Serine protease inhibitor Spi2 mediated apoptosis of olfactory neurons.

The olfactory epithelium of adult mouse, where primary sensory neurons are massively committed to apoptosis by removal of their synaptic target, was used as a model to determine in vivo mechanisms for neuronal cell death induction. A macro-array assay revealed that the death of olfactory neurons is accompanied with over-expression of the serine protease inhibitor Spi2. This over-expression is associated with decreased serine protease activity in the olfactory mucosa. Moreover, in vitro or in vivo inhibition of serine proteases induced apoptotic death of olfactory neuronal cells. Interestingly, Spi2 over-expression is not occurring in olfactory neurons but in cells of the lamina propria, suggesting that Spi2 may act extracellularly as a cell death inducer. In that sense, we present evidence that in vitro Spi2 overexpression generates a secreted signal for olfactory neuron death. Hence, taken together these results document a possible novel mechanism for apoptosis induction that might occur in response to neurodegenerative insults.

Cyclo-oxygenase 2 tissue distribution and developmental pattern of expression in the chicken.

1. Cyclo-oxygenase 2 (COX-2) is implicated in multiple physiopathological processes. We have studied its physiological expression during chicken embryogenesis. 2. An original procedure was set to prepare a COX-2 probe from red blood cells. In situ hybridization, reverse transcription-polymerase chain reaction and northern blots, were performed on chick embryos from embryonic day (E) 3 to postnatal day 15. 3. In the mesonephros, the signal detected in mesonephric tubules presented a slow increase from E5 to E9, a plateau up to E12 and then a decrease, while the signal increased in the metanephros and then decreased after hatching. Transient expression of COX-2 mRNA in endothelial cells of the infundibulum of the aorta was also detected between E12 and E17. 4. This enzyme may have important roles in kidney morphogenesis during early embryonic stages and in tubular functions during development and in adult life. In the cardiovascular system, its vasodilatory effect could modulate the vasoconstrictor effect of the systolic pressure between E12 and E17 and contribute to a normal morphogenesis of the arterial tree and heart.

Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at approximately 2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.

Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris.

A honeybee putative general odorant-binding protein ASP2 has been expressed in the methylotrophic yeast Pichia pastoris. It was secreted into the buffered minimal medium using either the alpha-factor preprosequence with and without the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or its native signal peptide. Whereas ASP2 secreted using the alpha-factor preprosequence with the spacer peptide showed N-terminal heterogeneity, the recombinant protein using the two other secretion peptides was correctly processed. Mass spectrometry showed that the protein secreted using the natural peptide sequence had a mass of 13,695.1 Da, in perfect agreement with the measured molecular mass of the native protein. These data showed a native-like processing and the three disulfide bridges formation confirmed by sulfhydryl titration analysis. After dialysis, the recombinant protein was purified by one-step anion-exchange chromatography in a highly pure form. The final expression yield after 7-day fermentation was approximately 150 mg/liter. To our knowledge, this is the first report of the use of a natural insect leader sequence for secretion with correct processing in P. pastoris. The overproduction of recombinant ASP2 should allow ligand binding and mutational analysis to understand the relationships between structure and biological function of the protein.

Identification and developmental profiles of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera.

Four distinct hexamerin subunits (referred to as "hexamerins" in the following text) have been identified in the developing honeybee, Apis mellifera, by N-terminal protein sequencing. Hexamerins are abundant in the hemolymph of late larval and early pupal stages, and gradually decline during metamorphosis and adult development. Three hexamerins in the 70 kDa range have been found (Hex70a, Hex70b, Hex70c). In worker and drone, Hex70a is the only hexamerin present in large amount in later adult stages. Hex70b and c exhibit a similar developmental profile, disappearing in the drone just before adult emergence, and in the worker just after. Hex70b or Hex70c are still detectable in the adult queen. Hex80/110 likely exist in at least 3 different subunits, 1 of 110 kDa, and 2 of around 80 kDa, which all share a common N-terminus. They disappear during metamorphosis earlier than Hex70b and c. All these hexamerins have been found also in the antenna, suggesting their utilization in building up of antennal cuticle structures.

Separation, characterization and sexual heterogeneity of multiple putative odorant-binding proteins in the honeybee Apis mellifera L. (Hymenoptera: Apidea).

According to precise molar mass determined by mass spectrometry and N-terminal sequence, some 25 odorant-binding-like proteins were characterized from the antennae and legs of worker and drone honeybees. Antennal specific proteins, composed of six different molecules, were classified into three subclasses according to N-terminal sequence homology. The major sexual difference was shown to lie in the relative abundance of these antennal specific proteins and in the occurrence of a drone-specific isoform. At least 19 other related proteins were found to occur in antennae and legs, forming another class showing homology with insect OBP. Genotype comparison of two honeybee races revealed a variability limited to this second class. Provided that these odorant-binding-like proteins are indeed able to bind odorants or pheromones, the question of whether their peculiar multiplicity contributes to the remarkable capacity of the honeybee to discriminate among a wide range of odor molecules is raised.

Biochemical characterization, molecular cloning and localization of a putative odorant-binding protein in the honey bee Apis mellifera L. (Hymenoptera : Apidea).

A honey bee antennal water-soluble protein, APS2, was purified and characterized as the first Hymenoptera putative odorant-binding protein. Comparison of its measured Mr (13695.2+/-1.6) to that of the corresponding cDNA clone shows it does not undergo any post-translational modification other than a 19-residue signal peptide cleavage and formation of three disulfide bridges. These biochemical features are close to those of Lepidoptera odorant-binding proteins. In situ hybridization experiments demonstrated its specific expression in olfactory areas. Based on its higher expression in the worker than in the drone, ASP2 might be more involved in general odorant than in sex pheromone detection.

Honeybees have putative olfactory receptor proteins similar to those of vertebrates.

Using nested-PCR amplification of honeybee cDNA with degenerate primers derived from mammalian sequences, we have cloned four DNA fragments from putative odour receptor genes. This conclusion is based on high amino acid sequence similarity with published sequences from vertebrates and hydrophilicity profiles in agreement with including these genes in the super family of seven transmembrane domain receptor genes. These results are discussed in the context of the evolution of the olfactory receptor genes.