The amphoterin (HMGB1)/receptor for advanced glycation end products (RAGE) pair modulates myoblast proliferation, apoptosis, adhesiveness, migration, and invasiveness. Functional inactivation of RAGE in L6 myoblasts results in tumor formation in vivo.

We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGEΔcyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mocktransfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21Waf1, and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGEΔcyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGEΔcyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation

S-100 protein, but not calmodulin, binds to the glial fibrillary acidic protein and inhibits its polymerization in a Ca(2+)-dependent manner.

S-100 protein, a Ca(2+)-binding protein of the EF-hand type, interacts with the glial fibrillary acidic protein (GFAP) in a Ca(2+)-dependent manner. The binding of S-100 protein to GFAP was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and cross-linking experiments using the bifunctional cross-linker, disuccinimidyl suberate. The binding affinity was observed to be in the nanomolar range with a stoichiometry of 2 mol of GFAP/mol of S-100 protein (dimer). S-100 protein was found to inhibit the polymerization of GFAP in a dose- and Ca(2+)-dependent manner, with a half-maximal effect at an S-100 protein/GFAP molar ratio of 0.2 and maximal effect at a molar ratio of 0.5. Identical results were obtained irrespective of whether the unfractionated bovine brain S-100 protein mixture (S-100a plus S-100b), S-100ao, S-100a, or S-100b was used. S-100 protein was observed to be maximally effective as an inhibitor of GFAP polymerization at approximately 3 microM free Ca2+. Calmodulin neither bound to GFAP nor inhibited its polymerization. Altogether, the present results suggest that S-100 protein might be involved in the regulation of the state of assembly of glial filaments by binding to and sequestering unpolymerized GFAP

Two novel brain proteins, CaBP33 and CaBP37, are calcium-dependent phospholipid- and membrane-binding proteins

AbstractTwo acidic Ca2+ -binding proteins (CaBP33 and CaBP37) purified from bovine brain have been characterized in terms of immunological properties, heat-sensitivity, electrophoretic mobility, and Ca2+-dependent binding to negatively charged phospholipids and to brain membranes. They were induced to bind to membranes by homogenization of brain tissue in the presence of CaCl2. The membrane-bound CaBP33/CaBP37 mixture resisted extraction with detergents and was solubilized with high concentrations of EGTA/KC1. However, apparent Ca2+-independent binding of the two proteins to membranes seemed to occur as well. This latter fraction of membrane-bound CaBP33 and CaBP37 could be solubilized with Triton X-100, indicating that brain membranes normally contain the two proteins as intrinsic components

S100B Increases Proliferation in PC12 Neuronal Cells and Reduces Their Responsiveness to Nerve Growth Factor via Akt Activation

S100B is a Ca2+-modulated protein of the EF-hand type expressed in high abundance in a restricted set of cell types including certain neuronal populations. S100B has been suggested to participate in cell cycle progression, and S100B levels are high in tumor cells, compared with normal parental cells. We expressed S100B in the neuronal cell line PC12, which normally does not express the protein, by the Tet-Off technique, and found the following: (i) proliferation was higher in S100B+ PC12 cells than in S100B- PC12 cells; (ii) nerve growth factor (NGF), which decreased the proliferation of S100B- PC12 cells, was less effective in the case of S100B+ PC12 cells; (iii) expression of S100B made PC12 cells resistant to the differentiating effect of NGF; and (iv) interruption of S100B expression did not result in an immediate restoration of PC12 cell sensitivity to the differentiating effect of NGF. Expression of S100B in PC12 cells resulted in activation of Akt; increased levels of p21WAF1, an inhibitor of cyclin-dependent kinase (cdk) 2 and a positive regulator of cdk4; increased p21WAF1-cyclin D1 complex formation; and increased phosphorylation of the retinoblastoma suppressor protein, Rb. These S100B-induced effects, as well as the reduced ability of S100B+ PC12 cells to respond to NGF, were dependent on Akt activation because they were remarkably reduced or abrogated in the presence of LY294002, an inhibitor of the Akt upstream kinase phosphatidylinositol 3-kinase. Thus, S100B might promote cell proliferation and interfere with NGF-induced PC12 cell differentiation by stimulating a p21WAF1/cyclin D1/cdk4/Rb/E2F pathway in an Akt-mediated manner

A Fuzzy Logic C-Means Clustering Algorithm to Enhance Microcalcifications Clusters in Digital Mammograms

The detection of microcalcifications is a hard task, since they are quite small and often poorly contrasted against the background of images. The Computer Aided Detection (CAD) systems could be very useful for breast cancer control. In this paper, we report a method to enhance microcalcifications cluster in digital mammograms. A Fuzzy Logic clustering algorithm with a set of features is used for clustering microcalcifications. The method described was tested on simulated clusters of microcalcifications, so that the location of the cluster within the breast and the exact number of microcalcifications is known

Characterization of mammalian heart annexins with special reference to CaBP33 (annexin V)

AbstractPorcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry. Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA-resistant, Triton X-100-soluble fraction from cardiac membranes. Including Ca2+ in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca2+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 μM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane-bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA-resistant and Triton X-100-extractable, and one Triton X-100-resistant and EGTA-extractable. Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane-related processes

S100 Calcium Binding Proteins and Ion Channels

S100 Ca2+-binding proteins have been associated with a multitude of intracellular Ca2+-dependent functions including regulation of the cell cycle, cell differentiation, cell motility and apoptosis, modulation of membrane–cytoskeletal interactions, transduction of intracellular Ca2+ signals, and in mediating learning and memory. S100 proteins are fine tuned to read the intracellular free Ca2+ concentration and affect protein phosphorylation, which makes them candidates to modulate certain ion channels and neuronal electrical behavior. Certain S100s are secreted from cells and are found in extracellular fluids where they exert unique extracellular functions. In addition to their neurotrophic activity, some S100 proteins modulate neuronal electrical discharge activity and appear to act directly on ion channels. The first reports regarding these effects suggested S100-mediated alterations in Ca2+ fluxes, K+ currents, and neuronal discharge activity. Recent reports revealed direct and indirect interactions with Ca2+, K+, Cl−, and ligand activated channels. This review focuses on studies of the physical and functional interactions of S100 proteins and ion channels

Automatic detection of lung nodules in CT datasets based on stable 3D mass–spring models

We propose a computer-aided detection (CAD) system which can detect small-sized (from 3 mm) pulmonary nodules in spiral CT scans. A pulmonary nodule is a small lesion in the lungs, round-shaped (parenchymal nodule) or worm-shaped (juxtapleural nodule). Both kinds of lesions have a radio-density greater than lung parenchyma, thus appearing white on the images. Lung nodules might indicate a lung cancer and their early stage detection arguably improves the patient survival rate. CT is considered to be the most accurate imaging modality for nodule detection. However, the large amount of data per examination makes the full analysis difficult, leading to omission of nodules by the radiologist. We developed an advanced computerized method for the automatic detection of internal and juxtapleural nodules on low-dose and thin-slice lung CT scan. This method consists of an initial selection of nodule candidates list, the segmentation of each candidate nodule and the classification of the features computed for each segmented nodule candidate.The presented CAD system is aimed to reduce the number of omissions and to decrease the radiologist scan examination time. Our system locates with the same scheme both internal and juxtapleural nodules. For a correct volume segmentation of the lung parenchyma, the system uses a Region Growing (RG) algorithm and an opening process for including the juxtapleural nodules. The segmentation and the extraction of the suspected nodular lesions from CT images by a lung CAD system constitutes a hard task. In order to solve this key problem, we use a new Stable 3D Mass–Spring Model (MSM) combined with a spline curves reconstruction process. Our model represents concurrently the characteristic gray value range, the directed contour information as well as shape knowledge, which leads to a much more robust and efficient segmentation process. For distinguishing the real nodules among nodule candidates, an additional classification step is applied; furthermore, a neural network is applied to reduce the false positives (FPs) after a double-threshold cut. The system performance was tested on a set of 84 scans made available by the Lung Image Database Consortium (LIDC) annotated by four expert radiologists. The detection rate of the system is 97% with 6.1 FPs/CT. A reduction to 2.5 FPs/CT is achieved at 88% sensitivity. We presented a new 3D segmentation technique for lung nodules in CT datasets, using deformable MSMs. The result is a efficient segmentation process able to converge, identifying the shape of the generic ROI, after a few iterations. Our suitable results show that the use of the 3D AC model and the feature analysis based FPs reduction process constitutes an accurate approach to the segmentation and the classification of lung nodules

S100B Protein, A Damage-Associated Molecular Pattern Protein in the Brain and Heart, and Beyond

S100B belongs to a multigenic family of Ca2+-binding proteins of the EF-hand type and is expressed in high abundance in the brain. S100B interacts with target proteins within cells thereby altering their functions once secreted/released with the multiligand receptor RAGE. As an intracellular regulator, S100B affects protein phosphorylation, energy metabolism, the dynamics of cytoskeleton constituents (and hence, of cell shape and migration), Ca2+ homeostasis, and cell proliferation and differentiation. As an extracellular signal, at low, physiological concentrations, S100B protects neurons against apoptosis, stimulates neurite outgrowth and astrocyte proliferation, and negatively regulates astrocytic and microglial responses to neurotoxic agents, while at high doses S100B causes neuronal death and exhibits properties of a damage-associated molecular pattern protein. S100B also exerts effects outside the brain; as an intracellular regulator, S100B inhibits the postinfarction hypertrophic response in cardiomyocytes, while as an extracellular signal, (high) S100B causes cardiomyocyte death, activates endothelial cells, and stimulates vascular smooth muscle cell proliferation