In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.     

DNA,FISH and complementation studies in ICF syndrome: DNA hypomethylation of repetitive and single copy loci and evidence for a trans acting factor

ICF syndrome (ICFS) is a rare immunodeficiency disorder characterized by instability of the pericentromeric heterochromatin predominantly of chromosomes 1 and 16. DNA methylation studies in two unrelated ICFS patients provide further evidence for a marked hypomethylation of satellite 2 DNA. The ICFS-specific disturbances of chromatin structure take place within the satellite 2 DNA regions, as demonstrated by fluorescence in situ hybridization analysis. Moreover, methylation studies of genomic imprinted loci D15S63, D15S9, and H19 have revealed hypomethylation to different degrees in both patients; this provides evidence for hypomethylation at autosomal single copy loci in ICFS. Cell fusion experiments have revealed a distinct reduction of chromosomal abnormalities in ICFS cells after fusion with normal cells, suggesting that the abnormalities are caused by the loss of function of an as yet unknown trans acting factor. Although it is now clear that wide-spread DNA hypomethylation is a characteristic feature of ICFS, neither the cause and mechanism of hypomethylation nor their relationship to the clinical symptoms is known. We speculate that a phenotypic effect might result from tissue-dependent abnormal gene expression and/or from a possible structural disturbance of DNA domains, which, with respect to the immunodeficiency, partially prevents the normal somatic recombinations in immunologically active cells.     

pH-dependency of iodothyronine metabolism in isolated perfused rat liver.

1. Isolated livers from fed male rats were perfused for 2 h with T4 (L-thyroxine), T3 (L-3,3',5-tri-iodothyronine) or rT3 (L-3,3',5'-tri-iodothyronine) at different pH values (7.1--7.6) in a fully synthetic medium, whereby normal metabolic functions were maintained without addition of rat blood constituents or albumin. 2. T3 output into the medium and net T3 production reached a maximum at a pH of the medium of 7.2 and significantly decreased with alteration of the pH when livers were perfused with T4 as a substrate. 3. However, the net T4 and T3 uptake by the liver, as well as the hepatic T4 and T3 content after perfusion, were not dependent on the pH of the perfusion when livers were offered T4 or T3 as substrates respectively. 4. Determination of intracellular pH by the analysis of the distribution of the weak acid dimethyloxazolidinedione allows the conclusion that the pH optimum of iodothyronine 5'-deiodinase in the intact perfused liver corresponds to the maximum determined in vitro for the membrane-bound enzyme localized in the endoplasmic reticulum. 5. The rapid 5'-deiodination of rT3 to 3,3'-T2 (L-3,3'-di-iodothyronine), the fast disappearance of 3,3'-T2, and the fact that no net rT3 production from T4 could be detected, supports the hypothesis that in rat liver iodothyronine 5'-deiodinase activity seems to predominate over iodothyronine 5-deiodinase activity. 6. Thus the rat liver can be considered in normal physiological situations as an organ forming T3 from T4 and deiodinating rT3 originating from extrahepatic tissues, whereby the cellular iodothyronine 5'-deiodination rate is controlled by the intracellular pH.     

Different sensitivity of diploid and trisomic cells from patients with Down syndrome mosaic after treatment with the trifunctional alkylating agent trenimon

Normal and trisomic cells of patients with Down syndrome mosaic offer the unique possibility to study the effect of an additional chromosome no. 21 against an identical genetic background. Here we show that a significant increase in the frequency of Trenimon induced sister chromatid exchanges (SCEs) and chromosome aberrations can be found in trisomic lymphocytes and fibroblasts as compared to disomic cells. The relative increase was clearly higher for chromosomal breaks than for SCEs.     

Steroid analysis and xenosteroid potentials in two small streams in southwest Germany

The presence of estrogenic substances in thewater of the small streams Körsch (Kö)and Krähenbach (Kr), Southwest Germany, wasdetermined by chemical and biological analysis.Because a large proportion of the Kö waternear its mouth consists of sewage treatmentplant (STPs) effluents, the impact of STPs onlevels of estrogens in surface water is anenvironmental issue of concern. In July 1996,water samples were taken from Kr and Kö(four sites) and tested in the E-Screen assaywith human MCF-7 breast cancer cells. AllKö samples induced estrogen-dependent cellproliferation resulting in 17-estradiolequivalent concentrations (EEQ) between 3.3 and9.7 ng/L while the Kr water showed no effect.In 1998/99 eight samples from Kö (near itsmouth) and nine samples from Kr were collectedand tested in the E-Screen after solid phaseextraction. Some estrogenicity was detectablein three Kr samples but Kö samples had amedian EEQ of 3.1 ng/L (range: 1.2–42 ng/L).GC/MS analysis revealed differences in thelevels of 17-estradiol and estronebetween the two streams. 17-estradiolwas detectable in five Kö samples only (0.7–1.8 ng/L). Estrone was found in the Köfrom 2.5 to 38 ng/L (median: 7.6 ng/L) and inthe Kr between 0.8 and 22 ng/L (median: 1.7 ng/L). Analysis for nine phenolic xenoestrogensrevealed rather low levels for five compoundswhich occurred more frequently and in higherconcentrations in the Kö. After asemi-field exposure of adult male rainbow troutfor 4 weeks in Kö water, plasmavitellogenin levels were significantly highercompared to those levels detected in the sameanimals before exposure.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.