Inhibition of in-stent restenosis by oral copper chelation in porcine coronary arteries.

Stress-induced release of IL-1alpha and fibroblast growth factor-1 is dependent on intracellular copper and is a major driver of neointimal hyperplasia. Therefore, we assessed the effect of tetrathiomolybdate (TTM), a clinically proven copper chelator, on in-stent restenosis. Nine pigs were treated with TTM (5 mg/kg po) twice daily for 2 wk before stent implantation and for 4 wk thereafter, and nine pigs served as controls. In-stent restenosis was assessed by quantitative coronary angiography (QCA), intravascular ultrasound (IVUS), and histomorphometry. Serum ceruloplasmin activity was used as a surrogate marker of copper bioavailability. In TTM-treated animals, ceruloplasmin dropped 70 +/- 10% below baseline levels. Baseline characteristics were comparable in TTM-treated and control animals. At 4-wk follow-up, all parameters relevant to in-stent restenosis were significantly reduced in TTM-treated animals: minimal lumen diameter by QCA was 2.03 +/- 0.57 and 1.47 +/- 0.45 mm in TTM-treated and control animals, respectively (P < 0.05), percent stenosis diameter was 39% less in TTM-treated animals (27.1 +/- 16.6% vs. 44.5 +/- 16.1%, P < 0.05), minimal lumen area by IVUS was 60% larger in TTM-treated animals (4.27 +/- 1.56 vs. 2.67 +/- 1.19 mm(2), P < 0.05), and neointimal volume by histomorphometry was 37% less in TTM-treated animals (34.9 +/- 11.5 vs. 55.2 +/- 19.6 mm(3), P < 0.05). We conclude that systemic copper chelation with a clinically approved chelator significantly inhibits in-stent restenosis.

Dynamics of membrane translocation of phosphatidylserine during apoptosis detected by a monoclonal antibody.

Translocation of phosphatidylserine (PS) to the outer leaflet of the cellular membrane seems to be a key step in apoptosis and cell activation. In this paper, the production and characterization of a monoclonal antibody designated as Mab 1H6 is described which does not show cross reactivity with others anionic phospholipids. It is demonstrated that Mab1H6 can recognize externalized PS at early stages after the induction of apoptosis shown by both flow cytometry and immunofluorescence. Our results show that translocation of PS can be detected as early as 5 min by immunofluorescence and 10 min by flow cytometry after the treatment of cells and a specific dynamics is observed concerning the location and distribution of the staining. These data prove that antibody Mab 1H6 can be used as a specific probe for detection of PS translocation.

The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1.

Although the extravesicular p40 domain of the transmembrane protein, p65 synaptotagmin (Syt) 1, is essential for the non-classical export of the signal peptide-less structure, FGF1, it was not possible to identify a specific intracellular protease responsible for the processing of p65 Syt1. Surprisingly, analysis of the p65 Syt1 coding sequence revealed the presence of two potential alternative ATG codons corresponding to Met103 and Met113 both of which were flanked by Kozak sequences. Indeed, in vitro translation of a Met103Ile but not a Met113Ile p65 Syt1 point mutant exhibited reduced expression of p40 Syt1 and the double p65 Syt1 Met103Ile and Met113Ile point mutant was unable to translate the p40 Syt1 isoform. Since the expression of the p65 Syt1 double point mutant inhibited the stress-induced release of FGF1, it is likely that the alternative translation of the p65 Syt1 transcript at Met103 may be involved in the generation of intracellular p40 Syt1, a critical component of the FGF1 release pathway.

Copper induces the assembly of a multiprotein aggregate implicated in the release of fibroblast growth factor 1 in response to stress.

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu2+-binding protein and Cu2+ is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu2+ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu2+ with similar affinity and to interact in the presence of Cu2+ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu2+ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu2+ with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu2+ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu2+ oxidation may be an essential post-translational intracellular modifier of this process.

Translocation of S100A1(1) calcium binding protein during heart surgery.

Myocardial ischemia during cardiopulmonary bypass terminated by reperfusion generally leads to different degrees of damage of the cardiomyocytes induced by transient cytosolic Ca(2+) overload. Recently, much attention has been paid to the role of heart-specific Ca(2+)-binding proteins in the pathogenesis of myocardial ischemia-reperfusion injury. S100A1 is a heart-specific EF-hand Ca(2+)-binding protein that is directly involved in a variety of Ca(2+)-mediated functions in myocytes. The aim of our study was to investigate the localization and translocation of S100A1 in the human heart under normal (baseline) conditions and after prolonged ischemia and reperfusion of the myocardium. Our data suggest that S100A1 is directly involved in the transient perioperative myocardial damage caused by ischemia during open heart surgery in humans. Given its role in the contractile function of muscle cells, this S100 protein could be an important "intracellular link" in ischemia-reperfusion injury of the heart.

Ultrastructural distribution of the S100A1 Ca2+-binding protein in the human heart.

Impaired calcium homeostasis and altered expression of Ca2+-binding proteins are associated with cardiomyopathies, myocardial hypertrophy, infarction or ischemia. S100A1 protein with its modulatory effect on different target proteins has been proposed as one of potential candidates which could participate in these pathological processes. The exact localization of S100A1 in human heart cells on the ultrastructural level accompanied with biochemical determination of its target proteins may help clarify the role of S100A1 in heart muscle. In the present study the distribution of the S100A1 protein using postembedding (Lowicryl K4M) immunocytochemical method in human heart muscle has been determined quantitatively, relating number of antigen sites to the unit area of a respective structural component. S100A1 antigen sites have been detected in elements of sarcoplasmic reticulum (SR), in myofibrils at all levels of sarcomere and in mitochondria, the density of immunolabeling at Z-lines being about 3 times and at SR more than 5 times higher than immunolabeling of remaining structural components. The presence of the S100A1 in SR and myofibrils may be related to the known target proteins for S100A1 at these sites.

The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import.

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.

Lack of nuclear apoptosis in cardiomyocytes and increased endothelin-1 levels in a rat heart model of myocardial stunning.

OBJECTIVE: Reperfusion injury may affect the cardiac NO and endothelin production. We investigated whether 20 min of total ischemia followed by 40 min of reperfusion can induce apoptosis in a Langendorff model of retrogradely perfused rat hearts (37 degrees C; paced at 300/'), and we attempted to correlate these findings with measured tissue NO and ET-1 levels. METHODS: An apoptosis detection system was utilized which catalytically incorporates fluorescein-12-dUTP at the 3'-OH DNA ends using the principle of the TUNEL assay, with direct visualization of the labeled DNA. ET-1 was measured by radioimmunoassay and NO3/NO2 by ion pairing HPLC on C18 reverse phase columns. RESULTS: None of the postischemic (n = 6) nor of the control perfused (90 min, n = 6) hearts showed signs of apoptosis, while those exposed to longer ischemia (40 min) and reperfusion (2 h) confirmed the presence of apoptotic cells. Myocardial ET-1 concentrations were 4.8 +/- 1.0 versus 8.3 +/- 2.5 pg/100 mg (control vs. ischemic hearts, respectively; mean +/- SD; p < 0.05). Myocardial NO contents showed no differences. CONCLUSION: These data suggest that the time window of apoptosis with detectable DNA fragmentation exceeds 20 min of global total ischemia and 40 min of reperfusion, a model frequently used for inducing myocardial stunning. While NO was not increased in postischemic hearts, increased ET-1 levels indirectly argue for a role of ET-1 as inducer of apoptosis, but only at a later stage of reperfusion.

Polymerization and structure of nucleotide-free actin filaments.

Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and approximately 99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA filaments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from filament ends. By electron microscopy of negatively stained specimens, NFA forms long filaments with a persistence length 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nm resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically significant less mass density between the two strands of NFA filaments. When compared with ADP-actin filaments, the major difference peak of NFA filaments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA filaments for rhodamine phalloidin is lower than that of native actin filaments, due to a lower association rate. This work confirms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of filaments through ATP hydrolysis and phosphate release, and to provide an internal timer for the age of the filament.

Structure, assembly, and dynamics of actin filaments in situ and in vitro.

Actin, though highly conserved, exhibits a myriad of diverse functions, most of which ultimately depend on its intrinsic ability to rapidly assemble and disassemble filamentous structures. Many organisms synthesize multiple actin isoforms even within the same cell. Tissue-specific expression patterns and tight developmental regulation as well as a high conservation across species emphasize the functional importance of isoforms. The detailed knowledge of the structure, assembly, and dynamic behavior of actin provides important pieces in solving the puzzle of how the different isoforms can be so versatile despite their extremely high sequence identity.

Isolation, electron microscopic imaging, and 3-D visualization of native cardiac thin myofilaments.

An increasing number of cardiac diseases are currently pinpointed to reside at the level of the thin myofilaments (e.g., cardiomyopathies, reperfusion injury). Hence the aim of our study was to develop a new method for the isolation of mammalian thin myofilaments suitable for subsequent high-resolution electron microscopic imaging. Native cardiac thin myofilaments were extracted from glycerinated porcine myocardial tissue in the presence of protease inhibitors. Separation of thick and thin myofilaments was achieved by addition of ATP and several centrifugation steps. Negative staining and subsequent conventional and scanning transmission electron microscopy (STEM) of thin myofilaments permitted visualization of molecular details; unlike conventional preparations of thin myofilaments, our method reveals the F-actin moiety and allows direct recognition of thin myofilament-associated porcine cardiac troponin complexes. They appear as "bulges" at regular intervals of approximately 36 nm along the actin filaments. Protein analysis using SDS-polyacrylamide gel electrophoresis revealed that only approximately 20% troponin I was lost during the isolation procedure. In a further step, 3-D helical reconstructions were calculated using STEM dark-field images. These 3-D reconstructions will allow further characterization of molecular details, and they will be useful for directly visualizing molecular alterations related to diseased cardiac thin myofilaments (e.g., reperfusion injury, alterations of Ca2+-mediated tropomyosin switch).

Distinct subcellular localization of calcium binding S100 proteins in human smooth muscle cells and their relocation in response to rises in intracellular calcium.

Changes in cytosolic Ca2+ concentration control a wide range of cellular responses, and intracellular Ca2+-binding proteins are the key molecules to transduce Ca2+ signaling via interactions with different types of target proteins. Among these, S100 Ca2+-binding proteins, characterized by a common structural motif, the EF-hand, have recently attracted major interest due to their cell- and tissue-specific expression pattern and involvement in various pathological processes. The aim of our study was to identify the subcellular localization of S100 proteins in vascular smooth muscle cell lines derived from human aorta and intestinal smooth muscles, and in primary cell cultures derived from arterial smooth muscle tissue under normal conditions and after stimulation of the intracellular Ca2+ concentration. Confocal laser scanning microscopy was used with a specially designed colocalization software. Distinct intracellular localization of S100 proteins was observed: S100A6 was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and S100A4 were found predominantly in the cytosol where they were strongly associated with the sarcoplasmic reticulum and with actin stress fibers. In contrast, S100A2 was located primarily in the cell nucleus. Using a sedimentation assay and subsequent electron microscopy after negative staining, we demonstrated that S100A1 directly interacts with filamentous actin in a Ca2+-dependent manner. After thapsigargin (1 microM) induced increase of the intracellular Ca2+ concentration, specific vesicular structures in the sarcoplasmic reticulum region of the cell were formed with high S100 protein content. In conclusion, we demonstrated a distinct subcellular localization pattern of S100 proteins and their interaction with actin filaments and the sarcoplasmic reticulum in human smooth muscle cells. The specific translocation of S100 proteins after intracellular Ca2+ increase supports the hypothesis that S100 proteins exert several important functions in the regulation of Ca2+ homeostasis in smooth muscle cells.