Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

Containment sensors for the determination of L-lactate and glucose

This paper reports some new results on enzyme based silicon containment sensors. For the first time an L-lactate sensor in containment technology is presented. Through optimization of the buffer system the stability of the lactate sensor was enhanced and the linear response of over 10 mM was achieved. The glucose sensor has also been optimized for a large linear measurement range exceeding 30 mM. A two-enzyme chip with glucose and lactate sensor elements which were integrated on one silicon chip is presented. The response behaviour of the two-enzyme chip was very similar to the single chip behaviour. No cross-talking effects could be observed. A fabrication process for mass-production is described.     

RAC protein directs the complete removal of the 3' external transcribed spacer by the Pac1 nuclease

In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein complex (RAC) which contains independent binding sites for each of the transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of the Pac1 nuclease, leading to the complete removal of the 3' ETS. Furthermore, the affinity of RAC protein for mutant 3' ETS correlates closely with in vivo effects on rRNA processing, and changes which disrupt RAC protein binding also inhibit Pac1 nuclease cleavage at the 3' end of the 25S rRNA sequence. The observations indicate that, in the presence of the RAC protein/3' ETS complex, cleavage by the RNase III-like homolog is not restricted to the known intermediate sites but also is directed at the 3' end of the 25S rRNA.     

Identification of functionally important acidic residues in transducin by group-specific labeling

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T alpha was combined with DCCD-treated T beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50% inhibited when DCCD-labeled T alpha was incubated with T beta gamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T alpha was combined with T beta gamma, and when EDC-treated T beta gamma was incubated with T alpha.     

Structural studies on a protein-binding zinc-finger domain of Eos reveal both similarities and differences to classical zinc fingers

The oligomerization domain that is present at the C terminus of Ikaros-family proteins and the protein Trps-1 is important for the proper regulation of developmental processes such as hematopoiesis. Remarkably, this domain is predicted to contain two classical zinc fingers (ZnFs), domains normally associated with the recognition of nucleic acids. The preference for protein binding by these predicted ZnFs is not well-understood. We have used a range of methods to gain insight into the structure of this domain. Circular dichroism, UV-vis, and NMR experiments carried out on the C-terminal domain of Eos (EosC) revealed that the two putative ZnFs (C1 and C2) are separable, i.e., capable of folding independently in the presence of Zn(II). We next determined the structure of EosC2 using NMR spectroscopy, revealing that, although the overall fold of EosC2 is similar to other classical ZnFs, a number of differences exist. For example, the conformation of the C terminus of EosC2 appears to be flexible and may result in a major rearrangement of the zinc ligands. Finally, alanine-scanning mutagenesis was used to identify the residues that are involved in the homo- and hetero-oligomerization of Eos, and these results are discussed in the context of the structure of EosC. These studies provide the first structural insights into how EosC mediates protein-protein interactions and contributes to our understanding of why it does not exhibit high-affinity DNA binding.     

Liver protein profiling in chronic hepatitis C: identification of potential predictive markers for interferon therapy outcome

The current anti-hepatitis C virus (HCV) therapy, based on pegylated-interferon alpha and ribavirin, has limited success rate and is accompanied by several side effects. The aim of this study was to identify protein profiles in pretreatment liver biopsies of HCV patients correlating with the outcome of antiviral therapy. Cytosolic or membrane/organelle-enriched protein extracts from liver biopsies of eight HCV patients were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Overall, this analysis identified 21 proteins whose expression levels correlate with therapy response. These factors are involved in interferon-mediated antiviral activity, stress response, and energy metabolism. Moreover, we found that post-translational modifications of dihydroxyacetone kinase were also associated with therapy outcome. Differential expression of the five best performing markers (STAT1, Mx1, DD4, DAK, and PD-ECGF) was confirmed by immunoblotting assays in an independent group of HCV patients. Finally, we showed that a prediction model based on the expression levels of these markers classifies responder and nonresponder patients with an accuracy of 85.7%. These results provide evidence that the analysis of pretreatment liver protein profiles is valuable for discriminating between responder and nonresponder HCV patients, and may contribute to reduce the number of nonresponder patients exposed to therapy-associated risks.