Purification and Identification of the Cardiac Sarcolemmal Na+Ca2+ Exchanger

The cardiac sarcolemmal Na+/Ca[superscript 2+] exchanger promotes the coupled movement of sodium and calcium across the cell membrane in a reversible, electrogenic manner. The goal of this study was to identify the protein(s) responsible for Na+/Ca[superscript 2+] exchange across cardiac sarcolemma. Purification of the Na+/Ca[superscript 2+] exchanger from canine ventricle was accomplished using the following sequence: 1) isolation of sarcolemma (SL); 2)alkaline extraction of membranes; 3) solubilization with CHAPS; 4) DEAE chromatography; 5) gel filtration HPLC; 6) addition of cholate, salt and phospholipids; 7) wheat germ agglutinin chromatography followed by 8)reconstitution into proteoliposomes by detergent dilution. Specific activity was increased from 5.2 (SL) to 3766 nmol/mg/sec with 100/0 recovery of activity and 0.02% recovery of protein. SDS-PAGE of the purified Na+/Ca[superscript 2+] exchanger preparation under reducing conditions revealed prominent proteins of 75, 120 and 140 kDa. One major protein with a molecular mass of 140 kDa was detected under nonreducing conditions. Polyclonal antibodies against the reconstituted, purified Na+/Ca[superscript 2+] exchanger preparation recognized the three proteins and immunoprecipitated 97.4%+1.3% (n=4) of the Na+/[Ca[superscript 2+] exchange activity from detergent-solubilized sarcolemma. Subsequently, antibodies against the 75, 120and 140 kDa proteins were antigen-purified and found to immunoprecipitate 92%, 91 % and 83%, respectively, of the Na +/Ca[superscript 2+] exchange activity from detergent-solubilized sarcolemma. Furthermore, the antigen-purified antibodies cross-reacted with each of the other two proteins on immunoblots of sarcolemmal protein. Immunoblots with samples prepared from isolated canine ventricular myocytes revealed predominantly the 140 kDa protein and trace amounts of the 75 kDa protein. The 120 kDa protein was not detected. The polyclonal antibodies were subsequently tested for the ability to affect Na +/Ca[superscript 2+] exchange activity manifested by sarcolemmal vesicles. IgG from immune serum stimulated exchange activity 3.5-fold in a concentration-dependent manner (ED[subscript 50] = 0.5 .µg IgG/µg sarcolemmal protein) while IgG from preimmune serum had little or no effect. Curiously, IgG from a rabbit immunized against total sarcolemmal protein stimulated activity nearly as much, but was unable to immunoprecipitate exchange activity. Stimulation appeared to be due to a small increase in V max and a larger decrease in the K[subscript 0.5] for extra vesicular calcium. Molecular cloning of the canine cardiac Na+/ Ca[superscript 2+] exchanger has recently been reported (Nicoll et al., 1990). RNA synthesized from the cDNA clone induced expression of Na +/Ca[superscript 2+] exchange activity when injected into Xenopus oocytes. The cDNA codes for a protein of 970 amino acids (~108 kDa). Antibodies developed against a synthetic peptide based on the deduced amino acid sequence reacted with sarcolemmal proteins of 70, 120 and 160 kDa. Antibodies generated against a partially purified canine cardiac Na+/Ca[superscript 2+] exchanger preparation also recognized proteins of 70, 120 and 160 kDa (Philipson et al., 1988). These antibodies (provided by K. D. Philipson) recognized proteins of 75, 120 and 140 kDa on immunoblots of sarcolemmal protein and the purified Na+/Ca[superscript 2+] exchanger preparation in this laboratory (Ambesi et al., 1991c). These data support the hypothesis that the 70-75, 120 and 140-160 kDa proteins are involved with Na+/Ca[superscript 2+] exchange across cardiac sarcolemma and that the 70-75 and 120 kDa proteins are fragments of the 140-160 kDa protein formed during isolation of the sarcolemmal preparation

Computational framework for the prediction of transcription factor binding sites by multiple data integration

Control of gene expression is essential to the establishment and maintenance of all cell types, and its dysregulation is involved in pathogenesis of several diseases. Accurate computational predictions of transcription factor regulation may thus help in understanding complex diseases, including mental disorders in which dysregulation of neural gene expression is thought to play a key role. However, biological mechanisms underlying the regulation of gene expression are not completely understood, and predictions via bioinformatics tools are typically poorly specific. We developed a bioinformatics workflow for the prediction of transcription factor binding sites from several independent datasets. We show the advantages of integrating information based on evolutionary conservation and gene expression, when tackling the problem of binding site prediction. Consistent results were obtained on a large simulated dataset consisting of 13050 in silico promoter sequences, on a set of 161 human gene promoters for which binding sites are known, and on a smaller set of promoters of Myc target genes. Our computational framework for binding site prediction can integrate multiple sources of data, and its performance was tested on different datasets. Our results show that integrating information from multiple data sources, such as genomic sequence of genes' promoters, conservation over multiple species, and gene expression data, indeed improves the accuracy of computational predictions

Isolation of native plasma membrane H⁺-ATPase (Pma1p) in both the active and basal activation states

The yeast plasma membrane H(+)-ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H(+)-ATPases are autoinhibited

Endostatin and anastellin inhibit distinct aspects of the angiogenic process

<p>Abstract</p> <p>Background</p> <p>Endostatin and anastellin, fragments of collagen type XVIII and fibronectin, respectively, belong to a family of endogenous inhibitors of angiogenesis which inhibit tumor growth and metastasis in a number of mouse models of human cancer. The mechanism of action of these inhibitors is not well understood, but they have great potential usefulness as non-toxic long-term therapy for cancer treatment.</p> <p>Methods</p> <p>In this study, we compare the anti-angiogenic properties of endostatin and anastellin using cell proliferation and transwell migration assays.</p> <p>Results</p> <p>Anastellin but not endostatin completely inhibited human dermal microvessel endothelial cell proliferation in response to serum stimulation. Both anastellin and endostatin additively inhibited endothelial cell migration in response to VEGF. Anastellin but not endostatin lowered basal levels of active ERK.</p> <p>Conclusion</p> <p>These data indicate that anastellin and endostatin exert their anti-angiogenic effects by modulating distinct steps in the angiogenic pathway and suggest that matrix-derived inhibitors of angiogenesis may exhibit higher efficacy when used in combination.</p

Radiation and Thyroid Cancer

Radiation-induced damage is a complex network of interlinked signaling pathways, which may result in apoptosis, cell cycle arrest, DNA repair, and cancer. The development of thyroid cancer in response to radiation, from nuclear catastrophes to chemotherapy, has long been an object of study. A basic overview of the ionizing and non-ionizing radiation effects of the sensitivity of the thyroid gland on radiation and cancer development has been provided. In this review, we focus our attention on experiments in cell cultures exposed to ionizing radiation, ultraviolet light, and proton beams. Studies on the involvement of specific genes, proteins, and lipids are also reported. This review also describes how lipids are regulated in response to the radiation-induced damage and how they are involved in thyroid cancer etiology, invasion, and migration and how they can be used as both diagnostic markers and drug targets

Spaceflight Induced Disorders: Potential Nutritional Countermeasures

Space travel is an extreme experience even for the astronaut who has received extensive basic training in various fields, from aeronautics to engineering, from medicine to physics and biology. Microgravity puts a strain on members of space crews, both physically and mentally: short-term or long-term travel in orbit the International Space Station may have serious repercussions on the human body, which may undergo physiological changes affecting almost all organs and systems, particularly at the muscular, cardiovascular and bone compartments. This review aims to highlight recent studies describing damages of human body induced by the space environment for microgravity, and radiation. All novel conditions, to ally unknown to the Darwinian selection strategies on Earth, to which we should add the psychological stress that astronauts suffer due to the inevitable forced cohabitation in claustrophobic environments, the deprivation from their affections and the need to adapt to a new lifestyle with molecular changes due to the confinement. In this context, significant nutritional deficiencies with consequent molecular mechanism changes in the cells that induce to the onset of physiological and cognitive impairment have been considered

Presynaptic Localization of Sodium/Calcium Exchangers in Neuromuscular Preparations

Calcium ions play a critical role in neurotransmitter release. The cytosolic Ca 2+ concentration ([Ca2+]cyt) at nerve terminals must therefore be carefully controlled. Several different mechanisms, including a plasmalemmal Na/Ca exchanger, are involved in regulating [Ca2+]cyta We employed immunofluorescence microscopy with polyclonal antiserum raised against dog cardiac sarcolemmal Na/Ca exchanger to determine the distribution of the exchanger in vertebrate neuromuscular preparations. Our data indicate that the Na/Ca exchanger is concentrated at the neuromuscular junctions of the rat diaphragm. The exchanger is also present in the nonjunctional sarcolemma, but at a much lower concentration than in the junctional regions. Denervation markedly lowers the concentration of the exchanger in the junctional regions; this implies that the Na/Ca exchanger is concentrated in the presynaptic nerve terminals. In Xenopus laevis nerve and muscle cell cocultures, high concentrations of the exchanger are observed along the neurites as well as at the nerve terminals. The high concentrations of Na/Ca exchanger at presynaptic nerve terminals in vertebrate neuromuscular preparations suggest that the exchanger may participate in the Ca-dependent regulation of neurotransmitter release. The Na/Ca exchanger is also abundant in developing neurites and growth cones, where it may also be important for Ca2+ homeostasis

How to infer gene networks from expression profiles

Inferring, or ‘reverse-engineering', gene networks can be defined as the process of identifying gene interactions from experimental data through computational analysis. Gene expression data from microarrays are typically used for this purpose. Here we compared different reverse-engineering algorithms for which ready-to-use software was available and that had been tested on experimental data sets. We show that reverse-engineering algorithms are indeed able to correctly infer regulatory interactions among genes, at least when one performs perturbation experiments complying with the algorithm requirements. These algorithms are superior to classic clustering algorithms for the purpose of finding regulatory interactions among genes, and, although further improvements are needed, have reached a discreet performance for being practically useful