Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family.

In an effort to identify psoriasis-associated genes, we compared gene expression in normal and psoriatic skin, using differential display RT-PCR technique. Sequence analysis of a 650-bp cDNA fragment (clone 110) that was highly up-regulated in lesional skin revealed homology to a noncoding cDNA (NICE-2). By subsequent cDNA cloning, using RNA from psoriatic skin, we have identified two alternatively spliced mRNA-isoforms (0.5 and 4.4 kb), which differ in composition of their untranslated regions. By sequence comparison, we have mapped the novel gene, named S100A15, to the S100 gene cluster within the epidermal differentiation complex (chromosome 1q21). Analysis of the deduced amino acid sequence revealed a protein of 101 amino acids containing two potential EF-hand motifs with high homology to the S100A7. Northern blot hybridization and semiquantitative RT-PCR analysis confirmed the S100A15 overexpression in psoriasis, showing different levels of expression of the S100A15 mRNA isoforms. In situ hybridization of the S100A15 revealed a markedly increased staining of basal and suprabasal epidermal layers of psoriatic skin compared with healthy tissue. Our data suggest an involvement of the novel S100A15 in epidermal differentiation and inflammation and might therefore be important for the pathogenesis of psoriasis and other diseases.

HAX-1, identified by differential display reverse transcription polymerase chain reaction, is overexpressed in lesional psoriasis.

Psoriasis is a chronic inflammatory disease characterized by epidermal hyperplasia and an inflammatory infiltrate. The normal differentiation from basal to granular keratinocytes with subsequent apoptosis and cornification is disturbed in the akanthotic epidermis. This could be due to both an excess of mitogenic stimuli with hyperproliferation and/or resistance to apoptosis. By mRNA differential display we found HAX-1 to be overexpressed in lesional psoriatic skin. The overexpression of HAX-1 was verified at the mRNA level by Northern blot and in situ hybridization, as well as at the protein level by Western blot and immunohistochemistry. Detection of HAX-1 in mRNA from different tissues showed strong expression in the brain, pancreas, skeletal muscle, and heart. In contrast to primary keratinocytes and melanocytes we found HAX-1 highly expressed in human immortalized keratinocytes (HaCaT) and different melanoma cell lines. In HaCaT cells as a model for psoriatic keratinocytes we found an increased ultraviolet-induced apoptosis after expression of HAX-1 antisense mRNA. In psoriasis, the epidermal differentiation could be disturbed due to the increased expression of HAX-1 and hence a prolonged resistance to terminal differentiation. Antiapoptotic mechanisms are an emerging concept for the understanding of the pathogenesis of this disease possibly leading to clinical applications.

Proteomics: state of the art and its application in cardiovascular research.

The cellular and molecular mechanisms underlying cardiovascular dysfunctions are widely unknown. Basically, pathological changes in the cardiovascular system arise from protein alterations. Proteomics comprises a set of tools for the large-scale study of gene expression at the protein level thereby allowing for the identification of protein alterations responsible for the development and the pathological outcome of diseases including those of the cardiovascular system. In principle these alterations include those of suitable candidates for drug targets and disease biomarkers as well as therapeutic proteins/peptides. Since gene therapy depends on the function of a therapeutic protein encoded by a "therapeutic" gene proteomic analyses also provide the basis for the design and application of gene therapies. Proteomic technologies allow to identify not only proteins but also the nature of their posttranslational modifications thus enabling the elucidation of signal transduction pathways and their deregulation under pathological conditions. The linkage of information about proteome changes with functional consequences lead to the development of functional proteomic studies. Functional proteomic analyses will particularly help to better understand the relations between proteome changes and cardiovascular dysfunctions. The storage and administration of experimental data obtained by the application of proteomic analyses is supported by species- and tissue-specific protein databases and specific software. Publications in this field are reviewed in this paper.

Molecular basis of basal cell carcinoma: analysis of differential gene expression by differential display PCR and expression array.

Basal cell carcinoma (BCC) is the most common tumor in the Caucasian population. Although BCC rarely metastasize and cause death, they are problematic due to their destructive growth and the frequent localization on the face. Until now the knowledge of genes differentially expressed in BCC has been incomplete. To elucidate the complex alterations in BCC-associated gene expression, we took advantage of 2 techniques: the differential display RT-PCR (DD-PCR) and the differential hybridization of cDNA arrays. Using DD-PCR, we showed differential expression of genes known from other biological contexts (e.g., rac, ubiquitin hydrolase), which could now be associated with BCC. In addition, we detected unknown genes possibly contributing to the carcinogenesis of BCC. Of the 588 genes screened by differential hybridization of the Atlas human cDNA array, differences in the expression levels of BCC were observed for 10 genes. These data were obtained with RNA probes pooled from several BCC of different donors and were subsequently confirmed by semiquantitative RT-PCR for Janus protein tyrosine kinase 3 (Jak3), microsomal glutathione S-transferase 1 (GST 12), teratocarcinoma-derived growth factor cripto, glutaredoxin and the monocyte chemoattractant protein 1 (MCP-1) in 10 individual BCC specimens, 2 squamous cell carcinoma (SCC), the cell line HaCaT and cultured normal human keratinocytes (NHK) in comparison to normal skin. These genes are candidates from gene families with known association to tumors, but they have not been reported in the carcinogenesis of BCC yet. In summary, both approaches allow the detection of differentially expressed genes possibly involved in the carcinogenesis of BCC.