Age dependent changes in histamine content of venom of queen and worker honey bees

We have compared the variation in histamine content of the venom of worker honey bees maintained in the field with those in an indoor flight room. We find that the temporal changes in venom histamine content are parallel under the different conditions.We have measured the histamine content of different regions of the venom system of virgin queen bees and found temporal changes in histamine concentration which parallel those seen in workers. While the absolute amounts of histamine in the queen venom system exceed those measured in workers, we estimate that the histamine concentration in queen and worker venom is similar.The LD₅₀ of histamine, injected into the haemocoele of worker bees in saline, is found to be 3.2 μg/worker.     

Insect protein synthesis in frog cells: the translation of honey bee promelittin messenger RNA in Xenopus oocytes

Venom glands of young queen bees (Apis mellifera) synthesize the toxic peptide melittin as their main product. Melittin is formed by proteolytic cleavage of a precursor, promelittin. Unfractionated RNA prepared from venom glands was injected into Xenopus oocytes and was shown to direct the synthesis of a promelittin-like substance. About half of the peptide chain made in oocytes has been sequenced; the 17 amino acid residues identified correspond exactly with sequences found in promelittin from venom gland cells. These results yield final proof that injected messenger RNAs can be read with great fidelity. The translation of a messenger from an insect gland shows that at least some of the translational systems within the oocyte are neither cell-type nor phylum specific. It seems likely that the oocyte can be used to assay any kind of eukaryotic mRNA.The conversion of promelittin to melittin could not be detected in oocytes. Moreover, the promelittin synthesized in oocytes differs at the carboxyl end from the product made in gland cells, for the latter terminates with glutamine amide while the oocyte material probably ends with an amino acid with a free α-carboxyl group. Some of the post-translational modifications characteristic of gland cells thus do not seem to take place in oocytes.     

Characterization of messenger RNA by direct translation from agarose gels

A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.     

Characterization of a messenger RNA transport protein

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.     

Translation of honeybee promelittin messenger RNA. Formation of a larger product in a mammalian cell-free system.

Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell-free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell-free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro. The larger polypeptide detected in vitro may represent a precursor of promelittin.     

Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees

The presence of myosin and dynein in the ovaries of both Apis mellifera and Scaptotrigona postica was investigated in extracts and in histological sections. In the ovary extracts, motor proteins, myosins V, VI and dynein were detected by Western blot. In histological sections, they were detected by immunocytochemistry, using a mouse monoclonal antibody against the intermediary chain of dynein and a rabbit polyclonal antibody against the myosin V head domain. The myosin VI tail domain was recognized by a pig polyclonal antibody. The results show that these molecular motors are expressed in the ovaries of both bee species with few differences in location and intensity, in regions where movement of substances is expected during oogenesis. The fact that antibodies against vertebrate proteins recognize proteins of bee species indicates that the specific epitopes are evolutionarily well preserved.     

Molecular weight of the thyroglobulin messenger RNA of sheep thyroid gland

Poly(A)+ mRNA from sheep thyroid total or thyroglobulin-specific polysomes obtained by immunological precipitation, was purified by two cycles of chromatography on oligo(dT)-cellulose. Upon electrophoresis in 98 % formamide-polyacrylamide gels, the purified RNA showed a major species of M_r 2.8×10⁶. The correlation found between the very high concentration of this species and its thyroglobulin messenger activity in the reticulocyte lysate protein synthesis system demonstrates that the thyroglobulin mRNA contains enough bases to code for the thyroglobulin peptide chain (M_r 300 000).     

Cellular energy metabolism maintains young status in old queen honey bees (Apis mellifera)

Trophocytes and oenocytes of queen honey bees are used in studies of cellular longevity, but their cellular energy metabolism with age is poorly understood. In this study, the molecules involved in cellular energy metabolism were evaluated in the trophocytes and oenocytes of young and old queen bees. The findings indicated that there were no significant differences between young and old queen bees in β‐oxidation, glycolysis, and protein synthesis. These results indicate that the cellular energy metabolism of trophocytes and oenocytes in old queen bees is similar to young queen bees and suggests that maintaining cellular energy metabolism in a young status may be associated with the longevity of queen bees. Fat and glycogen accumulation increased with age indicating that old queen bees are older than young queen bees.     

Characterization of messenger RNA populations of Crithidia fasciculata

Cells of Crithidia fasciculata taken from exponentially growing cultures display a relatively high content of poly(A) RNA (roughly 45% of the total) in a nonpolysomal compartment. The proportion of this fraction is significantly higher than that of the free ribosomal units. This RNA is translationally competent and is indistinguishable from polysomal mRNA by various criteria. The significance of these findings is discussed with respect to the protein synthesis capability of the cell and the metabolic relationships between the cytoplasmic compartments of mRNA.     

Isolation of active messenger RNA for s casein from bound polyribosomes of mammary gland

RNAs have been extracted from mammary bound polyribosomes and purified by sucrose gradient centrifugation. A cell free mammary system (homologous, except for the addition of initiation factors of reticulocyte origin) and a lysate of reticulocytes, have been used to assay biologically active messenger RNA. One of these messenger RNAs directs the synthesis of a product with electrophoretic and immunological properties analogous to α_S casein.     

Characterization of beta-adrenoceptors responsible for venom production in the venom gland of the snake Bothrops jararaca

We have shown that the stimulation of beta-adrenoceptors is an important step in venom production in the Bothrops jararaca venom gland. In the present study, the pharmacological profile of the beta-adrenoceptor present in Bothrops jararaca venom gland was characterized by radioligand binding assay and by the ability of isoprenaline to promote accumulation of cyclic AMP in dispersed secretory cells. In both cases, the venom glands were obtained from non-extracted snakes (quiescent stage) or from snakes which venom was extracted 4 days before sacrifice (venom production stimulated stage). [125I]-iodocyanopindolol ([125I]-ICYP) bound to extracted gland membranes in a concentration-dependent and saturable manner, but with low affinity. Propranolol, beta1- or beta2-selective adrenoceptors ligands displaced the [125I]-ICYP binding with low affinity, while selective beta3-adrenoceptor ligands did not displace the [125I]-ICYP binding. The displacement of [125I]-ICYP by propranolol was similar in non-extracted and extracted glands, showing the presence of beta-adrenoceptors in both stages. In dispersed secretory cells of non-extracted glands, isoprenaline (1 microM) increased the cyclic AMP production and propranolol (10 microM) was able to block this effect. On the other hand, in extracted glands, isoprenaline had no effect. The results suggest that the beta-adrenoceptors present in the Bothrops jararaca venom glands are different from those (beta1, beta2 or beta3) described in mammals, but are coupled to the Gs protein, like the known beta-adrenoceptor subtypes. Moreover, previous in vivo stimulation of venom production desensitizes the beta-adrenoceptors system and, although the receptors could be detected by binding studies, they are not coupled to the Gs protein, indicating that beta-adrenoceptors stimulation contributes to the initial steps of venom synthesis.     

First record of chemical signals from the queen during the oviposition process in stingless bees

In stingless bees, the cell provisioning and oviposition process consists of several integrated behavioral sequences and several stereotyped queen–worker interactions. This study aims to demonstrate that chemical signals originating from the queen may contribute as cues for the sequence of the oviposition process in Melipona marginata. For this, we analyzed the cell before and after queen laying, and compared them with the cuticular hydrocarbons of the queen’s abdomen, using a gas-chromatography and mass spectrometry system.     

Regulation of casein messenger RNA during the development of the rat mammary gland.

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.     

Characterization of presumptive histone messenger RNA from a cell line of Aedes aegypti.

Four presumptive histone messenger RNAs were characterized from a cell line of Aedes aegypti, and their molecular weights were determined by electrophoresis. They were shown to be associated with polysomes during the peak of DNA synthesis, but not when DNA synthesis was inhibited by cytosine arabinoside or when DNA was not being synthesized. These mRNAs are associated with polysomes containing less than 8 ribosomes and having a high ratio of incorporation of lysine to tryptophan into their nascent peptides. The mRNAs released from these polysomes were translated in vitro and histone products were synthesized. Histones were not synthesized when the mRNAs were obtained from large polysomes or from small polysomes during the non-DNA synthetic period.     

Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.