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The 55 kDa major protein of royal jelly, named apalbumin 1, is an authentic protein of honey and pollen pellet, and for its quantification an enzyme-linked immunosorbent assay (ELISA) was developed using specific polyclonal anti-apalbumin 1 antibody. The limit of detection for apalbumin 1 was 2 ng mL(-1). The floral honeys contained apalbumin 1 as follows: acacia, 0.011%; linden, 0.010%; chestnut, 0.029%; rape, 0.010%; and dandelion, 0.014%. The saccharose syrup honey contained only 0.001% of apalbumin 1. The average amount of apalbumin 1 relating to the total protein content of analyzed honey samples was 23.39%, whereas in SCCH apalbumin 1 presented only 4.81% of total proteins of honey. Apalbumin 1 is thermostabile in honey at 80 degrees C incubated for 40 min. ELISA results show good precision in the evaluation of apalbumin 1 quantity in honey (CV ranged from 0.69 to 4.25%).

This study illustrates multifunctionality of proteins of honeybee royal jelly (RJ) and how their neofunctionalization result from various PTMs of maternal proteins. Major proteins of RJ, designated as apalbumins belong to a protein family consisting of nine members with M(r) of 49-87 kDa and they are accompanied by high number of minority homologs derived from maternal apalbumins. In spite of many data on diversity of apalbumins, the molecular study of their individual minority homologous is still missing. This work is a contribution to functional proteomics of second most abundant protein of RJ apalbumin2 (M(r) 52.7 kDa). We have purified a minority protein from RJ; named as apalbumin2a, differ from apalbumin2 in M(r) (48.6 kDa), in N-terminal amino acids sequences - ENSPRN and in N-linked glycans. Characterization of apalbumin2a by LC-MALDI TOF/TOF MS revealed that it is a minority homolog of the major basic royal jelly protein, apalbumin2, carrying two fully occupied N-glycosylation sites, one with high-mannose structure, HexNAc2Hex9, and another carrying complex type antennary structures, HexNAc4Hex3 and HexNAc5Hex4. We have found that apalbumin2a inhibit growth of Paenibacillus larvae. The obtained data call attention to functional plasticity of RJ proteins with potential impact on functional proteomics in medicine.

Insect chitin possessing shell-like structure was prepared from the bumblebee corpses by a consequent treatment with 1M HCl and 1M NaOH. The bumblebee chitin was compared with crustacean (shrimp) chitin by using elemental analysis, Fourier-transform infrared (FT-IR) and solid-state (13)C cross-polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and confocal microscopy. Both chitins (bumblebee and shrimp) exhibited identical spectra, while the bumblebee chitin had a 5% lower degree of acetylation and was characterized by a fine membrane texture.

Apalbumin1 (Apa1) is the major royal jelly (RJ) and honey glycoprotein having various biological properties. We have previously demonstrated that Apa1 is a regular component of honey and honeybee pollen and stimulates macrophages to release tumor necrosis factor alpha (TNFalpha). The recombinant Apa1 (rApa1) and its four recombinant protein fragments derived on the basis of partial tryptic products of Apa1 were prepared by heterologous expression in Escherichia coli BL21-CodonPlus(DE3)-RIL. L-arginine at 50 mM concentration was used for improving the recombinant protein solubility. We report that the proteinous moiety of glycoprotein is responsible for stimulation of TNFalpha production by murine peritoneal macrophages. Moreover, we have shown that immunostimulatory effect is significantly increased after partial tryptic digestion of Apa1. It has been determined that recombinant N-terminal fragment of Apa1 is the most active elicitor of TNFalpha release in comparison to other three protein fragments of Apa1, as well as to the native Apa1 and rApa1. Furthermore, it was found that native honey was able to stimulate TNFalpha secretion from murine macrophages, whereas the deproteinized honey had no effect on the release of TNFalpha. This result suggests that immunostimulatory effect of honey is based on its RJ-protein content, primarily on its dominant protein Apa1.

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.

Two defensins showing high mutual similarity have previously been characterized in honeybee Apis mellifera: royalisin, a peptide isolated from the royal jelly, and defensin, found in the hemolymph of bacterially infected bees. Here we show that both these peptides are encoded by the same polymorphic gene, which we termed defensin1. Besides this gene, we identified an additional defensin gene coding for a novel honeybee defensin designated defensin2. The pre-pro-peptide sequence of defensin 2 was inferred from its cDNA. Mature defensin 2 peptide shows 55.8% identity with defensin 1. Sequences of genomic loci of the two defensin genes revealed their different structure. Defensin1 possesses an exon-intron structure unique among arthropoda defensin genes. Its second intron splits exactly the common structural module of defensins from a short amidated C-terminal extension found only in hymenopteran defensins. Transcription of defensin genes in some nurse honeybees tissues was studied by RT-PCR. Both defensins are expressed in heads and thoraces. Defensin1 but not defensin2 mRNA was detected in hyphopharyngeal, mandibular and thoracic salivary glands. Immune response elements were identified by computer analysis of the promoter regions of defensin genes. Their different representation in these genes reflects presumably observed tissue-specific expression of defensins.

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.

Mrjp1 gene belongs to the honeybee mrjp gene family encoding the major royal jelly proteins (MRJPs), secreted by nurse bees into the royal jelly. In this study, we have isolated the genomic clone containing the entire mrjp1 gene and determined its sequence. The mrjp1 gene sequence spans over 3038 bp and contains six exons separated by five introns. Seven mismatches between the mrjp1 gene sequence and two previously independently published cDNA sequences were found, but these differences do not lead to any change in the deduced amino acid sequence of MRJP1. With the aid of inverse polymerase chain reaction we obtained sequences flanking the 5' ends of other mrjp genes (mrjp2, mrjp3, mrjp4 and mrjp5). Putative promoters were predicted upstream of all mrjp genes (including mrjp1). The predicted promoters contain the TATA motif (TATATATT), highly conserved both in sequence and position. Ultraspiracle (USP) transcription factor (TF) binding sites in putative promoter regions and clusters of dead ringer TF binding sites upstream of these promoters were predicted computationally. We propose that USP, as a juvenile hormone (JH) binding TF, might possibly act as a mediator of mrjp expression in response to JH. Mrjp1's genomic locus is predicted to encode an antisense transcript, partially overlapping with five mrjp1 exons and entirely overlapping with the putative promoter and predicted transcriptional start point of mrjp1. This finding may shed light on the mechanisms of regulation of mrjps expression. Southern blot analysis of genomic DNA revealed that all so far known members of mrjp gene family (mrjp1, mrjp2, mrjp3, mrjp4 and mrjp5) are present as single-copy genes per haploid honeybee genome. Although MRJPs and the yellow protein of Drosophila melanogaster share a certain degree of similarity in aa sequence and although it has been shown that they share a common evolutionary origin, neither structural similarities in the gene organization, nor significant similarities between intron sequences of mrjp1 gene and fourteen yellow-like genes of D. melanogaster were found.

A peptide named apisimin was found in honeybee (Apis mellifera L.) royal jelly (RJ). N-terminal sequencing showed that this peptide corresponded to the sequence of a cDNA clone isolated from an expression cDNA library prepared from heads of nurse honeybees. No homology was found between the protein sequence of apisimin with a molecular mass of 5540.4 Da and sequences deposited in the Swiss-Prot database. The 54 amino acids of apisimin do not include Cys, Met, Pro, Arg, His, Tyr, and Trp residues. The peptide shows a well-defined secondary structure as observed by CD spectroscopy, and has the tendency to form oligomers. Isoelectrofocusing showed apisimin to be an acidic peptide.

A cDNA encoding a new member of the gene family of major royal jelly proteins (MRJPs) from the honeybee, Apis mellifera, was isolated and sequenced. Royal jelly (RJ) is a secretion of the cephalic glands of nurse bees. The origin and biological function of the protein component (12.5%, w/w) of RJ is unknown. We show that the MRJP gene family encodes a group of closely related proteins that share a common evolutionary origin with the yellow protein of Drosophila melanogaster. Yellow protein functions in cuticle pigmentation in D. melanogaster. The MRJPs appear to have evolved a novel nutritional function in the honeybee.

Major proteins of honey bee (Apis mellifera) royal jelly are members of the MRJP protein family. One MRJP protein termed MRJP3 exhibits a size polymorphism as detected by SDS-PAGE. In this report we show that polymorphism of the MRJP3 protein is a consequence of the polymorphism of a region with a variable number of tandem repeats (VNTR) located at the C-terminal part of the MRJP3 coding region. We present the characterization of five polymorphic alleles of MRJP3 by DNA sequencing. By PCR analyses, at least 10 alleles of distinct sizes were found in randomly sampled bees. Studies with nurse bees from a single honeybee colony revealed both Mendelian inheritance and very high variability of the MRJP3 genomic locus. The high variability and simple detection of the MRJP3 polymorphism may be useful for genotyping of individuals in studies of the honeybee.

The characterization of major proteins of honeybee larval jelly (49-87 kDa) was performed by the sequencing of new complementary DNAs (cDNAs) obtained from a honeybee head cDNA library, by the determination of N-terminal sequences of the proteins, and by analyses of the newly obtained and known sequence data concerning the proteins. It was found that royal jelly (RJ) and worker jelly (WJ) contain identical major proteins and that all the proteins belong to one protein family designated MRJP (from Major Royal Jelly Proteins). The family consists of five main members (MRJP1, MRJP2, MRJP3, MRJP4, MRJP5). The proteins MRJP3 and MRJP5 are polymorphic. MRJPs account for 82 to 90% of total larval jelly protein, and they contain a relatively high amount of essential amino acids. These findings support the idea that MRJPs play an important role in honeybee nutrition.

A plasmid encoding a fusion protein interlinked by thrombin recognition sequence between glutathione S-transferase and Japanese quail ovalbumin (without 40 amino acid residues from the 5'-end of the ORF) has been constructed, employing the expression system pGEX-2T. The deglycosylated fusion protein (64 kDa) was purified by affinity chromatography on glutathione agarose beads, analyzed by SDS-polyacrylamide gel electrophoresis, immunochemically detected with antiserum raised against Japanese quail ovalbumin and tested for its stability.

RNA polymerase from Streptomyces aureofaciens has been purified by polyethyleneimine precipitation followed by chromatography first on DEAE-cellulose, then heparin-Sepharose and finally on an aminooxybutylcellulose matrix containing immobilised S. aureofaciens DNA. The enzyme is composed of three subunits of approximately 145, 136 and 44 kDa that are in a ratio of approx. 1:1:2. In many isolations two additional subunits of approximately 68 and 39 kDa and some minor protein bands of approximately 110, 85 and 61 kDa are also present. Thus, the structure of this enzyme is very similar to other bacterial RNA polymerases, exhibiting an alpha 2 beta beta' core and the additional proteins rho and sigma.

Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation. Four clones containing 1900-1980 bp cDNAov were obtained. The cDNA ends in one of these clones were sequenced. Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned. They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease. A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov. The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed.

Several UV-absorbing substances inhibiting the DNA-dependent RNA polymerases of Escherichia coli and Streptomyces aureofaciens, as well as the restriction endonuclease Eco RI have been isolated from the water-soluble extract of Propolis by two-dimensional paper chromatography. The inhibition of bacterial RNA-polymerases by the components of Propolis was probably due to the loss of their ability to bind to DNA. The general characteristic of the UV-absorbing component of Propolis with the most pronounced inhibitory effect upon transcription in vitro is described.

The total mRNA was prepared from laying quail oviduct and, after denaturation with dimethyl sulphoxide and glyoxal, characterized by agarose gel electrophoresis. The electrophoresis showed that the preparation contains physically homogeneous molecules of mRNAs, which migrate as discrete bands. The molecular weights of four major oviduct mRNAs coding for conalbumin, ovalbumin, ovomucoid, and lysozyme were determined. The biological activity of mRNAs was assayed in a cell-free translation system derived from rabbit reticulocytes. One of the translation products, ovalbumin, was proved by immunoprecipitation.

1. Polynucleotide phosphorylase from a chlortetracycline-producing strain of Streptomyces aureofaciens was isolated by Polymin P fractionation. Using chromatography on DEAE-cellulose and Sephadex G-150 the enzyme, which appears homogeneous in gel chromatography and sedimentation analysis, was purified 2000-fole giving a final yield of 15%. 2. The sedimentation coefficient (s-o 20, w) of the native enzyme in 0.2 M NaCl is 9.15 S and its molecular weight is 210 000 plus or minus 15 000. Molecular weight estimated by sodium dodecylsulfate gel electrophoresis was about 100 000. 3. We have determined the optimal conditions for nucleoside 5'-diphosphate polymerization, their phosphate exchange and phosphorolysis of polyribonucleotides catalysed by polynucleotide phosphorylase from S. aureofaciens. 4. Chlortetracycline is a competitive inhibitor of S. aureofaciens polynucleotide phosphorylase. 5. Polynucleotide phosphorylase is activated in the polymerization reaction by ionic strength (K+, Na+, NH4+) while polyribonucleotide phosphorolysis is activated only by NH4+.

During the sudden decrease in RNA synthesis in Streptomyces aureofaciens, i.e. around the 6th hour of cultivation, synthesis of adenosine and guanosine tetraphosphates and pentaphosphates begins. The synthesis of these nucleotides is highest during the onset of chlortetracycline production, around the 20th hour of cultivation and continues. During this phase of growth of S. aureofaciens, RNA and protein synthesis are reduced by about one order of magnitude as compared to the rate which can be observed at the beginning of cultivation, but the synthesis is not inhibited by exogenous CTC.