Twist is substrate for caspase cleavage and proteasome-mediated degradation.

Twist is a member of the basic helix-loop-helix family of transcription factors. An aberrant Twist expression has been found in diverse types of cancer, including sarcomas, carcinomas and lymphomas, supporting a role for Twist in tumor progression. Twist is known to be essential for mesodermal development. However, since a prolonged Twist expression results in a block of muscle, cartilage and bone differentiation, Twist has to be excluded from somites during late embryogenesis for terminal differentiation to occur. This implies that Twist expression must be target of a tight control. Here we provide evidence that Twist undergoes post-transcriptional regulation. Twist is substrate for cleavage by caspases during apoptosis and its cleavage results in ubiquitin-mediated proteasome degradation. Our findings suggest that Twist post-transcriptional regulation may play an important role in tissue determination and raise the possibility that alterations in the protein turnover may account for Twist overexpression observed in tumors.

Cloning of the cDNAs coding for two novel molybdo-flavoproteins showing high similarity with aldehyde oxidase and xanthine oxidoreductase.

The cDNAs coding for two novel mouse molybdo-flavoproteins, AOH1 and AOH2 (aldehyde oxidase homolog 1 and 2), were isolated. The AOH1 and AOH2 cDNAs code for polypeptides of 1336 amino acids. The two proteins have similar primary structure and show striking amino acid identity with aldehyde oxidase and xanthine oxidoreductase, two other molybdo-flavoenzymes. AOH1 and AOH2 contain consensus sequences for a molybdopterin-binding site and two distinct 2Fe-2S redox centers. In its native conformation, AOH1 has a molecular weight consistent with a homotetrameric structure. Transfection of the AOH1 and AOH2 cDNAs results in the production of proteins with phenanthridine but not hypoxanthine oxidizing activity. Furthermore, the AOH1 protein has benzaldehyde oxidizing activity with electrophoretic characteristics identical to those of a previously identified aldehyde oxidase isoenzyme (Holmes, R. S. (1979) Biochem. Genet. 17, 517-528). The AOH1 transcript is expressed in the hepatocytes of the adult and fetal liver and in spermatogonia. In liver, the AOH1 protein is synthesized in a gender-specific fashion. The expression of AOH2 is limited to keratinized epithelia and the basal layer of the epidermis and hair folliculi. The selective cell and tissue distribution of AOH1 and AOH2 mRNAs is consistent with the localization of the respective protein products.

In vitro permeation through porcine buccal mucosa of Salvia desoleana Atzei & Picci essential oil from topical formulations.

In the light of recent studies, which have shown that the essential oil derived from some Lamiaceae species has appreciable anti-inflammatory activity, moderate anti-microbial action and the ability to inhibit induced hyperalgesia, an assessment of the diffusion and permeation of Salvia desoleana Atzei & Picci (S. desoleana) essential oil through porcine buccal mucosa was considered useful for a possible application in the stomatological field. Topical formulations (microemulsions, hydrogels and microemulsion-hydrogels) were prepared for application to the buccal mucosa. The mucosa permeation of the oil from the formulations was evaluated using Franz cells, with porcine buccal mucosa as septum between the formulations (donor compartment) and the receptor phase chambers. The study also aimed at optimising the permeability of the S. desoleana essential oil by means of an enhancer, the diethylene glycol monoethyl ether Transcutol. The diffusion of the oil through the membrane was determined by evaluating the amount of essential oil components present in the receiving solution, the flux and the permeation coefficient (at the steady state) in the different formulations at set intervals. Qualitative and quantitative determinations were done by gas chromatographic analysis. All the formulations allow a high permeability coefficient in comparison with the pure essential oil. In particular, the components with a terpenic structure (beta-pinene, cineole, alpha-terpineol and linalool) have the highest capacity to pass through the porcine buccal mucosa when compared to the other components (linalyl acetate and alpha-terpinil acetate). Moreover, the enhancer, diethylene glycol monoethyl ether largely increases the permeation of the essential oil components in relation to the concentration.

Molecular cloning of the cDNA coding for mouse aldehyde oxidase: tissue distribution and regulation in vivo by testosterone.

The cDNA coding for mouse aldehyde oxidase (AO), a molybdoflavoprotein, has been isolated and characterized. The cDNA is 4347 nt long and consists of an open reading frame predicting a polypeptide of 1333 amino acid residues, with 5' and 3' untranslated regions of 13 and 335 nt respectively. The apparent molecular mass of the translation product in vitro derived from the corresponding cRNA is consistent with that of the monomeric subunit of the AO holoenzyme. The cDNA codes for a catalytically active form of AO, as demonstrated by transient transfection experiments conducted in the HC11 mouse mammary epithelial cell line. The deduced primary structure of the AO protein contains consensus sequences for two distinct 2Fe-2S redox centres and a molybdopterin-binding site. The amino acid sequence of the mouse AO has a high degree of similarity with the human and bovine counterparts, and a significant degree of relatedness to AO proteins of plant origin. Northern blot and in situ hybridization analyses demonstrate that hepatocytes, cardiocytes, lung endothelial or epithelial cells and oesophagus epithelial cells express high levels of AO mRNA. In the various tissues and organs considered, the level of AO mRNA expression is not strictly correlated with the amount of the corresponding protein, suggesting that the synthesis of the AO enzyme is under translational or post-translational control. In addition, we observed sex-related regulation of AO protein synthesis. In the liver of male animals, despite similar amounts of AO mRNA, the levels of the AO enzyme and corresponding polypeptide are significantly higher than those in female animals. Treatment of female mice with testosterone increases the amounts of AO mRNA and of the relative translation product to levels similar to those in male animals.

The mouse aldehyde oxidase gene: molecular cloning, chromosomal mapping and functional characterization of the 5'-flanking region.

In this article, we report on the chromosome mapping and molecular cloning of the genetic locus encoding the mouse molybdo-iron/sulfur-flavoprotein aldehyde oxidase. The aldehyde oxidase locus maps to mouse chromosome 1 band C1-C2, as determined by fluorescence in situ hybridization experiments conducted on metaphase chromosomes. The gene is approximately 83 kb long and consists of 35 exons. The exon/intron boundaries are perfectly conserved relative to the corresponding human homolog and almost completely conserved relative to the mouse xanthine oxidoreductase gene. This further supports the concept that the aldehyde oxidase and xanthine oxidoreductase loci evolved from the same ancestral precursor by a gene duplication event. The position of a major transcription start site was defined by primer extension and RNase mapping analysis. The 5'-flanking region of the mouse aldehyde oxidase gene contains a functional and orientation-dependent promoter as well as several putative binding sites for known cell-specific and general transcription factors. Deletion analysis of the 5'-flanking region defines an approximately 470 bp DNA stretch which is necessary and sufficient for the transcription of the mouse aldehyde oxidase gene.

Isolation and characterization of the human aldehyde oxidase gene: conservation of intron/exon boundaries with the xanthine oxidoreductase gene indicates a common origin.

Aldehyde oxidase (AO) is a molybdo-flavo enzyme involved in the metabolism of various endogenous and exogenous N-heterocyclic compounds of pharmacological and toxicological importance. The enzyme is the product of a gene which is implicated in the aetio-pathogenesis of familial recessive amyotrophic lateral sclerosis. Here, we report the cloning and structural characterization of the human AO gene. AO is a single copy gene approximately 85 kb long with 35 transcribed exons. The transcription-initiation site and the sequence of the 5'-flanking region, containing several putative regulatory elements, were determined. The 5'-flanking region contains a functional promoter, as assessed by appropriate reporter constructs in transient transfection experiments. Comparison of the AO gene structure shows conservation of the position and type of exon/intron junctions relative to those observed in the gene coding for another molybdo-flavoprotein, i.e. xanthine oxidoreductase (XOR). As the two genes code for proteins with a high level of amino acid identity, our results strongly suggest that the AO and XOR genetic loci arose as the consequence of a duplication event. Southern blot analysis conducted on genomic DNA from various animal species with specific cDNA probes indicates that the AO gene is less conserved than the XOR gene during evolution.

Isolation and characterization of the gene coding for human cytidine deaminase.

The human gene coding for cytidine deaminase (CD), the enzyme which catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, was isolated and structurally characterized. CD is a single copy gene with a length of 31 kb and consists of four exons. Exon-intron junctions do not bracket functional domains of the encoded protein as the boundary between exons 2 and 3 interrupts the catalytically important zinc-finger domain, which is well conserved along phylogenesis. 5'-RACE and RNase mapping experiments identify one major and multiple other minor transcription initiation sites, which are present in placenta as well as in the myeloid cell lines, HL-60 and U937. The 5'-flanking region of the gene contains an orientation-dependent functional promoter and is characterized by the presence of several potential sites for the binding of known transcriptional factors.