Impedance and shear wave resonance analysis of ligand-receptor interactions at functionalized surfaces and of cell monolayers.

The present paper scrutinizes the application of impedance spectroscopy and quartz-crystal microbalance (QCM) measurements in the analysis of composite layers of receptor containing lipid bilayers, and their interaction with external ligands or pore-forming peptides. The formation of supramolecular structures and their analysis will be discussed. Impedance measurement allows one to follow the adsorption of proteins on artificial membranes. This method is even more suitable for quantifying changes in membrane conductivity induced by channel peptides incorporated into the lipid membrane. The QCM is another sophisticated method for analyzing ganglioside-lectin and ganglioside-toxin interactions. A critical comparison between both methods will be given. Moreover, we will demonstrate that the QCM method, especially in combination with impedance analysis, is a completely new approach for determining electrical and viscoelastic properties of epithelial and endothelial cell monolayers that form controlled barriers in vivo.

Cyclophilin A, the major intracellular receptor for the immunosuppressant cyclosporin A, maps to chromosome 7p11.2-p13: four pseudogenes map to chromosomes 3, 10, 14, and 18.

Cyclophilin A (CyP-A), the major intracellular receptor for the immunosuppressant cyclosporin A (CsA), is a member of the immunophilin class of proteins, which all possess peptidyl-prolyl cis-trans isomerase activity and, therefore, are believed to be involved in protein folding and/or intracellular protein transport. The CyP-A protein is encoded by a single gene; in addition, 15 pseudogenes have been identified. Recently, specific binding of CyP-A to the human immunodeficiency virus type 1 (HIV-1) gag protein has been reported. Interestingly, this interaction can be inhibited by the immunosuppressant CsA and also by nonimmunosuppressive, CyP-A-binding CsA derivatives, which were also shown to exhibit potent anti-HIV-1 activity. Results thus indicate that CyP-A may have an essential function in HIV-1 replication. Using a panel of somatic rodent-human cell hybrids and PCR technology, we localized the coding cyclophilin A gene (PPIA) on chromosome 7 and four pseudogenes (PPIP2, PPIP3, PPIP4, and PPIP6) on chromosomes 14, 10, 18, and 3, respectively. Using chromosome 7 and chromosome 10 deletion hybrid panels, we were able to localize further the coding gene to the region 7p11.2-p13, as confirmed by fluorescence in situ hybridization analysis, and one pseudogene (PPIP3) to the region 10q11.2-q23. This is the first report on the regional mapping of members of the CyP-A gene family.

Conserved high-affinity NF-kappa B binding site in the interferon regulatory factor-1 promoter is not occupied by NF-kappa B in vivo and is transcriptionally inactive.

The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast cells, despite the simultaneous induction of IRF-1 mRNA and NF-kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.

Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.

Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein.

Proteins from Sendai virus particles and from infected cells were analyzed in a protein-blotting protein-overlay assay for their interaction with in vitro-synthesized, [35S]methionine-labeled viral proteins NP, P, and M. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto polyvinylidene difluoride membranes, and renaturation, the immobilized proteins were found to interact specifically with radiolabeled proteins. NP proteins from virus particles and from infected cells retained 35S-P protein equally well. Conversely, P protein from virus particles and from infected cells retained 35S-NP protein. 35S-M protein was retained mainly by NP protein but also by several cellular proteins. To determine the domains on NP protein required for binding to immobilized P protein, a series of truncated and internally deleted 35S-NP proteins was constructed. The only deletion that did not affect binding resides between residues 426 and 497. The carboxyl-terminal 27 residues (positions 498 to 524) contribute significantly to the binding affinity. Removal of 20 residues (positions 225 to 244) in the hydrophobic middle part of NP protein completely abolished its binding to P protein.