两蜂种蜂王浆蛋白质组分析

采用蛋白质组双向电泳技术和已鉴定蜂王浆蛋白质组的比对,对王浆高产蜜蜂（浆蜂）和中华蜜蜂（中蜂）的蜂王浆蛋白质组进行了差异比较。结果表明,浆蜂蜂王浆的总蛋白质点数（93个）显著高于中蜂蜂王浆总蛋白质点数（70个）,它们的等电点主要集中在5-8之间,分子量在50-80kDa之间,其中共有蛋白质点为30个;通过与已鉴定的浆蜂蜂王浆蛋白质组比较,这些共有蛋白质点推断为蜂王浆主蛋白1、2、3和葡萄糖氧化酶。同时两蜂种蜂王浆的蛋白质丰度也存在较大差异,共有点中有4个蛋白质的丰度中蜂显著高于浆蜂,7个浆蜂显著高于中蜂中。结果表明浆蜂和中蜂蜂王浆蛋白质种类及丰度均有较大差异。     

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.     

Organization and potential function of the mrjp3 locus in four honeybee species

Royal jelly is a nutritious secretion produced by nurse honeybees to provision queens and growing larvae. Major proteins of royal jelly are mutually similar, and they all belong to the MRJP/yellow protein family (pfam03022). The mrjp3 loci in four traditional honeybee species (Apis mellifera, Apis cerana,Apis dorsata, and Apis florea) were sequenced and found to share high sequence and structural similarities. PCR analyses confirmed the presence of an extensive repetitive region, which showed size and sequence polymorphisms in all species. The evolutionary history of mrjp genes and their repetitive regions was reconstructed from their nucleotide sequences. The analyses proved that the repeat region appeared early in the evolution of the mrjp gene family and that the extreme elongation of the repeat is mrjp3 specific. In the MRJPs was documented a correlation between nitrogen content and repeat length. Therefore, it is argued that the repeat occurred due to a selection for an increase in nitrogen storage for a more efficient nutrition of queens and larvae.     

Expression of apalbumin1 of Apis cerana cerana in the larvae of silkworm, Bombyx mori

Royal jelly (RJ) is a thick, milky material produced by both the hypopharyngeal and the mandibular glands of nurse honeybees. The main proteins of RJ, named apalbumins or major royal jelly proteins (MRJPs), have multiple biological functions. Apalbumin1 is the most abundant glycoprotein of RJ. In this study, Bacmid- apalbumin1 was constructed for Apis cerana cerana using the newly established Bac-to-Bac/BmNPV baculovirus expression system (BES). This procedure allowed us to obtain the recombinant A. cerana cerana ( Acc) apalbumin1 (r Accapalbumin1) from the hemolymph of silkworm larvae through the BmNPV bacmid system, 96 h postinfection. The r Accapalbumin1 was then purified by Ni-NTA spin columns and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A 55 kDa protein with good solubility was then obtained. The peptide Ile-Phe was identified from trypsin production of r Accapalbumin1. Such a peptide has been reported to have an antihypertensive ability. Our results have therefore potential applications in biomedical research and open new perspectives for the study of apalbumins.     

不同采收时期蜂王浆抗氧化活性研究

为研究不同采收时间对蜂王浆品质的影响,以4-9月份采收的蜂王浆为研究对象,通过比较不同采收时间蜂王浆中蜂王浆主蛋白(MRJPs)、总水溶性蛋白和多酚的含量,分析了MRJPs与自由基清除能力和总抗氧化能力的相关性。结果表明,不同采收期蜂王浆中MRJPs含量存在显著差异(P<0.05),且6月份采收的MRJPs含量最低。水溶性蛋白及总酚含量差异不大(P>0.05)。蜂王浆自由基清除能力与MRJP1和 MRJP3含量的相关系数达0.828和0.847;总抗氧化能力与MRJP1和 MRJP3含量的相关系数分别为0.680和0.743。蜂王浆的抗氧化活性与其MRJPs存在一定程度的正相关,但与多酚含量相关性不明显,说明其自由基清除能力与总抗氧化活性可能是多种抗氧化性活性物质共同作用的结果。本研究为蜂王浆的抗氧化活性研究提供了参考。     

Novel royal jelly proteins identified by gel-based and gel-free proteomics

Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.     

Proteomic analysis of royal jelly from three strains of western honeybees (Apis mellifera)

To compare the protein complement of royal jelly (RJ) from high RJ producing honeybees ( Apis mellifera L.), a strain of A. mellifera artificially selected for increased RJ production from Italian honeybees in China for more than two decades was compared to those of native Italian honeybees ( A. mellifera L.) and Carnica honeybees ( A. mellifera C.); the protein in RJ from these three strains of honeybees was partially identified by using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), and a protein engine identification tool applied to the honeybee genome. The results showed that 152, 157, and 137 proteins were detected in the three species of RJ; among which 57, 57, 51 high abundant proteins ere identified, respectively. Most identifited spots, 45, 45, 41, were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. Also, 3-glucose oxidase, 1-peroxiredoxin (PRDX), and 1-glutathione S-transferase (GST) S1 were identified in three RJ samples. Furthermore, during the determination of the peptides mass fingerprinting (PMF) of each spot, for the first time, PRDX and GST S1 proteins have been identified in RJ. Thus, the results suggest that the protein complement of high RJ producing honeybees is not different compared to native Italian honeybees, while a difference remains between Carnica honeybees.     

蜂王浆三针夹取式拣虫机的设计与试验

蜂王浆拣虫是挖浆前必须完成的工序。由于蜜蜂幼虫个体较小、数目庞大,因此人工拣虫过程劳动强度非常大;然而中国养蜂人员老龄化日益严重,且目前蜂王浆拣虫机械化尚未成熟。该文针对中国养蜂现状提出一种以三针夹取的方式将王台中蜜蜂幼虫夹出的方法。以三针夹取的拣虫方式构建蜂王浆拣虫机整机方案。三根针在王台孔内壁同时做向心运动或分离运动,将蜜蜂幼虫夹住或松开。对传动机构中球面槽轮和凸轮的参数进行计算。通过有限元分析得出动力输入端脚踏板受力情况。计算得出球面槽轮圆柱销弧长7.5π,槽深20.68 mm;得出凸轮的理论廓线;有限元分析结果得出动力输入方式可靠。该机器在保留蜂王浆幼虫完整的前提下,一次性将整条王台(64孔)中蜜蜂幼虫全部拣出,可实现连续作业,拣虫效率为10条/min时为人工的10倍,克服了蜂场手工作业时间长和劳动强度大问题。对扩大中国蜂业养蜂规模有着极大的促进作用。     

Storage stability assessment of freeze-dried royal jelly by furosine determination

The effect of freeze-drying and the assessment of the storage stability of freeze-dried royal jelly (RJ) were investigated by the determination of furosine and blocked lysine. The level of furosine in the RJ samples collected from cells at different times (1, 2, and 3 days after grafting) showed that the Maillard reaction had already occurred in the hive as indicated by the increase in furosine: from 9.6 to 20.8 mg/100 g of protein. Freeze-dried RJ was more prone to the early stage of the Maillard reaction than fresh RJ, as confirmed by the significantly higher furosine values found after 12 months, both at 4 degrees C (253.4 versus 54.9 mg/100 g of protein) and at room temperature (884.3 versus 332.5 mg/100 g of protein). After 18 months at room temperature, the lyophilized samples reached a furosine level of 1440.4 mg/100 g of protein, which corresponded to the blocked lysine levels, amounting to 24% of total lysine.     

Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography

To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.     

Immunochemical approach to detection of adulteration in honey: physiologically active royal jelly protein stimulating TNF-alpha release is a regular component of honey

The presence of royal jelly (RJ) proteins in honey collected from nectars of different plants, origin, and regions and in honeybee's pollen was detected by Western-blot analysis using polyclonal antibodies raised against water-soluble RJ-proteins. The most abundant RJ-protein in honeybee products corresponded to a 55 kDa protein. The N-terminal amino acid sequence of 55 kDa protein was N-I-L-R-G-E. This sequence is identical to the apalbumin-1, the most abundant protein of RJ. Apalbumin-1 is a regular component of honeybee products and thus is a suitable marker tool for proving adulteration of honey by means of immunochemical detection. Its presence in all tested samples of honeys and honeybee pollen was confirmed also by Western-blot analysis using polyclonal antibodies raised against recombinant apalbumin-1. It has been found that major RJ-proteins, apalbumin-1, and apalbumin-2, stimulate mouse macrophages to release TNF-alpha, which demonstrates that physiologically active proteins of honey could be used for its biological valuation.     

Specific hydroxy fatty acids in royal jelly activate TRPA1

This is the first report of TRPA1 activation by fatty acids. Activation of TRPA1 and TRPV1 induces thermogenesis and energy expenditure enhancement. In this study, we searched for novel agonists of TRPA1 and TRPV1 from a nonpungent food, royal jelly (RJ). We measured the activation of human TRPA1 and TRPV1 by RJ extracts and found that the hexane extract contains TRPA1 agonists. The main functional compounds in the hexane extract were trans-10-hydroxy-2-decenoic acid (HDEA) and 10-hydroxydecanoic acid (HDAA). These are characteristic fatty acids of RJ. Their EC50 values were about 1,000 times larger than that of AITC, and their maximal responses were equal. They activated TRPA1 more strongly than TRPV1. Their EC50 values for TRPV1 were 2 times larger, and the maximal response was less than half of that for TRPA1. Next, we studied the potencies of other lipid components for both receptors. Most of them have higher affinity to TRPA1 than TRPV1. Among them, dicarboxylic acids showed equal efficacy for both receptors, but those are present in only small amounts in RJ. We concluded that the main function of RJ is TRPA1 activation by HDEA and HDAA, the major components of the RJ lipid fraction.     

Application of solid/liquid extraction for the gravimetric determination of lipids in royal jelly

Gravimetric lipid determination is a major parameter for the characterization and the authentication of royal jelly quality. A solid/liquid extraction was compared to the reference method, which is based on liquid/liquid extraction. The amount of royal jelly and the time of the extraction were optimized in comparison to the reference method. Boiling/rinsing ratio and spread of royal jelly onto the extraction thimble were identified as critical parameters, resulting in good accuracy and precision for the alternative method. Comparison of reproducibility and repeatability of both methods associated with gas chromatographic analysis of the composition of the extracted lipids showed no differences between the two methods. As the intra-laboratory validation tests were comparable to the reference method, while offering rapidity and a decrease in amount of solvent used, it was concluded that the proposed method should be used with no modification of quality criteria and norms established for royal jelly characterization.     

二氧化硫胁迫诱导拟南芥NIT2基因DNA甲基化修饰

DNA甲基化是一种重要的表观遗传修饰形式。利用亚硫酸氢盐修饰后测序法和甲基化敏感性限制性内切酶-PCR（MSRE-PCR）法,研究SO2胁迫对拟南芥腈水解酶（NIT2）基因序列中胞嘧啶甲基化状态的影响,分析甲基化特征改变在植物胁迫应答过程中的作用。研究发现,30 mg.m-3的SO2连续熏气3 d后,拟南芥植株地上组织细胞中NIT2基因启动子区域CG和CHH（H为C,A或T）位点甲基化水平下降,总甲基化水平降低,但未检出编码区5′端目的片段中CCGG位点甲基化状态的改变。RT-PCR分析表明,SO2胁迫组拟南芥植株地上组织细胞中NIT2基因的转录水平高于对照组。研究结果表明,SO2胁迫导致拟南芥NIT2基因启动子区甲基化水平降低,NIT2基因转录上调,说明SO2胁迫能诱发拟南芥基因胞嘧啶甲基化水平改变,启动子区甲基化水平的降低可能与防御基因的诱导表达有关,胞嘧啶甲基化修饰参与了植物的抗逆生理过程。     

Lead in women's and children's vitamins

A survey was conducted to determine the extent of lead (Pb) contamination in vitamins labeled for use by women and children. The Pb content of 324 multivitamin-mineral products was determined using microwave assisted nitric acid digestion and inductively coupled plasma mass spectrometry. Cryogenic grinding was used to composite soft samples such as oil filled capsules and candy-like products such as gummies and jelly beans. Estimates of Pb exposures from consumption of these products were derived for four population groups: young children (0-6 yrs), older children (7+ yrs), pregnant or lactating women, and adult women. The estimated median and maximum Pb exposures were 0.123 and 2.88 microg/day for young children, 0.356 and 1.78 microg/day for older children, 0.845 and 8.97 microg/day for pregnant and lactating women, and 0.842 and 4.92 microg/day for adult women. The overall median value for Pb exposure was 0.576 microg/day. Five samples would have provided exposures that exceeded 4 microg/day. Estimates of exposures were assessed with respect to safe/tolerable exposure levels that have been developed for the specific age and sex groups. These safe/tolerable levels are referred to as the provisional total tolerable intake levels (PTTI) and are 6, 15, 25, and 75 microg Pb/day for young children, older children, pregnant or lactating women, and adult women, respectively. Estimates of Pb exposures were below the PTTI levels for the four population groups. Median and maximum values were used instead of the mean and standard deviation because of the skewed distribution of results toward lower mass fraction and exposure.     

A Review and Bibliography of the Literature of Honey Bee Colony Collapse Disorder: A Poorly Understood Epidemic that Clearly Threatens the Successful Pollination of Billions of Dollars of Crops in America

ABSTRACT

This article briefs agricultural librarians on three key aspects of Colony Collapse Disorder (CCD), a disease involving the virtually total disappearance and presumed annihilation of the adult bee population of hives: (1) the yearly multibillion dollar importance of honeybees as the nation's key crop pollinators and the annual $200 million domestic production and export of honey; (2) the more common bee diseases and well-known hive stressors that current theories of CCD suggest interact with each other in some as yet undetermined combination, perhaps including new pathogens to which most American bees have limited or no immunity; and (3) several bees other than the honeybee that are not currently affected by CCD and that have some potential as partial substitute pollinators for some crops.     

新疆巴州幼年与成年牦牛瘤胃细菌区系多样性分析

为比较年龄对牦牛瘤胃细菌区系多样性的影响,以新疆巴州1岁幼年牦牛和4岁成年牦牛的瘤胃微生物总DNA为研究材料,利用16S rRNA基因序列分析技术对牦牛瘤胃细菌区系的操作归类单元(OTU)聚类、门和属水平上的物种丰度、Alpha多样性进行比较分析。结果表明,在门水平上,厚壁菌门(Firmicutes)相对丰度为66.96%~70.31%,拟杆菌门(Bacteroides)相对丰度为21.87%~24.11%,其他菌类所占比例极少。在属水平上,普雷沃菌属(Prevotella)在幼年牦牛瘤胃内相对丰度(4.69%)高于成年牦牛(2.68%);瘤胃球菌属(Ruminococcus)在幼年牦牛瘤胃内相对丰度(4.69%)比在成年牦牛瘤胃内(1.79%)高。幼年和成年牦牛的Chao1指数分别为783.50和544.00,Shannon指数分别为4.68和4.52。综上可知,在同一草地放牧饲养条件下,1岁和4岁巴州牦牛瘤胃细菌区系组成存在明显差异,幼年牦牛瘤胃消化吸收的功能正在转化建立的过程中,所以Chao1指数和Shannon指数高于成年牦牛,且瘤胃细菌物种丰度和多样性更高。本研究结果为该地区牦牛瘤胃微生物稳态及其营养调控研究提供了参考依据。     

小峰熊蜂工蜂蛹期发育蛋白质组分析

本文采用双向凝胶电泳法对小峰熊蜂工蜂蛹期的3个发育期进行蛋白质组研究,结果表明小峰熊蜂工蜂蛹期的白眼期（A期）、褐眼期（B期）和黑眼期（C期）分别检测到77、81和59个蛋白点,特有蛋白分别为7个、6个和1个,共有蛋白为48个,A期到B期显著上调的蛋白有9个,显著下调的蛋白有3个,B期到C期显著上调的蛋白有8个,显著下调的蛋白有3个,A期到C期显著上调的蛋白有15个,显著下调的蛋白有2个。研究还发现有17个蛋白是在A期和B期表达而在C期关闭;有2个蛋白是在A期表达,B期关闭,在C期又表达。本研究初步揭示了小峰熊蜂工蜂从蛹期发育到成蜂过程中,不仅需要一些保守蛋白来调控,而目．还需要一此特异蛋白。     

液压机械无级传动全功率换段过程排量比调节模型

为了解决液压机械换段过程中存在的转速波动和瞬时动力中断等问题,该文以两离合器结合重叠的五阶段全功率动力换段方法为基础,分析了液压机械全功率换段过程变排量液压元件排量比调节规律。以某等差两段式液压机械为研究对象,建立了液压机械全功率换段过程变排量液压元件排量比调节模型,通过仿真分析和全功率换段过程试验,获得了换段过程液压回路压力从当前段到目标段随排量比变化的动态响应过程。结果表明,排量比变化量的仿真与试验结果基本一致,最大偏差为8.93%,验证了模型的正确性;排量比调节模型能够根据当前段状态参量和目标段压力预测出目标段排量值;阶跃排量比调节规律能有效缩短液压回路建压时间,建压时间为0.93 s,压力波动量较小,为0.64 MPa;按阶跃调节排量比至目标值,能在换段过程完成液压回路高低压侧压力平稳互换,换段前后输出转速几乎无波动、转矩连续传递。经增速机后的输出转矩为100和150N·m时,换段时间分别为1.00和1.10s,该转矩的最大波动量分别为6.80和6.84N·m,换段过程中功率连续且平稳传递。研究结果可为实现液压机械无级传动全功率换段控制及后续研究提供参考。     

Protein/energy ratios of current diets in developed and developing countries compared with a safe protein/energy ratio: implications for recommended protein and amino acid intakes

Revised estimates of protein and amino acid requirements are under discussion by the Food and Agriculture Organization (FAO)/World Health Organizaion (WHO), and have been proposed in a recent report on Dietary Reference Intakes (DRIs) from the USA. The nature and magnitude of these requirements are not entirely resolved, and no consideration has been given to the potential influence of metabolic adaptation on dietary requirements. We have examined the implications of these new values, and of the conceptual metabolic framework in which they are used, for defining the nutritional adequacy of protein intakes in developed and developing countries. We have expressed proposed values for protein requirements in relation to energy requirements, predicted for physical activity levels of 1.5, 1.75 and 2.0 times basal metabolic rate, in order to generate reference ratios for protein energy/total energy (reference P/E ratio) as a function of age, body weight, gender and physical activity level. Proposed values for amino acid requirements have been used to adjust the available digestible P/E ratio of foods and diets for protein quality. Focusing on the diets of UK omnivores and vegetarians and on diets in India, the risk of protein deficiency is evaluated from a comparison of P/E ratios of metabolic requirements with protein-quality-adjusted P/E ratios of intakes. A qualitative and conservative estimate of risk of deficiency is made by comparing the adjusted P/E ratio of the intake with a reference P/E ratio calculated for age, body weight, gender and physical activity according to FAO/WHO/United Nations University. A semi-quantitative estimate of risk of deficiency has also been made by the cut point approach, calculated as the proportion of the intake distribution below the mean P/E ratio of the requirement. Values for the quality-adjusted P/E ratio of the diet range from 0.126 for the UK omnivore diet to 0.054 for a rice-based diet of adults in West Bengal, which is lysine-limited, falling to 0.050 for 1-year-old children. The reference P/E ratio for men and women increases with age, is higher for females than males, is higher for small compared with large adults at any age and decreases with physical activity. Thus if a particular diet is potentially limiting in protein, protein deficiency is most likely in large, elderly sedentary women followed by the adolescent female and least likely in moderately active young children, the opposite of what has usually been assumed. Within the currently accepted framework, the diets do not meet the protein needs of the entire population of the UK, have a significant risk of deficiency throughout India for all except extremely active small adults, and are grossly inadequate for all population groups, apart from physically active young children in West Bengal, regardless of body weight or level of food intake. The lysine limitation of the cereal-based Indian diets is dependent on the choice of lysine requirement values from the published range. We consider that the value selected is too high, because of uncertainties and inconsistencies in the approaches used. A more appropriate choice from the lower end of the range would remove the lysine limitation of cereal-based diets, and reduce some of the perceived risk of deficiency. However, diets remain limited by the amount of digestible protein for many population groups, especially in West Bengal. In the context of risk management, one option would be to accept the current values and the conceptual metabolic framework within which they have been derived. This would have major implications for the supplies of high-quality protein to the developing countries. An alternative option would be to re-evaluate the currently proposed values for the requirements for protein and amino acids. We conclude that the choice of values for the adult lysine requirement should be re-evaluated and that serious consideration should be given to the extent to which adaptive mechanisms might enable the metabolic requirement for protein to be met from current intakes. This will entail a better understanding of the relationships between dietary protein and health.