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Biomedical subjects

C G dos Remedios

Publications and source records attributed to C G dos Remedios.

At least 19 recordsLinked to original sources
PubMed2005

Cofilin, actin and their complex observed in vivo using fluorescence resonance energy transfer.

Actin is the principal component of microfilaments. Its assembly/disassembly is essential for cell motility, cytokinesis, and a range of other functions. Recent evidence suggests that actin is present in the nucleus where it may be involved in the regulation of gene expression and that cofilin binds actin and can translocate into the nucleus during times of stress. In this report, we combine fluorescence resonance energy transfer and confocal microscopy to analyze the interactions of cofilin and G-actin within the nucleus and cytoplasm. By measuring the rate of photobleaching of fluorescein-labeled actin in the presence and absence of Cy5-labeled cofilin, we determined that almost all G-actin in the nucleus is bound to cofilin, whereas approximately (1/2) is bound in the cytoplasm. Using fluorescence resonance energy transfer imaging techniques we observed that a significant proportion of fluorescein-labeled cofilin in both the nucleus and cytoplasm binds added tetramethylrhodamine-labeled G-actin. Our data suggest there is significantly more cofilin-G-actin complex and less free cofilin in the nucleus than in the cytoplasm.

Actins↗
PubMed2005

A novel biosensor for mercuric ions based on motor proteins.

We explored the potential of contractile proteins, actin and myosin, as biosensors of solutions containing mercuric ions. We demonstrate that the reaction of HgCl2 with myosin rapidly inhibits actin-activated myosin ATPase activity. Mercuric ions inhibit the in vitro analog of contraction, namely the ATP-initiated superprecipitation of the reconstituted actomyosin complex. Hg reduces both the rate and extent of this reaction. Direct observation of the propulsive movement of actin filaments (10 nm in diameter and 1 microm long) in a motility assay driven by a proteolytic fragment of myosin (heavy meromyosin or HMM) is also inhibited by mercuric ions. Thus, we have demonstrated the biochemical, biophysical and nanotechnological basis of what may prove to be a useful nano-device.

Biosensing Techniques↗
PubMed2004

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin.

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.

Actin Cytoskeleton↗
PubMed2003

Apparatus for exposing cell membranes to rapid temperature transients.

We seek to determine whether cell membranes contain sensors that trigger a downstream response to temperature excursions. To do this, we have developed a novel apparatus for exposing a cell membrane to an extremely rapid temperature excursion in the nanosecond range. Cells are plated on a gold surface that is back-heated by a pulsed laser and cooled by conduction of heat into the glass substrate and the liquid medium. Analysis using the heat diffusion equation shows that the greatest temperature rise is localized within a region tens of nanometres thick, suitable for specifically heating a cell membrane without heating the remainder of a cell. We refer to this device as a nanosecond hotplate.

Cell Culture Techniques↗
PubMed2003

Cardiomyocyte apoptosis is associated with increased wall stress in chronic failing left ventricle.

AIMS: We examined cardiomyocyte apoptosis in chronic heart failure (HF) and its possible link to elevated wall stress. METHODS AND RESULTS: Moderate HF was produced in sheep by sequential coronary microembolization. Six months later, the animals remained in a stable compensated haemodynamic state of HF. Apoptosis of cardiomyocytes in left ventricles was verified using Western blotting based on increased expression of: the apoptosis-associated death receptor Fas (1.5-fold); its ligand (FasL, 2.0-fold); and an upstream protease caspase-8 (2.7-fold) as well as its active cleavage peptide, p20 (5.6-fold). Previously we have reported the elevated expression of caspase-3 in the same animal model. The occurrence of apoptotic cardiomyocytes (0.3%) was quantified by TUNEL assays. Haemodynamic analysis indicated that ventricular dilatation, without wall thickening, caused a 2-fold increase in LV wall stress which, together with LV end-diastolic pressure, was linearly correlated with expression of Fas/FasL. Immunohistochemical studies localized FasL and caspase-8 to intercalated discs, suggesting that wall stress may play a role in initiating cardiomyocyte apoptosis. CONCLUSION: Apoptosis of cardiomyocytes in chronic HF is associated with increased wall stress, which may be responsible for the activation of a Fas/FasL and caspase-8 interaction in the region of intercalated discs.

Animals↗
PubMed2003

Actin binding proteins: regulation of cytoskeletal microfilaments.

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions. In 2001, significant advances were made to our understanding of the structure and function of actin monomers. Many of these are likely to help us understand and distinguish between the structural models of actin microfilaments. In particular, 1) the structure of actin was resolved from crystals in the absence of cocrystallized actin binding proteins (ABPs), 2) the prokaryotic ancestral gene of actin was crystallized and its function as a bacterial cytoskeleton was revealed, and 3) the structure of the Arp2/3 complex was described for the first time. In this review we selected several ABPs (ADF/cofilin, profilin, gelsolin, thymosin beta4, DNase I, CapZ, tropomodulin, and Arp2/3) that regulate actin-driven assembly, i.e., movement that is independent of motor proteins. They were chosen because 1) they represent a family of related proteins, 2) they are widely distributed in nature, 3) an atomic structure (or at least a plausible model) is available for each of them, and 4) each is expressed in significant quantities in cells. These ABPs perform the following cellular functions: 1) they maintain the population of unassembled but assembly-ready actin monomers (profilin), 2) they regulate the state of polymerization of filaments (ADF/cofilin, profilin), 3) they bind to and block the growing ends of actin filaments (gelsolin), 4) they nucleate actin assembly (gelsolin, Arp2/3, cofilin), 5) they sever actin filaments (gelsolin, ADF/cofilin), 6) they bind to the sides of actin filaments (gelsolin, Arp2/3), and 7) they cross-link actin filaments (Arp2/3). Some of these ABPs are essential, whereas others may form regulatory ternary complexes. Some play crucial roles in human disorders, and for all of them, there are good reasons why investigations into their structures and functions should continue.

Actins↗
PubMed2001

Immunophenotyping of leukemias using a cluster of differentiation antibody microarray.

Different leukemias express on their plasma membranes particular subsets of the 247 defined cluster of differentiation (CD) antigens, which may resemble those of precursor cells along the lineages of differentiation to mature myeloid and lymphoid leukocytes. The extent of use of CD antigen expression (immunophenotyping) for identification of leukemias has been constrained by the technique used, flow cytometry, which commonly specifies only three CD antigens in any one assay. Currently, leukemias and lymphomas are diagnosed using a combination of morphology, immunophenotype, cytochemistry, and karyotype. We have developed a rapid, simple procedure, which enables concurrent determination of 50 or more CD antigens on leukocytes or leukemia cells in a single analysis using a microarray of antibodies. A suspension of cells is applied to the array, and cells only bind to antibody dots for which they express the corresponding CD antigen. For patients with significantly raised leukocyte counts, the resulting dot pattern then represents the immunophenotype of those cells. For patients at earlier stages of disease, the diagnosis depends on recognition of dot patterns distinct from the background of normal leukocytes. Distinctive and reproducible dot patterns have been obtained for normal peripheral blood leukocytes, chronic lymphocytic leukemia (CLL), hairy cell leukemia, mantle cell lymphoma, acute myeloid leukemia, and T-cell acute lymphoblastic leukemia. The consensus pattern for CD antigen expression found on CLL cells taken from 20 patients in descending order of cells bound was CD44, HLA-DR, CD37, CD19, CD20, CD5, CD52, CD45RA, CD22, CD24, CD45, CD23, CD21, CD71, CD11c, and CD9. The antigens that provided the best discrimination between CLL and normal peripheral blood leukocytes were CD19, CD20, CD21, CD22, CD23, CD24, CD25, and CD37. Results obtained for the expression of 48 CD antigens from the microarray compared well with flow cytometry. The microarray enables extensive immunophenotyping, and the intact cells captured on antibody dots can be further characterized using soluble, fluorescently labeled antibodies.

Acute Disease↗
PubMed2001

Heart failure and apoptosis: electrophoretic methods support data from micro- and macro-arrays. A critical review of genomics and proteomics.

The multiple causes and multiple consequences of mammalian heart failure make it an attractive proposition for analysis using gene array technology, especially where the failure is idiopathic in nature. However, gene arrays also hold potential artefacts, particularly when gene expression levels are low, and where changes in expression levels are modest. Also, at present, the number of genes available on arrays is not large enough to prevent potential sampling deficiencies. Thus, it may not be wise to place too much reliance on quantitative interpretations of gene array data. Also, recently doubts were raised about the qualitative reliability of array genes. Electrophoretic methods are slow, cumbersome and complex but they can provide confirmation that the trends and numbers arising from the new gene arrays are reliable. In this overview, we compare gene array data with data from protein activity assays such as zymograms, Western blots, two-dimensional electrophoresis, and immunohistochemistry. Similar or complementary data from the same heart tissues analyzed by either microarrays or macroarrays can be reassuring to those interested in reliable molecular analyses of normal and failing hearts. Similar principles will apply to other tissues and cells.

Animals↗
PubMed2001

Nuclear membrane proteins in failing human dilated cardiomyopathy.

Emerin (34 kDa) is a 254 amino acid protein located on the cytoplasmic surface of the inner nuclear membrane in cardiac muscle. It interacts with nuclear lamins and nuclear actin. Emerin is usually completely absent in Emery-Dreifuss muscular dystrophy, a condition that also manifests in the heart. Nuclear lamins are specialized nuclear proteins that line the inner nuclear membrane. Two isoforms, lamin A and C, differ in their C-terminal amino acids. Both are important in apoptosis and are degraded by caspase enzymes. Mutations in the rod domain of the lamin A/C gene are known to cause dilated cardiomyopathy (DCM) (Fatkin et al. New Engl. J. Med. 1999, 351, 1715-1724). We have used Western blots to detect emerin and lamin A/C in left ventricles from both nondiseased and failing DCM samples. The lamins form dimers, however it is not known if they are homodimers and / or heterodimers. In this report we compare and quantify expression levels of emerin from samples of left ventricles from ten failing DCM patients and five nondiseased (donor) hearts. We observed three lamin bands that suggest the expression of the three isoforms of the A-type lamin gene, lamin A, lamin C and lamin C2 (Ye et al. Subcellular Biochem. 1998, 31, 587-610). Preliminary data show that both lamin isoforms and emerin are present.

Blotting, Western↗
PubMed2001

The affinity of chick cofilin for actin increases when actin is complexed with DNase I: formation of a cofilin-actin-DNase I ternary complex.

Cofilin, an actin-binding protein, regulates the rate, nature and extent of assembly of the actin cytoskeleton. Native Phast gels show that the addition of cofilin to an actin-DNase I complex (74 kDa) results in the formation of a ternary complex of 94 kDa indicating an equimolar stoichiometry in the ternary complex. Furthermore, native gels show that the addition of cofilin to a solution containing free actin and actin-DNase I and run at pH 8.3 results in cofilin complexing preferentially to the actin-DNase I complex. Conversely, the addition of DNase I to a solution containing an actin-cofilin complex and free actin results in the preferential binding of DNase I to the actin-cofilin complex. These results show that the affinity of cofilin for actin can be increased when actin forms binary complexes. When native gels were run at pH 6.8 the affinity of cofilin for monomeric actin was greater than for the actin-DNase I complex indicating that the cofilin-actin interaction can be regulated by changes in pH. The addition of cofilin to actin resulted in the polymerisation of actin at pH 6.8 whereas at alkaline pH a stable cofilin-actin binary complex could be formed. The biological implications are discussed.

Actin Depolymerizing Factors↗
PubMed2001

Apoptosis of ventricular and atrial myocytes from pacing-induced canine heart failure.

OBJECTIVE: Rapid ventricular pacing in dogs results in a low output cardiomyopathic state similar to idiopathic dilated cardiomyopathy in man. Cell death by apoptosis may play an important role in the loss of cardiac function. This study investigates the molecular pathways involved in the regulation of apoptosis in dogs with pacing-induced heart failure. METHODS: Apoptosis was identified by terminal transferase nick end-labelling (TUNEL) in the ventricles and atria of dog hearts affected by rapid-ventricular pacing. Western blots were used to determine expression of the components involved in the initiation (Fas, Fas-Ligand, FADD), regulation (Bcl-2, Bax) and execution (caspase-2 and caspase-3) of apoptosis. RESULTS: Pacing-induced heart failure resulted in a significant increase in the number of ventricular and atrial myocyte nuclei undergoing apoptosis as measured by TUNEL. Compared to the samples from control hearts (n=6) the expression of Bcl-2, an inhibitor of apoptosis, was significantly reduced in ventricles from five dogs with pacing-induced heart failure. No change in the expression of the apoptotic inducer Bax was detected. Fas and FADD were significantly elevated in all paced ventricles, and Fas-L was only detected in the paced hearts. Both caspase-2 and caspase-3 were elevated following ventricular pacing. CONCLUSIONS: We have identified components of the signalling pathways along which apoptosis proceeds following the induction of heart failure in dogs. Apoptosis was also detected in the atria raising the possibility that, like human dilated cardiomyopathy, the molecular changes are global.

Animals↗
PubMed2000

Determination of P2X1alpha-sarcoglycan (adhalin) expression levels in failing human dilated cardiomyopathic left ventricles.

This study is concerned with the molecular basis of human idiopathic dilated cardiomyopathy (DCM). This disorder affects the entire heart including both atria and ventricles. It is characterized by a progressive dilatation of the ventricles and loss of contractile power that results in an impaired cardiac output. Changes in cellular levels of dystrophin have been reported in patients with muscular dystrophies (Beckers and Duchenne) which manifest as DCM. However, previous studies using Western blots dos Remedios et al., Electrophoresis 1996, 17, 235-238) of samples of left ventricles from DCM patients showed no abnormalities in dystrophin content. P2X receptors are ATP-gated cation channels located in the sarcolemma. They are upregulated by a factor of about two in the atria of DCM patients compared with nondiseased control samples. A dystrophin-associated protein, alpha-sarcoglycan, has recently been shown to be an ecto-ATPase (an extracellular ATPase) capable of regulating ATP concentrations in the space between the cardiomyocytes. In this report we examine the relationship between changes in P2X1 receptors in left ventricle samples from DCM patients and the concentration of alpha-sarcoglycan. We found no evidence for upregulation of P2X1 receptors nor was the expression of alpha-sarcoglycan significantly altered.

Cardiomyopathy, Dilated↗
PubMed2000

The role of ATP, ADP and divalent cations in the formation of binary and ternary complexes of actin, cofilin and DNase I.

Actin is the major cytoskeletal protein of virtually all eukaryotic cells. Actin assembly/disassembly is involved in a variety of cellular processes and actin-binding proteins are essential in regulation of the pool of actin monomers. Cofilin and DNase I are actin-binding proteins, which form both binary (actin-DNase 1, cofilin-actin) and ternary (cofilin-actin-DNase I) complexes with actin. Here we use native gel electrophoresis to examine the roles of ATP, ADP, Ca2+ and Mg2+ in the formation of these complexes as well as on the ability of actin to self-assemble. Conditions which favour actin polymerisation are: ATP (no Me2+) > or = ADP (no Me2+) > ADP-Ca2+ = ADP-Mg2+ > ATP-Mg2+ > ATP-Ca2+. Preferential conditions for the formation of the binary actin-cofilin complex are: ADP-Mg2+ > or = ADP-Ca2+ >> ATP-Ca2+ approximately equals ATP-Mg2+ approximately equals ADP-No Me2+ approximately equals ATP-No Me2+. Actin forms a very tight complex with DNase I in the order: ATP-Ca2+ > or = ATP-Mg2+ approximately equals ADP-Mg2+ approximately equals ADP-Ca2+ > or = ADP-(no Me2+) > ATP-(no Me2+). Effectively, the complex does not form in the presence of ATP and the absence of free Me2+. Finally, the conditions which favour the formation of a ternary complex of cofilin-actin-DNase I resemble the actin-DNase I, namely: ATP-Ca2+ approximately equals ADP-Ca2+ approximately equals ADP-Mg2+ approximately equals ATPMg2+ ADP (no Me2+) > ATP-(no Me2+).

Acrylic Resins↗
PubMed1999

Elevated DNase activity and caspase expression in association with apoptosis in failing ischemic sheep left ventricles.

We examined the DNase activity as well as the expression of the caspases in left ventricles of failing sheep hearts and compared it with samples from normal controls. The sheep model of chronic ischemic heart failure was developed by intracoronary microembolisation. Moderate heart failure was defined as a decrease of left ventricular (LV) ejection fraction (EF) to 35% or less and was stable for four weeks after the last embolisation. The failing hearts were harvested from these animals six months later. Bovine pancreas DNase I was used as a standard for the DNase zymograms. In normal sheep left ventricles, a low level of the DNase I-like activity was detected (1.16 +/- 0.17 pg/100 microg protein, n = 20), whereas it was elevated (3.64 +/- 0.49 pg/100 microg, n = 32, p < 0.001) in the ischemic failing LV samples. The identity of the DNase in sheep is yet to be fully characterised. However, we also detected the expression of caspase-2 and caspase-3 by Western blotting. There was a 2.2-fold increased expression of caspase-2 p < 0.001 = and a 2.6-fold increased expression of caspase-3 p < 0.001) in failing left ventricles compared to normal samples.

Animals↗
PubMed1999

Electrophoretic monitoring of pollutants: effect of cations and organic compounds on protein interactions monitored by native gel electrophoresis.

We describe how the interaction between actin and its protein ligands can be used to evaluate the presence of certain metal (Cd, Cu, Hg, Zn) ions and organic compounds (2,4-dioxin or Picloram) which are common components of environmental pollution. The assay detects the high-affinity binding of actin to actin-binding proteins (ABPs), cofilin or DNase I. The actin-ABP complex was analyzed using native polyacrylamide gel electrophoresis and quantified by scanning densitometry. These proteins are widely distributed in animals and plant cells. The assay involves allowing the proteins to form an actin-ABP complex into which increasing amounts of pollutants are titrated. Thus, the assay directly tests for inhibition of protein-protein interaction. It is sensitive to common pollutants using concentration ranges over which they are known to exert a biological toxicity. A convenient feature of the assay is the fact that all the proteins can be stored in freeze-dried form, and can be purchased commercially. We suggest that if this molecular assay is sensitive to a wide range of environmental pollutants, it could be used as a rapid and convenient assay of the environment in combination with currently available tests.

Actin Depolymerizing Factors↗