Biomedical subjects

H Bouhin

Publications and source records attributed to H Bouhin.

10 recordsLinked to original sources

PubMed2000

Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysone.

Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by lambda-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response.

Amino Acid Sequence↗

PubMed1999

cDNA cloning of an adult male putative lipocalin specific to tergal gland aphrodisiac secretion in an insect (Leucophaea maderae).

Lma-P22 is a cuticular surface protein specific to the tergal gland secretion of Leucophaea maderae adult males which is ingested by females just before copulation. The complete Lma-P22 cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. The recombinant protein expressed in Escherichia coli was recognized by an anti-Lma-P22 antibody. Northern blot analysis indicates that the corresponding mRNA is transcribed only in the epidermis of male tergites. Sequence analysis indicated that Lma-P22 deduced protein belongs to the lipocalin family. Lipocalins are extracellular proteins which carry hydrophobic compounds and some of them can bind sexual pheromone in vertebrates. Lma-P22 is the first example of a lipocalin-like protein involved in insect sexual behavior.

Amino Acid Sequence↗

PubMed1999

DEC: a new miniature inverted-repeat transposable element from the genome of the beetle Tenebrio molitor.

In this paper we describe a novel family of miniature inverted-repeat transposable elements (MITEs), named DEC, isolated from the genome of the beetle Tenebrio molitor. These elements are highly reiterated and their number is estimated to be around 3500 per haploid genome. Two of them have been isolated and the two sequences are 84% identical. Like other MITEs, they are characterized by their small size, their A + T richness, the presence of terminal inverted repeats and the absence of open reading frames. These data suggest that MITEs are probably widely distributed in arthropods.

Animals↗

PubMed1998

Characterization of two new cuticular genes specifically expressed during the post-ecdysial molting period in Tenebrio molitor.

In a previous study, we have isolated a cDNA, TM-ACP17, coding for a post-ecdysial adult protein of Tenebrio molitor. After screening of a genomic library with TM-ACP17, we report isolation and sequencing of TM-ACP17 gene and a new gene, TM-LPCP29, coding for a larval-pupal protein. These two genes exhibit a common sequence of 15 nucleotides and a characteristic of most cuticular protein genes so far described: an intron interrupting the signal peptide. The deduced aa sequence of TM-LPCP29 exhibits a high percentage of Ala (26.5%) and Val (17.5%) and is highly hydrophobic. In the N-terminal part, the motif VAAPV is repeated ten times. Numerous histidine residues are present in the C- and N-terminal regions. A comparison is made with other cuticle protein sequences. Northern hybridization analysis showed that TM-LPCP29 is present during larval and mainly pupal post-ecdysial cuticle secretion. In-situ hybridization revealed that TM-LPCP29 mRNA is expressed in epidermis and not in muscles or fat body.

Amino Acid Sequence↗

PubMed1996

Up-regulation of an adult cuticular gene by 20-hydroxyecdysone in insect metamorphosing epidermis cultured in vitro.

Pupal forewing epidermis of the Coleoptera, Tenebrio molitor, was used to develop an in vitro system to study the hormonal control of metamorphosis at the cellular and molecular levels. Exposure to 1 microM 20-hydroxyecdysone for 48 h caused the formation of a typical adult cuticle. Under these conditions the expression of ACP-20, an adult-specific cuticular gene, was fivefold higher than in absence of exogenous hormone. This stimulation was also observed when a higher level of 20-hydroxyecdysone was maintained, and prevented by protein inhibitors, indicating that 20-hydroxyecdysone does not act directly on this gene. Exposure to 20-hydroxyecdysone followed by exposure in hormone-free medium caused the cessation of this stimulation, showing the requirement of the 20-hydroxyecdysone continuous presence for stimulating ACP-20 gene expression. Thus, unlike the other cuticular protein genes so far studied, its expression is not repressed by 20-hydroxyecdysone, and does not need the decline in ecdysteroids titer.

Animals↗

PubMed1995

Identification, sequence and mRNA expression pattern during metamorphosis of a cDNA encoding a glycine-rich cuticular protein in Tenebrio molitor.

The study of insect cuticular proteins and their sequences is of interest because they are involved in protein-protein and protein-chitin interactions which confer the mechanical properties and fine architecture of the cuticle. Moreover, in the coleopteran Tenebrio molitor there is a dramatic change in cuticular architecture between pre- and postecdysial secretion. We report the isolation, by differential screening, and the sequence characterization of a cDNA clone encoding a cuticular protein of T. molitor, ACP17. After insertion in the expression vector pEX1, the recognition of the fusion protein by an anti-cuticular monoclonal antibody confirmed the cuticular nature of ACP17. Northern hybridization analysis showed that ACP17 mRNA expression begins weakly 3 days before adult ecdysis and strongly increases during the secretion of postecdysial adult cuticle, with a maximum just after ecdysis. In situ hybridization revealed that the ACP17 mRNA is only present in the epidermis which secretes hard cuticle. The deduced amino acid (aa) composition exhibits a high content of Gly (28%) and Ala (20%) and, particularly, two poly(Gx) stretches separated by repetitive motifs with proline AAPVA. A comparison is made with other cuticle aa sequences.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence of an adult-specific cuticular protein gene from the beetle Tenebrio molitor: effects of 20-hydroxyecdysone on mRNA accumulation.

The accumulation of transcripts from two adult-specific cuticular genes (ACP-20 and ACP-22) is shown to be modified after addition of exogenous 20-hydroxyecdysone. In the continuous presence of high levels of the hormone, the expression of ACP-20 gene is significantly weaker than that of untreated controls, while ACP-22 expression is 2.5-fold increased. During active synthesis of the ACP messages, a 0.5 microg 20-hydroxyecdysone injection causes a rapid 2-fold increase in ACP-22 mRNA and is not able to repress ACP-20 mRNA accumulation. We conclude that these genes whose transcripts appear in an almost coordinated manner in epidermal cells during the moulting cycle are regulated by ecdysteroids in a different way. In order to undertake a functional dissection of the promoter regions of ACP-22 gene, we have isolated and sequenced a genomic clone. The sequence similarities with other cuticular protein genes are discussed.

Amino Acid Sequence↗

PubMed1992

cDNA cloning and deduced amino acid sequence of a major, glycine-rich cuticular protein from the coleopteran Tenebrio molitor. Temporal and spatial distribution of the transcript during metamorphosis.

In Coleoptera, the elytra (forewings), with a very hard and thick cuticle, protect the membranous and delicate hindwings against mechanical stress. We have isolated and characterized a cDNA encoding a major cuticle protein in Tenebrio molitor, named ACP-20. The deduced amino acid sequence is roughly tripartite, with two terminal glycine-rich domains and a central region showing pronounced similarities with some other hard cuticle proteins. Northern blot and in situ hybridization analyses reveal that ACP-20 gene expression is developmentally regulated since transcript accumulation occurs only in epidermal regions synthesizing hard cuticle and is restricted to the period of preecdysial adult cuticle deposition. Moreover, application of a juvenile hormone analogue prevents the appearance of the transcript, indicating that juvenile hormone, a key molecule involved in the control of insect metamorphosis, negatively regulates the expression of the ACP-20 gene.

Amino Acid Sequence↗

PubMed1992

Developmental profiles of epidermal mRNAs during the pupal-adult molt of Tenebrio molitor and isolation of a cDNA clone encoding an adult cuticular protein: effects of a juvenile hormone analogue.

Changes in translatable mRNAs from the wing epidermis of the Coleoptera Tenebrio molitor have been investigated during metamorphosis by analysis of in vitro translated products. Striking differences between the patterns obtained from mRNAs extracted during pupal and adult cuticle secretion indicated that a drastic change in gene expression occurs during the pupal-adult transition. In addition to these stage-specific modifications, the mRNA patterns changed within each cuticular synthesis program (pupal or adult), especially at ecdysis. After tritiated leucine incorporation, some of the major radiolabeled cuticular proteins showed similar changes suggesting that the sequential appearance of mRNAs corresponds to sequential deposition of cuticular proteins. In supernumerary pupae obtained after juvenile hormone analogue (JHA) application on newly ecdysed pupae, translatable mRNA were very similar to those of pharate pupae. The JHA seemed, therefore, to prevent the expression of the adult program. By immunoblotting in vitro translated products with a monoclonal antibody recognizing an adult-specific cuticular protein, the developmental profile of the corresponding mRNA was studied. This mRNA was detected in anterior wing epidermis during the first 80 hr of the pharate adult stage. Using the same antibody, a cDNA clone was isolated from epidermal mRNA. The hybrid selected mRNA coded for only one protein with an apparent MW of 22 kDa which was, furthermore, recognized by the antibody. The Northern blot analysis performed with the clone confirmed the Western blot analysis of the in vitro translation products. JHA application at the beginning of the pupal-adult reprograming prevented the appearance of this mRNA; however, this transcript was present during the following molting cycle. This reversibility of the JHA action was confirmed by immunogold labeling of the cuticles formed in treated animals.

Animals↗

PubMed1992

Characterization of a cDNA clone encoding a glycine-rich cuticular protein of Tenebrio molitor: developmental expression and effect of a juvenile hormone analogue.

The complete sequence of a cDNA clone, isolated from epidermal mRNA of Tenebrio molitor using a monoclonal antibody raised against an adult-specific cuticular antigen only present in the hard cuticle, was obtained after primer extension at the 5' end. From this cDNA sequence, the deduced protein encompasses 199 amino acids (including a signal peptide) with a total molecular weight of 20.7 kDa. The protein exhibits a bipartite structure: glycine-rich region located in its NH2-terminal part and a carboxy-terminal domain sharing homologies with other cuticular proteins of Orthoptera, Diptera and Lepidoptera. In-situ hybridization analysis shows that the corresponding mRNA is present only in epidermal cells secreting the adult fibrous cuticle destined to become heavily sclerotized. In supernumerary pupae obtained after the application of the juvenile hormone analogue (JHA) to newly ecdysed pupae, the mRNA was undetectable, indicating that JHA can prevent the switch to the adult programme. However, in pupal-adult intermediates, obtained when JHA is applied later, the mRNA is detected.

Amino Acid Sequence↗