Peter P. Mueller

Review: Activities of IRF-1

Interferon (IFN) regulatory factor-1 (IRF-1) was isolated by virtue of its affinity to specific DNA sequences in the IFN-β promoter that mediate virus responsiveness. IRF-1 was the first factor identified of the IRF family and was most extensively characterized at the molecular level. Also, its physiologic role in host defense against pathogens, tumor prevention, and development of the immune system was investigated in detail. Even though some of the functions first associated with IRF-1 were later found to be mediated in part or predominantly by other activators of the IRF family of transcription factors, IRF-1 has remained a central paradigm in the transcriptional regulation of the IFN response.

Genetic optimization of recombinant glycoprotein production by mammalian cells

Genetically modified mammalian cells are the preferred system for the production of recombinant therapeutic glycoproteins. Other applications include engineering of cell lines for drug screening and cell-based therapies, and the construction of recombinant viruses for gene therapy. This article highlights contemporary core genetic technologies and emerging strategies for genetically engineering mammalian cells for optimal recombinant-protein expression.

Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors.

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture.

Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms

We cloned the GCD5 gene of S. cerevisiae and found it to be identical to KRS1, which encodes lysyl-tRNA synthetase (LysRS). The mutation gcd5-1 changes a conserved residue in the putative lysine-binding domain of LysRS. This leads to a defect in lysine binding and, consequently, to reduced charging of tRNA(Lys). Mutant gcd5-1 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level. GCN4 protein in turn stimulates transcription of GCD5, leading to increased LysRS activity. We propose an autoregulatory model in which uncharged tRNA(Lys) stimulates the protein kinase GCN2, a translational activator of GCN4, and thereby increases transcription of GCD5 and other genes regulated by GCN4.

Mesoporous silica coatings for controlled release of the antibiotic ciprofloxacin from implants

To generate bioactive coatings for medical implants, a novel procedure has been developed using a coating of mesoporous silica for controlled drug delivery. Plain glass slides were used as substrates. The mesoporous coatings were then loaded with the antibacterial drug ciprofloxacin. The drug release kinetics were investigated in a physiological buffered solution. The drug loading capacity of the unmodified mesoporous coatings was low but could be increased nearly ten-fold (to about 2 µg cm−2 of the macroscopic surface) by functionalizing the mesoporous surface with sulfonic acid groups. To achieve a controlled drug release over an extended time period, further coatings were added. Covering the surface of the drug loaded mesoporous silica layer by dip-coating with bis(trimethoxysilyl)hexane resulted in an organosiloxane layer which retarded the release for up to 30 days. By an additional evaporation coating with dioctyltetramethyldisilazane, the release of ciprofloxacin was prolonged for up to 60 days. The biocompatibility of the different coatings was tested in cell culture assays. The presence of the additional silane-derived hydrophobic coatings somewhat reduced the biocompatibility. The antibacterial efficacy of the materials was demonstrated by using clinically relevant biofilm-forming pathogenic bacteria. A test where the sequential release of ciprofloxacin (in 2 days intervals) and the bacterial viability were tested in parallel showed good concordance in the results. The material where a sulfonate-functionalized mesoporous silica layer is loaded with ciprofloxacin and then coated by an organosiloxane layer derived from bis(trimethoxysilyl)hexane showed the best results with regard to antibacterial efficacy and will further be tested in animal experiments.

Histological and molecular evaluation of iron as degradable medical implant material in a murine animal model

A small animal model was established to evaluate the potential of iron as a degradable implant material. After insertion into the tail of mice, the implants gradually degraded over a clinically relevant time period of several months. Histological analysis and gene expression data from whole-genome microarray analyses indicated a limited inflammatory reaction. No evidence of cellular responses to excess iron ions was detected, suggesting that the iron degradation products were metabolically inactive. Iron-rich compounds could be detected in the vicinity of the implant and in individual cells distant from the implantation site. These results demonstrate that the mouse model could be useful for the primary in vivo evaluation of novel implant materials and that iron degradation products can accumulate in diverse organs of the body.

Nucleolar localization and mobility analysis of the NF-κB repressing factor NRF

NF-κB plays a central role in mediating pathogen and cytokine-stimulated gene transcription. NF-κB repressing factor (NRF) has been shown to interact with specific negative regulatory DNA elements (NRE) to mediate transcriptional repression by inhibition of the NF-κB activity at certain promoters. mRNA ablation experiments demonstrated that the trans-acting NRF protein is involved in constitutive but not post-stimulated silencing of IFN-β, IL-8 and iNOS genes by binding to cis-acting NRE elements in their promoters. We have examined the subcellular localization and mobility of the NRF protein. Since neither tagging nor overexpression perturbs NRF localization the GFP-tagged protein was used for detailed localization and mobility studies. Owing to an N-terminal nuclear localization sequence, all NRF fragments that contain this signal show a constitutive nuclear accumulation. C-terminal NRF fragments also localize to the nucleus although no canonical NLS motifs were detected. Full-length NRF is highly enriched in nucleoli and only a small fraction of NRF is found in the nucleoplasm and cytoplasm. This relationship was found to be independent of the protein expression rate. FRAP analysis proved to be a sensitive method to determine protein mobility and made it possible to differentiate between the NRF protein fragments. Nucleolar localization correlated inversely with mobility. The data demonstrate that a series of neighboring fragments in a large central domain of the protein contribute to the strong nucleolar affinity. These properties were not altered by viral infection or LPS treatment. Several sequence motifs for RNA binding were predicted by computer-mediated databank searches. We found that NRF binds to double stranded RNA (dsRNA). This property mapped to several NRF fragments which correlate with the nucleolar affinity domain. Since treatment with actinomycin D releases NRF from nucleoli the identified RNA binding motifs might act as nucleolar localization signals.

The formation of an organic coat and the release of corrosion microparticles from metallic magnesium implants.

Magnesium alloys have been proposed as prospective degradable implant materials. To elucidate the complex interactions between the corroding implants and the tissue, magnesium implants were analyzed in a mouse model and the response was compared to that induced by Ti and by the resorbable polymer polyglactin, respectively. One month after implantation, distinct traces of corrosion were apparent but the magnesium implants were still intact, whereas resorbable polymeric wound suture implants were already fragmented. Analysis of magnesium implants 2weeks after implantation by energy-dispersive X-ray spectroscopy indicated that magnesium, oxygen, calcium and phosphate were present at the implant surface. One month after implantation, the element composition of the outermost layer of the implant was indicative of tissue without detectable levels of magnesium, indicating a protective barrier function of this organic layer. In agreement with this notion, gene expression patterns in the surrounding tissue were highly similar for all implant materials investigated. However, high-resolution imaging using energy-filtered transmission electron microscopy revealed magnesium-containing microparticles in the tissue in the proximity of the implant. The release of such corrosion particles may contribute to the accumulation of calcium phosphate in the nearby tissue and to bone conductive activities of magnesium implants.

Differential fine-tuning of cochlear implant material–cell interactions by femtosecond laser microstructuring

Cochlear implants (CIs) can restore hearing in deaf patients by electrical stimulation of the auditory nerve. To optimize the electrical stimulation, the number of independent channels must be increased by reduction of connective tissue growth on the electrode surface and selective neuronal cell contact. The femtosecond laser microstructuring of the electrode surfaces was performed to investigate the effect of fibroblast growth on the implant material. A cell culture model system was established to evaluate cell-material interactions on these microstructured CI-electrode materials. Fibroblasts were used as a cell culture model for connective tissue formation, and differentiating neuronal-like cells were employed to represent neuronal cells. For nondestructive microscopic examination of living cells on the structured surfaces, the cells were genetically modified to express green fluorescent protein. To investigate the special interaction between the electrode material and the tissue we used electrode material which is originally used for manufacturing CI for human applications, namely platinum (contact material) and silicone carrier material (LSR 30, HCRP 50). Microstructures of various dimensions (groove width 1-10 microm) were generated by using femtosecond laser ablation. The highest fibroblast growth rate was observed on platinum, but cell growth rates on the silicone carrier material were lower. Microstructuring reduced fibroblast cell growth on platinum significantly. On the microstructured silicone, a trend to lower cell growth rates was observed. In addition, microgrooves on platinum surfaces can direct neurite outgrowth parallel to the grooves. The implications of the results are discussed with respect to the design of a microstructured CI surface.

Recombinant glycoprotein product quality in proliferation‐controlled BHK‐21 cells

We analyzed product quality to determine the applicability of proliferation-controlled mammalian cells for recombinant pharmaceutical protein production. Baby hamster kidney (BHK)-21 cells were engineered to express a dicistronic, stabilized, self-selecting growth control system consisting of a beta-estradiol-activatable transcription factor IRF-1 fusion protein. IRF-1 activity led to a reduced growth rate, whereas productivity, protein integrity, and glycosylation pattern of the industrially relevant secreted pharmaceutical glycoprotein erythropoietin remained consistent, showing that this technique has the potential for improving the consistency of high-quality pharmaceutical products and thus warrants further development.