No abstract

The chicken lysozyme gene is constitutively expressed in macrophages. Transfection of recombinant genes containing different portions of the lysozyme 5′ upstream region revealed the existence of two negative transcriptional elements within 1 kb upstream of the start sites. Both elements placed upstream or downstream of a heterologous promoter‐gene unit repress transcription independent of their orientation and are therefore called silencer elements, although their repressing activities 3′ of the gene are reduced. One silencer (N‐1.0 kb) at position −1 kb consists of the central region of the chicken middle repetitive sequence element CR1 and can be divided into two functional domains. N‐1.0 kb is active in all cell types tested. The other silencer (N‐0.25 kb) at position −0.25 kb shows reduced activity in primary macrophages. Despite their different specificities, the activity of both silencer elements can be influenced similarly. An inverse linear relationship between the transcriptional activity of the tested constructs and the potential inhibition by the silencer elements was found: weak transcription units can be strongly repressed, whereas strong transcription units can be only weakly repressed. Such a mechanism may help to turn off completely a particular gene in situations or tissues where strong positive regulators are inactive.

Expression of the lysozyme gene is a marker for the differentiation of macrophages, lysozyme transcription being gradually increased during maturation. We have analyzed the fine structure and function of two macrophage-specific enhancer elements of the chicken lysozyme gene (E-2.7 kb and E-0.2 kb). Both increase their activities upon LPS induction, both contain multiple binding sites for similar or identical nuclear factors and both can be divided into two functional modules. For the E-0.2 kb enhancer we found a synergistic activity of the modules to be dependent on their distance. Binding sites for nuclear proteins within enhancer E-0.2 kb overlap substantially with the previously identified progesterone/glucocorticoid receptor binding site, which is required for steroid induction of lysozyme transcription in the oviduct.

Individualized cancer therapy, as part of personalized medicine, aims for an optimal treatment with minimal side effects. In chemotherapy, one approach is to screen for the most potent combination of chemotherapeutics (chemotherapeutic scheme) to improve quality of life and achieve a high therapy efficacy. Due to high personnel costs associated with the first generation of manual chemosensitivity assays most health insurance providers do not cover this type of therapy. Also, the outcome of such personalized assays has to be proven first. The fully automated DiagnoSYS platform technology is a system effectively using the potential of chemosensitivity assays. It was shown that with an adequate degree of automation, higher reproducibility was achieved. An ATP/TCA assay was used as the first demonstrator assay. Integrated tissue preparation, based on precise regulation of a Miltenyi Biotec M-or C-Tube, combined with magnetic cell enrichment and depletion via EpCAM and CD90 labeling and luminescence based cell vitality measurements, made it possible to enhance the signal to noise ratio of luminescence readings. The platform technology is based on the internationally accepted SBS-format. Therefore, all processing steps, from tissue preparation to luminescence or fluorescence readings, could rapidly and easily be exchanged, and allows for processing different assay approaches, such as ATP/TCA or prognostic biomarkers as uPA/PAI-1, on the same platform. As a result of modular programming, further processing steps could also be implemented without difficulty. Besides the optimization and standardization of personalized assays, cost reduction, which goes hand in hand with automation, will make the platform affordable for research groups and clinical personnel, amplifying the acceptance of personalized medicine approaches in the future.

No abstract