佳复合血清中添加不同的细胞因子, 再通过同样的比较分析, 得到适合的细胞因子组合。结 论MRJPs 对多种人体细胞具有促进分裂作用, 可部分替代FBS 培养人体细胞。MRJPs 与FBS 的最佳配比为: 60% MRJPs 溶液和40% FBS(复合血清); 该复合血清与细胞因子的最佳组合为: 复合血清+表皮生长因子+胰岛素-转铁蛋白-硒。