ABSTRACT
Until now, the properties of honey have been defined based exclusively on the content of plant components in the nectar of given plant. We showed that apalbumin1, the major royal jelly (RJ) protein, is an authentic and regular component of honey. Apalbumin1 and other RJ proteins and peptides are responsible for the immunostimulatory properties and antibiotic activity of honey. For the quantification of apalbumin1, an enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal anti-apalbumin1 antibody. The method is suitable for honey authenticity determination; moreover it is useful for detection of the honey, honeybee pollen and RJ in products of medicine, pharmacy, cosmetics, and food industry, where presences of these honeybee products are declared. Results from the analysis for presence and amount of apalbumin1 in honeys will be used for high-throughput screening of honey samples over the world. On the basis of our experiments which show that royal jelly proteins are regular and physiologically active components of honey we propose to change the definition of honey (according to the EU Honey Directive 2001/110/EC) as follows: Honey is a natural sweet substance produced by honey bees from nectar of plants or from secretions of plants, or excretions of plant sucking insects, which honey bees collect, transform by combining with major royal jelly proteins and other specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature.
Subject(s)
Fatty Acids/analysis , Fatty Acids/chemistry , Glycoproteins/analysis , Glycoproteins/chemistry , Honey/analysis , Insect Proteins/analysis , Insect Proteins/chemistry , Animals , Anti-Bacterial Agents/analysis , Anti-Bacterial Agents/chemistry , Bees/chemistry , Biomarkers/analysis , Biomarkers/chemistry , Enzyme-Linked Immunosorbent Assay , Food Quality , Plants/anatomy & histology , Plants/chemistryABSTRACT
Neprilysin (NEP) is one of the candidate amyloid ß protein (Aß) degrading enzymes affecting brain Aß clearance. This enzyme declines in the brain with age, which leads to the increased Aß deposition in Alzheimer's disease (AD). Pharmacological activation of NEP during the aging process, therefore, represents a potential strategy to prevent the development of AD. To examine the influence of nobiletin on neprilysin activity, we measured cellular NEP activity in SK-N-SH cells. Moreover, NEP expression was examined by using reverse transcription - polymerase chain reaction and Western blotting. Measurement of cellular NEP activity showed that nobiletin stimulated this in a dose- and time-dependent manner in SK-N-SH cells. Moreover, nobiletin increased the expression of NEP mRNA, and then the levels of NEP protein, also in a dose- and time-dependent manner. Our findings showed that nobiletin promoted NEP gene and protein expression, resulting in enhancement of cellular NEP activity in SK-N-SH cells. This compound could be a novel Aß-degrading compound for use in the development of disease-modifying drugs to prevent and (or) cure AD.
Subject(s)
Antioxidants/pharmacology , Citrus , Flavones/pharmacology , Neprilysin/metabolism , Cell Line , Gene Expression/drug effects , Humans , Neprilysin/geneticsABSTRACT
Rabbit polyclonal antibody produced by a major royal jelly protein 1 (MRJP1) specific peptide reacted only with a MRJP1. Indirect ELISA with the antibody revealed a MRJP1 level of 4.12-4.67 g/100 g in different company's royal jelly, which almost agreed with that of a hexametric form of MRJP1 (apisin) measured by high performance liquid chromatography. These results suggest that MRJP1 exists mainly as apisin in royal jelly.
Subject(s)
Antibodies/immunology , Fatty Acids/chemistry , Glycoproteins/isolation & purification , Insect Proteins/isolation & purification , Animals , Annexin A2/chemistry , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Fatty Acids/metabolism , Glycoproteins/chemistry , Glycoproteins/immunology , Insect Proteins/chemistry , Insect Proteins/immunologyABSTRACT
Soluble royal jelly (RJ) proteins (SRJPs) include the major RJ protein (MRJP) family, which contribute to the physiological actions of RJ. Although SRJPs are prepared using conventional methods involving dialysis and centrifugation, dialysis is a time-consuming process. We have therefore developed a simple method to isolate SRJPs from RJ. This new method produces 20-fold higher levels of SRJPs than that of the conventional procedure; hence, the levels obtained by the new and existing methods were compared. A 1-h ultracentrifugation separated SRJPs in the supernatant into upper, middle and lower layers. Each layer was analyzed by size-exclusion HPLC, SDS-PAGE and 2-DE. The upper and middle layers contained MRJP2 (52 kDa) and MRJP3 (60-70 kDa), while the lower layer contained MRJP1 (290 kDa). In nature, MRJP1 is a monomer and/or oligomer. When the lower layer was analyzed by Superose 12 HPLC, MRJP1 was predominantly an oligomer. Our MRJP isolation method reduces the procedure time by using ultracentrifugation without dialysis to obtain SRJPs and produces layers containing MRJP1 oligomers, MRJP2 and MRJP3.
Subject(s)
Fatty Acids/chemistry , Insect Proteins/isolation & purification , Ultracentrifugation/methods , Chromatography, High Pressure Liquid , Insect Proteins/chemistry , Molecular WeightABSTRACT
To prove the pharmacological actions of honeybee royal jelly (RJ) on the nervous system, we examined the effects of RJ on CRE-mediated transcription. RJ increased CRE-mediated transcription in PC12D cells. Moreover, CRE-mediated transcriptional activity by RJ was enhanced by nobiletin. U0126, a MEK inhibitor, inhibited CRE-mediated transcription by combining RJ plus nobiletin without affecting transcription by RJ alone. These results suggest that RJ stimulates CRE-mediated transcription via an ERK-independent cascade, whereas the increasing CRE-mediated transcriptional effect by nobiletin is dependent on ERK phosphorylation. Combining RJ plus nobiletin may activate effectively neuronal functions via enhancement of CRE-mediated transcription.
Subject(s)
Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fatty Acids/pharmacology , Flavones/pharmacology , Transcription, Genetic/drug effects , Animals , Bees , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/metabolism , Nervous System/drug effects , Nervous System/metabolism , PC12 Cells , Phosphorylation/drug effects , RatsABSTRACT
Royal jelly contains numerous components, including proteins. Major royal jelly protein (MRJP) 1 is the most abundant protein among the soluble royal jelly proteins. In its physiological state, MRJP 1 exists as a monomer and/or oligomer. This study focuses the molecular characteristics and functions of MRJP 1 oligomer. MRJP 1 oligomer purified using HPLC techniques was subjected to the following analyses. The molecular weight of MRJP 1 oligomer was found to be 290 kDa using blue native-PAGE. MRJP 1 oligomer was separated into 55 and 5 kDa spots on 2-D blue native/SDS-PAGE. The 55 kDa protein was identified as MRJP 1 monomer by proteome analysis, whereas the 5 kDa protein was identified as Apisimin by N-terminal amino acid sequencing, and this protein may function as a subunit-joining protein within MRJP 1 oligomer. We also found that the oligomeric form included noncovalent bonds and was stable under heat treatment at 56 degrees C. Furthermore, MRJP 1 oligomer dose dependently enhanced and sustained cell proliferation in the human lymphoid cell line Jurkat. In conclusion, MRJP 1 oligomer is a heat-resistant protein comprising MRJP 1 monomer and Apisimin, and has cell proliferation activity. These findings will contribute to further studies analyzing the effects of MRJP 1 in humans.
