Glial fibrillary acidic protein and vimentin expression in the frog olfactory system during metamorphosis.

In the present study, we investigated glial cell organization in the olfactory system of adult and tadpole Xenopus laevis using glial fibrillary acidic protein and vimentin antibodies. Our results showed for the first time that glial fibrillary acidic protein was strongly expressed at the level of the olfactory nerve from tadpole to adult and was likely to be expressed by ensheathing glia. In the olfactory bulb, the nerve layer was stained, and no staining was observed in glomeruli. By contrast, vimentin decorated radial glia in the bulb but faintly stained the olfactory nerve. Interestingly, glial fibrillary acidic protein and vimentin presented complementary staining patterns, with glial fibrillary acidic protein being expressed in the peripheral olfactory system and vimentin being expressed in the central part of the olfactory system.

Specific expression of olfactory binding protein in the aerial olfactory cavity of adult and developing Xenopus.

Olfactory binding proteins (OBP), commonly associated with aerial olfaction, are found in the olfactory mucus of mammals but have never been identified in fish. It is still not clear whether the presence of OBP in aerial olfactory systems is due to phylogenetic or to functional differences linked to the adaptation of the olfactory system to an aerial environment. To test this alternative, the olfactory system of Xenopus offers a unique opportunity because it includes two olfactory cavities, one of which is thought to be devoted to aquatic olfaction and the other to aerial olfaction. We therefore purified and cloned OBPs in two Xenopus species. Xenopus laevis OBP (XlaeOBP) and Xenopus tropicalis OBP (XtroOBP) exhibit 158 and 160 amino acids, respectively, sharing 89 residues. cRNA probes allowed us to demonstrate that XlaeOBP and XtroOBP are expressed at the level of Bowman's gland specifically in the aerial olfactory cavity, as confirmed using anti-XlaeOBP antiserum. OBP mRNA transcription occurs early during metamorphosis, as early as stage 57. This is the first study to demonstrate that OBPs are exclusively present in the aerial chamber and are only expressed as the tadpole becomes an adult in species which possess both aquatic and aerial olfactory organs.

Orchestin, a calcium-binding phosphoprotein, is a matrix component of two successive transitory calcified biomineralizations cyclically elaborated by a terrestrial crustacean.

Orchestia cavimana is a crustacean that cyclically replaces its calcified cuticle during molting cycles in order to grow. Its terrestrial way of life requires storage of calcium during each premolt period, as calcareous concretions, in tubular diverticula of the midgut. During the postmolt period the stored calcium is reabsorbed and is translocated through the storage organ epithelium as calcified small spherules. In a previous study, we sequenced and characterized a remarkable component of the organic matrix of the premolt storage structures, Orchestin, which is a calcium-binding phosphoprotein. In this paper, we analyzed the spatiotemporal expression of the orchestin gene by Northern blotting and in situ hybridization, and its translated product by immunocytochemistry. We found evidence that the gene and the protein are expressed specifically during premolt in the storage organs. More interestingly, we demonstrated that the protein is synthesized also during the postmolt period, as a component of the organic matrix of the calcium resorption spherules. Thus, Orchestin is a matrix component that is synthesized by the same cells to contribute alternately to the elaboration of two different calcifications. These results, in addition to the physical and chemical features of the protein, suggest that Orchestin is probably a key molecule in the calcium carbonate precipitation process leading to the cyclic elaboration of two transitory calcified mineralizations by the crustacean Orchestia.

Molecular characterization of a male-specific glycosyl hydrolase, Lma-p72, secreted on to the abdominal surface of the Madeira cockroach Leucophaea maderae (Blaberidae, Oxyhaloinae).

The epicuticular surface protein Lma-p72 is specific to the abdominal secretions of Leucophaea maderae (Madeira cockroach) adult males. Natural Lma-p72 was purified and the complete cDNA sequence determined by reverse-transcription PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p72 was expressed in the tergal and sternal glands. Sequence alignment indicates that Lma-p72 is closely related to the family 1 glycosyl hydrolases (EC 3.2.1). Native Lma-p72 was proved to be active in the abdominal secretions and exhibit a beta-galactosidase-like activity. However, weak specificity with respect to the C-4 configuration of the substrate was observed. Two main hypotheses were proposed concerning the function of this enzyme: Lma-p72 could hydrolyse oligosaccharides from the male abdominal secretions, making them more phagostimulatory for the female during the precopulatory behaviour. The protein could also cleave a pheromone-sugar conjugate to release the pheromonal compounds on to the cuticular surface. Such a sugar conjugate could be a transport form. Data from the first in vivo inhibition tests indicate that a glycosidase could be directly involved in the production process of some pheromonal compounds in L. maderae males.

Characterization and spatiotemporal expression of orchestin, a gene encoding an ecdysone-inducible protein from a crustacean organic matrix.

We report the characterization of a new gene encoding an acidic protein named Orchestin. This protein is a component of the organic matrix of calcium storage structures (calcareous concretions) elaborated during the moulting cycles of the terrestrial crustacean Orchestia cavimana. The deduced molecular mass of Orchestin is estimated to be 12.4 kDa and the pI to be 4.4, whereas the native protein extracted from the calcium deposits migrates as a 23 kDa band on SDS/PAGE. This discrepancy is probably due to the richness of this protein in acidic amino acids (approx. 30%). The protein obtained by expressing the Orchestin cDNA in Escherichia coli presents an electrophoretic mobility of 25 kDa. Antibodies raised against the recombinant protein recognize the 23 kDa native protein exclusively among the organic-matrix components. Spatiotemporal analysis of the expression of the orchestin gene shows that it is expressed only in the storage organ cells when the concretions are elaborated during the premoult period and also, to a smaller extent, during the postmoult period. The translation products are expressed in accordance with the transcript expression during both the premoult and postmoult periods. Study of the hormonal stimulation of orchestin reveals that 20-hydroxyecdysone induces this gene as a secondary-response or late-response gene.

A pheromone-binding protein from the cockroach Leucophaea maderae: cloning, expression and pheromone binding.

Odorant-binding proteins (OBPs) are thought to transport volatile compounds from air to their receptors through the sensillary lymph. In this protein family, the subgroup of pheromone-binding proteins (PBPs) is specifically tuned to the perception of the sexual pheromone. To date, the description of OBPs has been restricted to Endopterygota and Paraneoptera. Their expression in Orthopteroid has been hypothesized, but no evidence of OBP has been produced in this assemblage to date. In the present study, we describe the first OBP from a Dictyopteran insect that belongs to the cockroach Leucophaea maderae. The PBP of L. maderae (PBPLma) shares all the hallmarks of the OBP family and is expressed specifically in the female adult antennae, the sex that perceives the sexual pheromone. The affinity of the recombinant PBPLma produced in the Escherichia coli periplasm for the pheromonal compounds has been tested by displacement of a fluorophore, 8-anilino-1-naphtalenesulphonic acid (ANS). Our results suggest that two chemically close compounds of the pheromonal blend (3-hydroxy-butan-2-one and butane-2,3-diol) are capable of displacing ANS, whereas two other pheromone components (E-2-octenoic acid and senecioic acid) and other alkyl volatile compounds are not capable of displacing ANS, indicating a certain filtering of binding, which can be correlated with the putative function.

Isolation and characterization of Urbain, a 20-hydroxyecdysone-inducible gene expressed during morphogenesis of Bombyx mori wing imaginal discs.

In insects, wing imaginal discs respond to the steroid hormone 20-hydroxyecdysone by initiating morphogenesis leading to the formation of the adult flight appendages. In this work we analyse the expression of a Bombyx gene, referred to as Urbain, whose cDNA had been previously isolated from wing discs (Chareyre et al. 1993). Accumulation of the 1.8 kb transcript occurs concomitantly with the increase of 20-hydroxyecdysone titer at every stage examined during post-embryonic development. In vitro, its accumulation is delayed 6-9 h after exposure to 20-hydroxyecdysone. Studies in the presence of cycloheximide have established that Urbain is a secondary response gene. The sequence of the mRNA contains a putative open reading frame of 1656 nucleotides encoding a secreted hydrophilic protein, and we suggest that the Urbain gene product plays a role in cellular mechanisms involved in morphogenesis of the epithelium.