Study of housekeeping gene expression in human keratinocytes using OLISA, a long-oligonucleotide microarray and q RT-PCR.

In recent years, applications of microarray platforms have been extended to different areas of research including cosmetic and pharmaceutical. Although microarray technology is still improving its sensitivity and flexibility, researchers often turn toward quantitative RT-PCR for data validation. Assessment of messenger RNA quantity by these methods is based on comparison with internal standard genes, mainly housekeeping genes, so called because their synthesis occurs normally at a constant level. However, numerous studies showed that expression of these genes could vary in given situations. Here, we report results on four housekeeping genes (GAPDH, beta-2 microglobulin, S40 and S26 ribosomal sub-units) with constant expression levels established on OLISA microarray using different keratinocyte cultures. Moreover, qRT-PCR validation demonstrates that S26 ribosomal is a good housekeeping gene on keratinocytes and skin studies. Our data indicate that S26 gene can be routinely used to standardize results to investigate differentially expressed genes in a healthy human skin.

Pyrrolidone carboxyl peptidase (Pcp): an enzyme that removes pyroglutamic acid (pGlu) from pGlu-peptides and pGlu-proteins.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) is an exopeptidase commonly called PYRase, which hydrolytically removes the pGlu-proteins. pGlu also known as pyrrolidone carboxylic acid may occur naturally by an enzymatic procedure or may occur as an artifact in proteins or peptides. The enzymatic synthesis of pGlu suggests that this residue may have important biological and physiological functions. Several studies are consistent with this supposition. PYRase has been found in a variety of bacteria, and in plant, animal, and human tissues. For over two decades, biochemical and enzymatic properties of PYRase have been investigated. At least two classes of PYRase have been characterized. The first one includes the bacterial and animal type I PYRases and the second one the animal type II and serum PYRases. Enzymes from these two classes present differences in their molecular weight and in their enzymatic properties. Recently, the genes of PYRases from four bacteria have been cloned and characterized, allowing the study of the primary structure of these enzymes, and their over-expression in heterelogous organisms. Comparison of the primary structure of these enzymes revealed striking homologies. Type I PYRases and bacterial PYRases are generally soluble enzymes, whereas type II PYRases are membrane-bound enzymes. PYRase II appears to play as important a physiological role as other neuropeptide degrading enzymes. However, the role of type I and bacterial PYRases remains unclear. The primary application of PYRase has been its utilization for some protein or peptide sequencing. Development of chromogenic substrates for this enzyme has allowed its use in bacterial diagnosis.

One step purification and characterization of the pyrrolidone carboxyl peptidase of Streptococcus pyogenes over-expressed in Escherichia coli.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA-peptides and -proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over-expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non-denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over-expressed in E. coli disclosed properties similar to those found for the enzyme extracted from S. pyogenes or for some other Pcps studied previously. Thus the over-expressed enzyme should serve as a suitable source for N-terminal unblocking prior to some PCA protein sequencing.

Molecular characterization of pcp, the structural gene encoding the pyrrolidone carboxylyl peptidase from Streptococcus pyogenes.

This paper describes the cloning of a gene (pcp) coding for pyrrolidone carboxylyl peptidase (PYRase), an enzyme which selectively removes N-terminal pyroglutamic acid residues from polypeptides. This gene was isolated from Streptococcus pyogenes by construction of a gene library with a bacteriophage lambda-derived cosmid-Escherichia coli host system. Nucleotide sequence determination of a 1.3 kb restriction fragment revealed a 645 bp open reading frame encoding a 215-amino-acid product of M(r) 23,135 consistent with the 26 kDa polypeptide obtained from in vivo overexpression in E. coli. Southern hybridization confirmed that pcp is a single-copy gene on the S. pyogenes chromosome. 5' and 3' endpoint mapping of the 0.7 kb specific transcript observed by Northern analysis permitted the identification of transcriptional initiation and termination signals. Structural features of the pcp gene product from S. pyogenes are discussed and compared with that from Bacillus subtilis. The lack of sequence identity with any other known protein or nucleotide sequence suggests that this enzyme belongs to a new class of peptidase.

Characterization of the pcp gene encoding the pyrrolidone carboxyl peptidase of Bacillus subtilis.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp) is an enzyme that catalyzes the removal of the N-terminal pyroglutamyl group from some peptides or proteins. Its value in protein chemistry and bacterial diagnosis makes this enzyme an interesting subject of study. The present paper reports for the first time the cloning and characterization of a pyrrolidone carboxyl peptidase gene (pcp). This gene is present in a single copy in the genome of Bacillus subtilis as indicated by Southern blot hybridization analysis. The pcp transcripts were analyzed in Escherichia coli by Northern blot hybridization and S1 nuclease mapping. The deduced amino acid sequence predicts a protein of 215 amino acids with a calculated molecular weight of 23,777 Da. The pcp gene has been over-expressed in E. coli, allowing the identification and partial characterization of Pcp protein.

Specific detection of Escherichia coli and Shigella species using fragments of genes coding for beta-glucuronidase.

The occurrence of beta-glucuronidase activity, a main characteristic of Escherichia coli and the presence of the uid chromosomal region of E. coli, coding for this enzyme, were tested on representative members of enteric bacteria. DNA hybridization techniques using uid probes and amplification experiments of uidA gene by the polymerase chain reaction (PCR) confirmed the specificity of uid genes for E. coli and Shigella spp. (i.e., S. boydii, S. dysenteriae, S. flexneri and S. sonnei), independent of the beta-glucuronidase phenotype of bacterial strains. This specificity seemed to be conserved when studies were extended to a wide range of bacteria. It was not possible to distinguish E. coli from Shigella spp. The detection sensitivity using double stranded DNA radiolabeled probes was 3 x 10(4) bacteria and could be brought down to 8 bacteria by PCR. Thus, the uid genes appeared to be ideal candidates for DNA probes technology to detect E. coli-Shigella species.