%0 Journal Article %~ Pubmed %A Li, Amy %A Ponten, Fredrik %A dos Remedios, Cristobal G %T The interactome of LIM domain proteins: the contributions of LIM domain proteins to heart failure and heart development. %B Proteomics %D 2012 %V 12 %N 2 %P 203-25 %@ 1615-9861 %X LIM domain proteins all contain at least one double zinc-finger motif. They belong to a large family and here we review those expressed mainly in mammalian hearts, but particularly in cardiomyocytes. These proteins contain between one and five LIM domains and usually these proteins contain other domains that have specific functions such as actin-binding, kinases and nuclear translocation motifs. While several recent reviews have summarised the importance of individual LIM domain proteins, this is the first review of its kind to cover all LIMs associated with the heart. Here we examine 33 LIM proteins (including three that bind to, but do not themselves contain, LIM domains) that are implicated in either the development of the heart, heart disorders and failure, or both. Our analysis is consistent with the view that cardiac LIM domain proteins form multiple extensive networks of multi-protein complexes within the myocardium. This multiplicity of binding partners probably protects the heart as it is challenged to maintain cardiac output, until the imbalance reaches a turning point that results in failure. We believe that the complexity of LIM interactions is properly described by the term LIM interactome. %Z FOR Codes: 110106 %0 Journal Article %~ Pubmed %A van Dijk, Sabine J %A Paalberends, E Rosalie %A Najafi, Aref %A Michels, Michelle %A Sadayappan, Sakthivel %A Carrier, Lucie %A Boontje, Nicky M %A Kuster, Diederik W D %A van Slegtenhorst, Marjon %A Dooijes, Dennis %A dos Remedios, Cris %A ten Cate, Folkert J %A Stienen, Ger J M %A van der Velden, Jolanda %T Contractile dysfunction irrespective of the mutant protein in human hypertrophic cardiomyopathy with normal systolic function. %B %D 2012 %V 5 %N 1 %P 36-46 %@ 1941-3297 %X Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. %Z FOR Codes: 111602 60199 110201 %0 Journal Article %A Wolfe, James Erle %A Ishiwata, Shin?ichi %A Braet, Filip %A Whan, Renee %A Su, Yingying %A Lal, Sean %A dos Remedios, Cristobal %T SPontaneous Oscillatory Contraction (SPOC): auto-oscillations observed in striated muscle at partial activation %B Biophysical Reviews %D 2011 %C Germany %I Springer %V 3 %N 2 %P 53-62 %@ 1867-2450 %X %Z FOR Codes: 111602 %0 Journal Article %~ Pubmed %A Maeder, Micha T %A Khammy, Ouda %A dos Remedios, Cris %A Kaye, David M %T Myocardial and systemic iron depletion in heart failure implications for anemia accompanying heart failure. %B Journal of the American College of Cardiology %D 2011 %V 58 %N 5 %P 474-80 %@ 1558-3597 %X This study sought to determine the potential pathophysiological link between anemia and disease severity, and adverse outcome in heart failure (HF). %Z FOR Codes: 110201 %0 Journal Article %~ Pubmed %A Polden, Julie %A McManus, Ciara A %A Dos Remedios, Cris %A Dunn, Michael J %T A 2-D gel reference map of the basic human heart proteome. %B Proteomics %D 2011 %V 11 %N 17 %P 3582-6 %@ 1615-9861 %X We have undertaken the identification of basic proteins (pH 6-11) of the human heart using 2-DE. Tissue from the left ventricle of human heart was lysed and proteins were separated in the first dimension on pH 6-11 IPG strips using paper-bridge loading followed by separation on 12% SDS polyacrylamide gels in the second dimension. Proteins were then identified by mass spectrometry and analysed using Proline, a proteomic data analysis platform that was developed in-house. The proteome map contains 176 identified spots with 151 unique proteins and has been made available as part of the UCD-2DPAGE database at http://proteomics-portal.ucd.ie:8082. The associated mass spectrometry data have been submitted to PRIDE (Accession number ?10098). This reference map, and the other heart reference maps available through the UCD-2DPAGE database, will aid further proteomic studies of heart diseases such as dilated cardiomyopathy and ischaemic heart disease. %Z FOR Codes: 60109 %0 Journal Article %A Hirsh, S. L. %A Nosworthy, N. J. %A Kondyurin, A. %A dos Remedios, Cristobal %A McKenzie, D. R. %A Bilek, M. M. M. %T Linker-free covalent thermophilic β-glucosidase functionalized polymeric surfaces %B Journal of Materials Chemistry %D 2011 %C United Kingdom %I Royal Society of Chemistry %V 21 %N 44 %P 17832?17841 %@ 0959-9428 %X %Z FOR Codes: 60109 %0 Journal Article %~ Pubmed %A Foreman, Amy L %A Phillips, Leo %A Kanellis, Vangelis G %A Hammoudeh, Daoud %A Naumann, Christoph %A Wong, Henri %A Chisari, Robert %A Hibbert, D Brynn %A Lee, Garry S H %A Patra, Ronald %A Julli, Moreno %A Chapman, John %A Cooke, A Roger %A Dos Remedios, Cristobal G %T A DNA-based assay for toxic chemicals in wastewater. %B Environmental toxicology and chemistry / SETAC %D 2011 %V 30 %N 8 %P 1810-8 %@ 1552-8618 %X Chemical toxicants, particularly metal ions, are a major contaminant in global waterways. Live-organism bioassays used to monitor chemical toxicants commonly involve measurements of activity or survival of a freshwater cladoceran (Ceriodaphnia dubia) or light emitted by the marine bacterium Vibrio fischeri, used in the commercial Microtox?? bioassay. Here we describe a novel molecule-based assay system employing DNA as the chemical biosensor. Metals bind to DNA, causing structural changes that expel a bound (intercalated) fluorescent reporter dye. Analyses of test data using 48 wastewater samples potentially contaminated by metal ions show that the DNA-dye assay results correlate with those from C. dubia and Microtox bioassays. All three assays exhibit additive, antagonistic, and synergistic responses that cannot be predicted by knowing individual metal concentrations. Analyses of metals in these samples imply the presence of chemical toxicants other than metal ions. The DNA-dye assay is robust, has a 12-month shelf life, and is only slightly affected by sample pH in the range 4 to 9. The assay is completed in a matter of minutes, and its portability makes it well suited as a screening assay for use in the field. We conclude that the DNA-dye test is a surrogate bioassay suitable for screening chemical toxicity. %Z FOR Codes: 1004 %0 Journal Article %~ Pubmed %A Bilek, Marcela M M %A Bax, Daniel V %A Kondyurin, Alexey %A Yin, Yongbai %A Nosworthy, Neil J %A Fisher, Keith %A Waterhouse, Anna %A Weiss, Anthony S %A Dos Remedios, Cristobal G %A McKenzie, David R %T Free radical functionalization of surfaces to prevent adverse responses to biomedical devices. %B Proceedings of the National Academy of Sciences %D 2011 %V 108 %N 35 %P 14405-10 %@ 1091-6490 %X Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines. %Z FOR Codes: 30401 60106 %0 Journal Article %A Morales, Marcelo M. %A dos Remedios, Cristobal %T Biophysical educational experiment: science and goodwill in Latin America and Africa %B Biophysical Reviews %D 2011 %C Germany %I Springer %V 3 %N %P 101?106 %@ 1867-2469 %X %Z FOR Codes: 130209 %0 Journal Article %~ Pubmed %A Changsirivathanathamrong, Dechaboon %A Wang, Yutang %A Rajbhandari, Dorrilyn %A Maghzal, Ghassan J %A Mak, Wendy M %A Woolfe, Clive %A Duflou, Johan %A Gebski, Val %A Dos Remedios, Cris G %A Celermajer, David S %A Stocker, Roland %T Tryptophan metabolism to kynurenine is a potential novel contributor to hypotension in human sepsis. %B Critical Care Medicine %D 2011 %V %N %P %@ 1530-0293 %X OBJECTIVES:: To determine whether tryptophan metabolism to kynurenine contributes to the direct regulation of vascular tone in human septic shock. BACKGROUND:: Indoleamine 2,3-dioxygenase 1 is an inducible enzyme that converts tryptophan to kynurenine and shares functional similarities with inducible nitric oxide synthase. Recently, kynurenine has been identified as an endothelium-derived relaxing factor produced during inflammation, raising the possibility that this novel pathway may contribute to hypotension in human sepsis. DESIGN:: Prospective, matched, single-center, cohort study. SETTINGS:: Intensive care unit of a tertiary teaching hospital matched to control subjects from the general medical ward and healthy volunteers. SUBJECTS:: Patients (n = 16) with septic shock had indoleamine 2,3-dioxygenase 1 activity assessed as the kynurenine-to-tryptophan ratio, and the severity of hypotension was determined by their inotrope requirements. Healthy and blood pressure-matched nonseptic control subjects were also studied. INTERVENTIONS:: None. MEASUREMENTS AND MAIN RESULTS:: Tissues from septic and control patients were stained for the presence of indoleamine 2,3-dioxygenase 1. Indoleamine 2,3-dioxygenase 1 activity increased up to ninefold in patients with septic shock and was significantly higher than in the two control groups (p < .005). Indoleamine 2,3-dioxygenase 1 activity was strongly correlated with inotrope requirements (p < .001). Indoleamine 2,3-dioxygenase 1 protein was expressed in inflamed cardiac tissue as well as in endothelial cells of resistance vessels in hearts and kidneys from subjects who died from sepsis. CONCLUSIONS:: Indoleamine 2,3-dioxygenase 1 is expressed in resistance vessels in human sepsis and Indoleamine 2,3-dioxygenase 1 activity correlates with hypotension in human septic shock. Indoleamine 2,3-dioxygenase 1 is thus a potential novel contributor to hypotension in sepsis. %Z FOR Codes: 1114 %0 Journal Article %~ Pubmed %A Wijnker, Paul J M %A Boknik, Peter %A Gergs, Ulrich %A M?ller, Frank U %A Neumann, Joachim %A dos Remedios, Cris %A Schmitz, Wilhelm %A Sindermann, J?rgen R %A Stienen, Ger J M %A van der Velden, Jolanda %A Kirchhefer, Uwe %T Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium. %B Journal of muscle research and cell motility %D 2011 %V 32 %N 3 %P 221-33 %@ 1573-2657 %X Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation-contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (?pCa50=0.05?0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ?pCa50=0.10?0.03) and dilated (DCM, ?pCa50=0.06?0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56? was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts. %Z FOR Codes: 1102 601 %0 Journal Article %~ Pubmed %A Lu, Zhongbing %A Xu, Xin %A Hu, Xinli %A Lee, Sangjin %A Traverse, Jay H %A Zhu, Guangshuo %A Fassett, John %A Tao, Yi %A Zhang, Ping %A dos Remedios, Cris %A Pritzker, Marc %A Hall, Jennifer L %A Garry, Daniel J %A Chen, Yingjie %T Oxidative stress regulates left ventricular PDE5 expression in the failing heart. %B Circulation %D 2010 %V 121 %N 13 %P 1474-83 %@ 1524-4539 %X BACKGROUND: Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF. METHODS AND RESULTS: Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)-induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased approximately equal 4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3'-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (SOD) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase 3 knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF. CONCLUSIONS: Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF. %Z FOR Codes: 110201 %0 Journal Article %~ Pubmed %A Hirsh, S L %A Bilek, M M M %A Nosworthy, N J %A Kondyurin, A %A Dos Remedios, C G %A McKenzie, D R %T A comparison of covalent immobilization and physical adsorption of a cellulase enzyme mixture. %B Langmuir : the ACS journal of surfaces and colloids %D 2010 %V 26 %N 17 %P 14380-8 %@ 1520-5827 %X This paper reports the first use of a linker-free covalent approach for immobilizing an enzyme mixture. Adsorption from a mixture is difficult to control due to varying kinetics of adsorption, variations in the degree of unfolding and competitive binding effects. We show that surface activation by plasma immersion ion implantation (PIII) produces a mildly hydrophilic surface that covalently couples to protein molecules and avoids these issues, allowing the attachment of a uniform monolayer from a cellulase enzyme mixture. Atomic force microscopy (AFM) showed that the surface layer of the physically adsorbed cellulase layer on the mildly hydrophobic surface (without PIII) consisted of aggregated enzymes that changed conformation with incubation time. The evolution observed is consistent with the existence of transient complexes previously postulated to explain the long time constants for competitive displacement effects in adsorption from enzyme mixtures. AFM indicated that the covalently coupled bound layer to the PIII-treated surface consisted of a stable monolayer without enzyme aggregates, and became a double layer at longer incubation times. Light scattering analysis showed no indication of aggregates in the solution at room temperature, which indicates that the surface without PIII-treatment induced enzyme aggregation. A model for the attachment process of a protein mixture that includes the adsorption kinetics for both surfaces is presented. %Z FOR Codes: 90301 903 %0 Book Section %A Mohamed, Alana %A Lal, Sean %A Ho, Joshua WK %A Brown, Angus %A Lui, Rodney %A Nguyen, Lisa %A Yong, Andy %A Su, YingYing %A Braet, Philip %A Dyer, Wayne %A Junius, Frank %A Cumming, Robert %A Freedman, Saul %A Kritharides, Leonard %A dos Remedios, Cristobal %T How to interrogate the cellular immune system in patients with ischemic heart disease %B Myocardial ischemia: Causes, symptoms and treatment %D 2010 %C United States %I Nova Publishers %V %N %P 195-216 %@ 9781608766109 %E Vukovic, Dmitry %E Kiyan, Vladimir %X %Z FOR Codes: 110201 110704 %0 Journal Article %~ Pubmed %A Kooij, Viola %A Boontje, Nicky %A Zaremba, Ruud %A Jaquet, Kornelia %A Dos Remedios, Cris %A Stienen, Ger %A van der Velden, Jolanda %T Protein kinase C alpha and epsilon phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium. %B Basic research in cardiology %D 2010 %V 105 %N 2 %P 289-300 %@ 1435-1803 %X Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCalpha- and PKCepsilon-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCalpha and PKCepsilon equally reduced Ca2+ sensitivity in failing cardiomyocytes (DeltapCa50 = 0.08 +/- 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca2+ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCalpha resulted in a modest significant reduction in passive force. Effects of PKCalpha were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCalpha. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca2+ sensitivity. Subsequently, PKCalpha treatment of donor cardiomyocytes reduced Ca2+ sensitivity (DeltapCa50 = 0.08 +/- 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCalpha- and PKCepsilon-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca2+ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium. %Z FOR Codes: 110201 %0 Journal Article %~ Pubmed %A Kong, Sek Won %A Hu, Yong Wu %A Ho, Joshua W K %A Ikeda, Sadakatsu %A Polster, Sean %A John, Ranjit %A Hall, Jennifer L %A Bisping, Egbert %A Pieske, Burkert %A dos Remedios, Cristobal G %A Pu, William T %T Heart failure-associated changes in RNA splicing of sarcomere genes. %B %D 2010 %V 3 %N 2 %P 138-46 %@ 1942-3268 %X BACKGROUND: Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts. METHODS AND RESULTS: We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy. CONCLUSIONS: Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes. %Z FOR Codes: 110201 %0 Journal Article %A Kwok, Dixon TK %A Tong, Liping %A Yeung, Che Yan %A dos Remedios, Cristobal %A Chu, Paul K %T Hybrid plasma surface modification and ion implantation of biopolymers %B Surface and Coatings Technology %D 2010 %C Switzerland %I Elsevier Inc. %V 204 %N 18-19 %P 2892-2897 %@ 0257-8972 %X %Z FOR Codes: 30499 %0 Journal Article %A Ho, Joshua %A Lin, Ming-Wei %A Adelstein, Stephen %A dos Remedios, Cristobal %T Customising an antibody leukocyte capture microarray for systemic lupus erythematosus: Beyond biomarker discovery %B Proteomics - Clinical Applications %D 2010 %C Germany %I Wiley - VCH Verlag %V 4 %N 2 %P 179-189 %@ 1862-8346 %X %Z FOR Codes: 60109 %0 Journal Article %~ Pubmed %A Lal, Sean %A Dos Remedios, Cris G %T Is there a connection between ablation of atrial fibrillation and reduced apoptosis? %B Cardiology %D 2010 %V 117 %N 3 %P 161-2 %@ 1421-9751 %X %Z FOR Codes: 1102 %0 Journal Article %~ Pubmed %A Hoskins, Anita C %A Jacques, Adam %A Bardswell, Sonya C %A McKenna, William J %A Tsang, Victor %A Dos Remedios, Cristobal G %A Ehler, Elisabeth %A Adams, Kim %A Jalilzadeh, Shapour %A Avkiran, Metin %A Watkins, Hugh %A Redwood, Charles %A Marston, Steven B %A Kentish, Jonathan C %T Normal passive viscoelasticity but abnormal myofibrillar force generation in human hypertrophic cardiomyopathy. %B Journal of Molecular and Cellular Cardiology %D 2010 %V 49 %N 5 %P 737-45 %@ 1095-8584 %X Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca(2+)-activated force was much lower in HCM myocytes (14 ?? 1 kN/m(2)) than in donor myocytes (23 ?? 3 kN/m(2); P<0.01), though cross-bridge kinetics (k(tr)) during maximal Ca(2)(+) activation were 10% faster in HCM myocytes. Myofibrillar Ca(2)(+) sensitivity in HCM myocytes (pCa(50)=6.40 ?? 0.05) was higher than for donor myocytes (pCa(50)=6.09 ?? 0.02; P<0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca(2+) concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca(2+)-activated force and high Ca(2+) sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients. %Z FOR Codes: 110201 %0 Journal Article %~ Pubmed %A Iskratsch, Thomas %A Lange, Stephan %A Dwyer, Joseph %A Kho, Ay Lin %A dos Remedios, Cris %A Ehler, Elisabeth %T Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance. %B The Journal of cell biology %D 2010 %V 191 %N 6 %P 1159-72 %@ 1540-8140 %X Members of the formin family are important for actin filament nucleation and elongation. We have identified a novel striated muscle-specific splice variant of the formin FHOD3 that introduces a casein kinase 2 (CK2) phosphorylation site. The specific targeting of muscle FHOD3 to the myofibrils in cardiomyocytes is abolished in phosphomutants or by the inhibition of CK2. Phosphorylation of muscle FHOD3 also prevents its interaction with p62/sequestosome 1 and its recruitment to autophagosomes. Furthermore, we show that muscle FHOD3 efficiently promotes the polymerization of actin filaments in cardiomyocytes and that the down-regulation of its expression severely affects myofibril integrity. In murine and human cardiomyopathy, we observe reduced FHOD3 expression with a concomitant isoform switch and change of subcellular targeting. Collectively, our data suggest that a muscle-specific isoform of FHOD3 is required for the maintenance of the contractile structures in heart muscle and that its function is regulated by posttranslational modification. %Z FOR Codes: 60106 %0 Journal Article %~ Pubmed %A Robinson, Aisling A %A Dunn, Michael J %A McCormack, Ann %A dos Remedios, Cris %A Rose, Marlene L %T Protective effect of phosphorylated Hsp27 in coronary arteries through actin stabilization. %B Journal of Molecular and Cellular Cardiology %D 2010 %V 49 %N 3 %P 370-9 %@ 1095-8584 %X There is evidence for an inverse association between cellular expression of Hsp27 and vascular disease with carotid plaques, endarterectomy specimens, and cardiac biopsies investigated to date. Here we compare non-diseased coronary arteries from human heart transplant donors and patients with dilated cardiomyopathy (DCM) with no evidence of coronary artery disease, to coronary arteries from patients with ischemic heart disease (IHD) in order to determine abundance of phosphorylated Hsp27 (phospho-Hsp27) in plaque-free diseased vessels and elucidate how this protective effect is brought about through protein regulation. Western blotting identified phospho-Hsp27, phosphorylated on Ser82, Ser78, and Ser15, to be specifically decreased in IHD, but not DCM, compared to non-diseased vessels. Immunohistochemistry confirmed these results and revealed phospho-Hsp27 was located within both smooth muscle and endothelial cells. Disease-free coronary arteries and from patients with IHD were then subjected to 2-Dimensional Difference Gel Electrophoresis (2D-DIGE) analysis to detect proteins with altered abundance, which were subsequently identified by mass spectrometry. Hsp27 showed decreased abundance in ischemic vessels as expected. The expression of cytoskeletal proteins, namely vimentin was significantly reduced, while transgelin and tropomyosin showed significantly increased abundance in vessels with IHD. Immunohistochemistry studies suggested an increase in G-actin abundance to be present within IHD vessels. The results are consistent with the hypothesis that phospho-Hsp27 protects against vascular disease possibly by stabilizing the actin cytoskeleton within endothelial and/or smooth muscle cells. %Z FOR Codes: 110201 60106 %0 Journal Article %~ Pubmed %A Hamdani, Nazha %A Borb?ly, Attila %A Veenstra, Sophie P G R %A Kooij, Viola %A Vrydag, Wim %A Zaremba, Ruud %A Dos Remedios, Cris %A Niessen, Hans W M %A Michel, Martin C %A Paulus, Walter J %A Stienen, Ger J M %A van der Velden, Jolanda %T More severe cellular phenotype in human idiopathic dilated cardiomyopathy compared to ischemic heart disease. %B Journal of muscle research and cell motility %D 2010 %V 31 %N 4 %P 289-301 %@ 1573-2657 %X Activation of the ?-adrenergic receptor (?AR) pathway is the main mechanism of the heart to increase cardiac output via protein kinase A (PKA)-mediated phosphorylation of cellular target proteins, and perturbations therein may contribute to cardiac dysfunction in heart failure. In the present study a comprehensive analysis was made of mediators of the ?AR pathway, myofilament properties and cardiac structure in patients with idiopathic (IDCM; n?=?13) and ischemic (ISHD; n?=?10) cardiomyopathy in comparison to non-failing hearts (donor; n?=?10) for the following parameters: ?AR density, G-coupled receptor kinases 2 and 5, stimulatory and inhibitory G-proteins, phosphorylation of myofilament targets of PKA, protein phosphatase 1, phospholamban, SERCA2a and single myocyte contractility. All parameters exhibited the expected alterations of heart failure, but for most of them the extent of alteration was greater in IDCM than in ISHD. Histological analysis also revealed higher collagen in IDCM compared to ISHD. Alterations in the ?AR pathway are more pronounced in IDCM than in ISHD and may reflect sequential changes in cellular protein composition and function. Our data indicate that cellular dysfunction is more severe in IDCM than in ISHD. %Z FOR Codes: 60103 110201 %0 Journal Article %~ Pubmed %A Kooij, Viola %A Saes, Martina %A Jaquet, Kornelia %A Zaremba, Ruud %A Foster, D Brian %A Murphy, Anne M %A Dos Remedios, Cris %A van der Velden, Jolanda %A Stienen, Ger J M %T Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background. %B Journal of Molecular and Cellular Cardiology %D 2010 %V 48 %N 5 %P 954-63 %@ 1095-8584 %X Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca(2+)-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca(2+)-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66+/-1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca(2+)-sensitivity (pCa(50)) to 5.50+/-0.02 relative to the donor control value (pCa(50)=5.43+/-0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca(2+)-sensitivity relative to the failing control (pCa(50)=5.60+/-0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca(2+)-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca(2+)-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca(2+)-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca(2+)-sensitivity. %Z FOR Codes: 110201 %0 Journal Article %A Estigoy, Colleen B %A Ponten, Fredrik %A Odeberg, Jacob %A Herbert, Benjamin %A Guilhaus, Michael %A Charleston, Michael %A Ho, Joshua W K %A Cameron, Darryl %A dos Remedios, Cristobal %T Intercalated discs: multiple proteins perform multiple functions in non-failing and failing human hearts %B Biophysical Reviews %D 2009 %C Germany %I Springer %V 1 %N 1 %P 43-49 %@ 1867-2450 %X %Z FOR Codes: 110106 110299 %0 Journal Article %~ Pubmed %A Lal, Sean %A Brown, Angus %A Nguyen, Lisa %A Braet, Filip %A Dyer, Wayne %A Dos Remedios, Cris %T Using antibody arrays to detect microparticles from acute coronary syndrome patients based on cluster of differentiation (CD) antigen expression. %B Molecular & cellular proteomics : MCP %D 2009 %V 8 %N 4 %P 799-804 %@ 1535-9484 %X Microparticles circulate in plasma and have recently emerged as potential inflammatory markers in cardiovascular disease. They are fragments of cell membranes that express cluster of differentiation (CD) antigens and are present at elevated levels in patients with acute coronary syndrome. We have developed a novel method for the rapid detection of microparticles in plasma using a fluorescence-based antibody array system. Isolated microparticles are captured on anti-CD antibody spots immobilized on a nitrocellulose membrane. These CD antibodies are directed against extracellular epitopes, whereas the intracellular exposed surface of the microparticles is labeled with a fluorescent anti-annexin antibody. The array is then scanned and quantified. A pilot study was undertaken to compare microparticle CD antigen expression in acute coronary syndrome and healthy subjects. Ten CD antigens (44, 45, 54, 62E, 79, 102, 117, 130, 138, and 154) had significantly increased expression in the disease group relative to the healthy controls. These results were then verified using flow cytometry and scanning electron microscopy. Although we have focused our analysis on changes in microparticle CD antigen expression, this technique is amenable to analyzing other surface markers. Microparticles can be derived from a wide variety of cell types, so selection of the primary antibody can be tailored to the cell origin that is to be investigated. %Z FOR Codes: 110201 %0 Journal Article %~ Isi %A Messer, A. E. %A Gallon, C. E. %A McKenna, W. J. %A Dos Remedios, C. G. %A Marston, S. B. %T The use of phosphate-affinity SDS-PAGE to measure the cardiac troponin I phosphorylation site distribution in human heart muscle %B Proteomics Clinical Applications %D 2009 %C Germany %I Wiley - VCH Verlag GmbH & Co. KGaA %V 3 %N %P 1371-1382 %@ 1862-8346 %X %Z FOR Codes: 110201 %0 Journal Article %~ Pubmed %A van Dijk, Sabine J %A Holewijn, Rozemarije A %A Tebeest, Anouk %A Dos Remedios, Cris %A Stienen, Ger J M %A van der Velden, Jolanda %T A piece of the human heart: variance of protein phosphorylation in left ventricular samples from end-stage primary cardiomyopathy patients. %B Journal of muscle research and cell motility %D 2009 %V 30 %N 7-8 %P 299-302 %@ 1573-2657 %X Cardiomyocyte contraction is regulated by phosphorylation of sarcomeric proteins. Throughout the heart regional and transmural differences may exist in protein phosphorylation. In addition, phosphorylation of sarcomeric proteins is altered in cardiac disease. Heterogeneity in protein phosphorylation may be larger in hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) as it may be caused by multiple mutations in genes encoding different sarcomeric proteins. Moreover, HCM is characterized by asymmetric remodelling of the heart. In the present study we assessed if local differences in sarcomeric protein phosphorylation are more evident in primary HCM or DCM than in non-failing donors. Thereto, phosphorylation of the two main target proteins of the beta-adrenergic receptor pathway, troponin I (cTnI) and myosin binding protein C (cMyBP-C) was analysed in different parts in the free left ventricular wall of end-stage failing HCM and DCM patients and donors obtained during transplant surgery. Intra-patient variability in protein phosphorylation within tissue samples of approximately 2 g wet weight was comparable between donor, HCM and DCM samples and could partly be attributed to the precision of the technique. Thus, our data indicate that within the precision of the measurements small, biopsy-sized cardiac tissue samples are representative for the region of the free left ventricular wall from which they were obtained. %Z FOR Codes: 110201 %0 Journal Article %~ Pubmed %A Ho, Joshua W K %A Stefani, Maurizio %A dos Remedios, Cristobal G %A Charleston, Michael A %T A model selection approach to discover age-dependent gene expression patterns using quantile regression models. %B BMC genomics [electronic resource] %D 2009 %V 10 Suppl 3 %N %P S16 %@ 1471-2164 %X BACKGROUND: It has been a long-standing biological challenge to understand the molecular regulatory mechanisms behind mammalian ageing. Harnessing the availability of many ageing microarray datasets, a number of studies have shown that it is possible to identify genes that have age-dependent differential expression (DE) or differential variability (DV) patterns. The majority of the studies identify "interesting" genes using a linear regression approach, which is known to perform poorly in the presence of outliers or if the underlying age-dependent pattern is non-linear. Clearly a more robust and flexible approach is needed to identify genes with various age-dependent gene expression patterns. RESULTS: Here we present a novel model selection approach to discover genes with linear or non-linear age-dependent gene expression patterns from microarray data. To identify DE genes, our method fits three quantile regression models (constant, linear and piecewise linear models) to the expression profile of each gene, and selects the least complex model that best fits the available data. Similarly, DV genes are identified by fitting and comparing two quantile regression models (non-DV and the DV models) to the expression profile of each gene. We show that our approach is much more robust than the standard linear regression approach in discovering age-dependent patterns. We also applied our approach to analyze two human brain ageing datasets and found many biologically interesting gene expression patterns, including some very interesting DV patterns, that have been overlooked in the original studies. Furthermore, we propose that our model selection approach can be extended to discover DE and DV genes from microarray datasets with discrete class labels, by considering different quantile regression models. CONCLUSION: In this paper, we present a novel application of quantile regression models to identify genes that have interesting linear or non-linear age-dependent expression patterns. One important contribution of this paper is to introduce a model selection approach to DE and DV gene identification, which is most commonly tackled by null hypothesis testing approaches. We show that our approach is robust in analyzing real and simulated datasets. We believe that our approach is applicable in many ageing or time-series data analysis tasks. %Z FOR Codes: 100402 %0 Journal Article %~ Pubmed %A van Dijk, Sabine J %A Dooijes, Dennis %A dos Remedios, Cris %A Michels, Michelle %A Lamers, Jos M J %A Winegrad, Saul %A Schlossarek, Saskia %A Carrier, Lucie %A ten Cate, Folkert J %A Stienen, Ger J M %A van der Velden, Jolanda %T Cardiac myosin-binding protein C mutations and hypertrophic cardiomyopathy: haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction. %B Circulation %D 2009 %V 119 %N 11 %P 1473-83 %@ 1524-4539 %X BACKGROUND: Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3(mut)). METHODS AND RESULTS: Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3(mut). Protein expression of cMyBP-C was significantly reduced in MYBPC3(mut) by 33+/-5%. Cardiac MyBP-C phosphorylation in MYBPC3(mut) samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84+/-5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the beta-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3(mut) (20.2+/-2.7 kN/m(2)) compared with donor (34.5+/-1.1 kN/m(2)). Moreover, Ca(2+) sensitivity was higher in MYBPC3(mut) (pCa(50)=5.62+/-0.04) than in donor (pCa(50)=5.54+/-0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca(2+) sensitivity between MYBPC3(mut) (pCa(50)=5.46+/-0.03) and donor (pCa(50)=5.48+/-0.02). CONCLUSIONS: Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca(2+) sensitivity in MYBPC3(mut) is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction. %Z FOR Codes: 110299 %0 Book Section %A dos Remedios, Cristobal %A Nosworthy, N %T The role of PIP2 in actin, actin-binding proteins and disease %B Actin-binding proteins and disease %D 2008 %C United Kingdom %I Springer %V %N %P 290-297 %@ 978-0-387-71747-0 %X %0 Journal Article %~ Pubmed %A Fermin, David R %A Barac, Ana %A Lee, Sangjin %A Polster, Sean P %A Hannenhalli, Sridhar %A Bergemann, Tracy L %A Grindle, Suzanne %A Dyke, David B %A Pagani, Francis %A Miller, Leslie W %A Tan, Sarah %A Dos Remedios, Cris %A Cappola, Thomas P %A Margulies, Kenneth B %A Hall, Jennifer L %T Sex and age dimorphism of myocardial gene expression in nonischemic human heart failure. %B %D 2008 %V 1 %N 2 %P 117-25 %@ 1942-3268 %X BACKGROUND: We report the first comprehensive analysis of gene expression differences by sex and age in left ventricular samples from 102 patients with dilated cardiomyopathy. METHODS AND RESULTS: Gene expression data (HG-U133A gene chip, Affymetrix) were analyzed from 30 females and 72 males from 3 separate centers. More than 1800 genes displayed sexual dimorphism in the heart (adjusted P value <0.05). A significant number of these genes were highly represented in gene ontology pathways involved in ion transport and G-protein-coupled receptor signaling. Localization of these genes revealed enrichment on both the sex chromosomes as well as chromosomes 3, 4, and 14. The second goal of this study was to determine the effect of age on gene expression. Within the female cohort, >140 genes were differentially expressed in the <55 years age group compared with the >55 years age group. These genes were highly represented in gene ontology pathways involved in DNA damage. In contrast, zero genes in the male cohort <55 years met statistical significance when compared with the >55 years age group. CONCLUSIONS: Gene expression in dilated cardiomyopathy displayed evidence of sexual dimorphism similar to other somatic tissues and age dimorphism within the female cohort. %Z FOR Codes: 110201 %0 Journal Article %~ Pubmed %A Kr?ger, Martina %A K?tter, Sebastian %A Gr?tzner, Anika %A Lang, Patrick %A Andresen, Christian %A Redfield, Margaret M %A Butt, Elke %A Dos Remedios, Cris G %A Linke, Wolfgang A %T Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs. %B Circulation research %D 2008 %V 104 %N 1 %P 87-94 %@ 1524-4571 %X The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure. %Z FOR Codes: 110299 %0 Book Section %A Lui, P %A Brown, Andrew %A Wu, Ben %A Velauthen, C %A Lin, M-W %A Thompson, John %A Braet, F %A Dyer, Wayne %A Macdonald, P %A Hayward, C %A Adelstein, Stephen %A dos Remedios, Cristobal %T Use of antibody microarrays in the analysis of inflammation, autoimmunity, viral infection, and cancer metastases %B Clinical Proteomics: From Diagnosis to Therapy %D 2008 %C United States %I John Wiley & Sons %V %N %P 571-592 %@ 978-3-527-31637-3 %E Van Eyk, Jennifer E %E Dunn, Michael J %X %Z FOR Codes: 110316 110701 %0 Journal Article %~ Pubmed %A Martinez-Neira, R %A Dos Remedios, C G %A Mackay, I R %T An actin-myosin functional assay for analysis of smooth muscle (anti-microfilament) autoantibodies in human plasma. %B Journal of Immunological Methods %D 2008 %V 338 %N 1-2 %P 63-6 %@ 0022-1759 %X The detection of serum autoantibodies to smooth muscle (SMA) on rodent gastric mucosa by indirect immunofluorescence (IIF) has long been an immunodiagnostic marker for autoimmune hepatitis type 1 (AIH-1). The reactive antigenic moieties are cytoskeletal proteins which include polymeric F-actin as judged by the staining of microfilaments of tissue by IIF. However, their specificity for actin in AIH-1 can be and usually is uncertain. Using an in vitro functional assay, we compared the effects of Fab fragments of immunoglobulin (IgG) prepared from SMA-positive plasma from two patients with the effects of Fabs from 10 healthy subjects. Fabs are incorporated into an assay where actin (the putative antigen) activates skeletal muscle heavy meromyosin (HMM) ATPase activity. The data from these functional assays provide new insights into the significance of anti-microfilament assays in the diagnosis, and perhaps also pathogenesis, of AIH-1. %Z FOR Codes: 110302 %0 Journal Article %~ Pubmed %A Hamdani, Nazha %A de Waard, Monique %A Messer, Andrew E %A Boontje, Nicky M %A Kooij, Viola %A van Dijk, Sabine %A Versteilen, Amanda %A Lamberts, Regis %A Merkus, Daphne %A Dos Remedios, Cris %A Duncker, Dirk J %A Borbely, Attila %A Papp, Zoltan %A Paulus, Walter %A Stienen, Ger J M %A Marston, Steven B %A van der Velden, Jolanda %T Myofilament dysfunction in cardiac disease from mice to men. %B Journal of muscle research and cell motility %D 2008 %V 29 %N 6-8 %P 189-201 %@ 1573-2657 %X In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation. %Z FOR Codes: 110299 %0 Journal Article %A Brown, Angus %A Lattimore, Jo-Dee %A McGrady, Michele %A Sullivan, David %A Dyer, Wayne %A Braet, Filip %A dos Remedios, Cristobal %T Stable and unstable angina: Identifying novel markers on circulating leukocytes %B PROTEOMICS - Clinical Applications %D 2008 %C Germany %I Wiley - VCH Verlag GmbH & Co. KGaA %V 2 %N 1 %P 90-98 %@ 1862-8354 %X %Z FOR Codes: 110201 %0 Book Section %A Stefani, M %A Tsubakihara, M %A Allen, PD %A Macdonald, PS %A dos Remedios, Cristobal %T Actin and its binding proteins in heart failure %B Actin-binding proteins and disease %D 2008 %C United Kingdom %I Springer %V %N %P 318-334 %@ 978-0-387-71747-0 %X %Z FOR Codes: 1101 %0 Journal Article %~ Pubmed %A Ho, Joshua W K %A Stefani, Maurizio %A Dos Remedios, Cristobal G %A Charleston, Michael A %T Differential variability analysis of gene expression and its application to human diseases. %B Bioinformatics (Oxford, England) %D 2008 %V 24 %N 13 %P i390-i398 %@ 1460-2059 %X MOTIVATION: Current microarray analyses focus on identifying sets of genes that are differentially expressed (DE) or differentially coexpressed (DC) in different biological states (e.g. diseased versus non-diseased). We observed that in many human diseases, some genes have a significantincrease or decrease in expression variability (variance). Asthese observed changes in expression variability may be caused by alteration of the underlying expression dynamics, such differential variability (DV) patterns are also biologically interesting. RESULTS: Here we propose a novel analysis for changes in gene expression variability between groups of amples, which we call differential variability analysis. We introduce the concept of differential variability (DV), and present a simple procedure for identifying DV genes from microarray data. Our procedure is evaluated with simulated and real microarray datasets. The effect of data preprocessing methods on identification of DV gene is investigated. The biological significance of DV analysis is demonstrated with four human disease datasets. The relationships among DV, DE and DC genes are investigated. The results suggest that changes in expression variability are associated with changes in coexpression pattern, which imply that DV is not merely stochastic noise, but informative signal. AVAILABILITY: The R source code for differential variability analysis is available from the contact authors upon request. CONTACT: joshua@it.usyd.edu.au; mcharleston@it.usyd.edu.au. %Z FOR Codes: 110706 %0 Journal Article %~ Pubmed %A Ho, Joshua W K %A Koundinya, Rajeev %A Caetano, Tib?rio S %A dos Remedios, Cristobal G %A Charleston, Michael A %T Inferring differential leukocyte activity from antibody microarrays using a latent variable model. %B %D 2008 %V 21 %N %P 126-37 %@ 0919-9454 %X Recent development of cluster of differentiation (CD) antibody arrays has enabled expression levels of many leukocyte surface CD antigens to be monitored simultaneously. Such membrane-proteome surveys have provided a powerful means to detect changes in leukocyte activity in various human diseases, such as cancer and cardiovascular diseases. The challenge is to devise a computational method to infer differential leukocyte activity among multiple biological states based on antigen expression profiles. Standard DNA microarray analysis methods cannot accurately infer differential leukocyte activity because they often fail to take the cell-to-antigen relationships into account. Here we present a novel latent variable model (LVM) approach to tackle this problem. The idea is to model each cell type as a latent variable, and represent the class-to-cell and cell-to-antigen relationships as a LVM. Once the parameters of the LVM are learned from the data, differentially active leukocytes can be easily identified from the model. We describe the model formulation and assumptions which lead to an efficient expectation-maximization algorithm. Our LVM method was applied to re-analyze two cardiovascular disease datasets. We show that our results match existing biological knowledge better than other methods such as gene set enrichment analysis. Furthermore, we discuss how our approach can be extended to become a general framework for gene set analysis for DNA microarrays. %Z FOR Codes: 80608 %0 Journal Article %~ Pubmed %A Hamdani, Nazha %A Kooij, Viola %A van Dijk, Sabine %A Merkus, Daphne %A Paulus, Walter J %A Remedios, Cris Dos %A Duncker, Dirk J %A Stienen, Ger J M %A van der Velden, Jolanda %T Sarcomeric dysfunction in heart failure. %B Cardiovascular research %D 2008 %V 77 %N 4 %P 649-58 %@ 0008-6363 %X Sarcomeric dysfunction plays a central role in reduced cardiac pump function in heart failure. This review focuses on the alterations in sarcomeric proteins in diseased myocardium that range from altered isoform expression to post-translational protein changes such as proteolysis and phosphorylation. Recent studies in animal models of heart failure and human failing myocardium converge and indicate that sarcomeric dysfunction, including altered maximum force development, Ca(2+) sensitivity, and increased passive stiffness, largely originates from altered protein phosphorylation, caused by neurohumoral-induced alterations in the kinase-phosphatase balance inside the cardiomyocytes. Novel therapies, which specifically target phosphorylation sites within sarcomeric proteins or the kinases and phosphatases involved, might improve cardiac function in heart failure. %Z FOR Codes: 110299 %0 Book %A dos Remedios, Cristobal %A Chhabra, Deepak %T Actin-Binding Proteins and Disease %B %D 2008 %C United Kingdom %I Springer %V %N %P %@ 978-0-387-71747-0 %X %0 Book Section %A Chhabra, Deepak %A dos Remedios, Cristobal %T Actin: an overview of its structure and function %B Actin-binding proteins and disease %D 2008 %C United Kingdom %I Springer %V %N %P 1-16 %@ 978-0-387-71747-0 %X %0 Book Section %A dos Remedios, Cristobal %A Chhabra, Deepak %T Foreword %B Actin-binding proteins and disease %D 2008 %C United Kingdom %I Springer %V %N %P v-vii %@ 978-0-387-71747-0 %X %0 Journal Article %~ Pubmed %A Dos Remedios, Cristobal G %T The regulation of muscle contraction: as in life, it keeps getting more complex. %B Biophysical Journal %D 2007 %V 93 %N 12 %P 4097-8 %@ 1542-0086 %X %Z FOR Codes: 110106 %0 Journal Article %~ Pubmed %A Gan, B K %A Nosworthy, N J %A McKenzie, D R %A Dos Remedios, C G %A Bilek, M M M %T Plasma immersion ion implantation treatment of polyethylene for enhanced binding of active horseradish peroxidase. %B %D 2007 %V 85 %N 3 %P 605-10 %@ 1552-4965 %X Robust attachment of active proteins to synthetic surfaces underpins the development of biosensors and protein arrays. This paper presents the results of experiments in which energetic ions, extracted from an inductively coupled nitrogen plasma, are used to modify the surface of ultrahigh molecular weight polyethylene (UHMWPE). The ability of the surface to bind active horseradish peroxidase (HRP) is significantly enhanced by the plasma treatment. The amide signal in infrared spectroscopy indicates an increased quantity of surface-attached protein on the modified surface. The activity of the bound HRP remains high compared with that of protein attached to the untreated surface, after repeated washing in buffer solution. Although Tween 20 was an effective blocking agent for the unmodified polyethylene surface, binding of HRP to the modified surface is not inhibited by its presence. We propose that the treatment produces new binding sites on the surface and that the function of the HRP is retained because the treated surface is substantially more hydrophilic. %0 Journal Article %A Zaremba, Ruud %A Merkus, Daphne %A Hamdani, Nazha %A Lamers, Jos %A Paulus, Walter %A dos Remedios, Cristobal %A Duncker, Dirk %A Stienen, Ger %A van der Velden, Jolanda %T Quantitative analysis of myofilament protein phosphorylation in small cardiac biopsies %B Proteomics - Clinical Applications %D 2007 %C Germany %I Wiley - V C H Verlag %V 1 %N %P 1285 - 1290 %@ 1862-8346 %X %0 Journal Article %A Ho, Joan P. Y. %A Nosworthy, Neil J. %A Bilek, Marcela M. M. %A Gan, Bee K. %A McKenzie, David R. %A Chu, Paul K. %A dos Remedios, Cristobal %T Plasma-Treated Polyethylene Surfaces for Improved Binding of Active Protein %B Plasma Processes and Polymers %D 2007 %C Germany %I WILEY-VCH Verlag %V 4 %N 5 %P 583-590 %@ 1612-8850 %X %0 Journal Article %~ Pubmed %A Narolska, Nadiya A %A Piroddi, Nicoletta %A Belus, Alexandra %A Boontje, Nicky M %A Scellini, Beatrice %A Deppermann, Sascha %A Zaremba, Ruud %A Musters, Rene J %A Dos Remedios, Cris %A Jaquet, Kornelia %A Foster, D Brian %A Murphy, Anne M %A van Eyk, Jennifer E %A Tesi, Chiara %A Poggesi, Corrado %A van der Velden, Jolanda %A Stienen, Ger J M %T Impaired diastolic function after exchange of endogenous troponin I with C-terminal truncated troponin I in human cardiac muscle. %B Circulation research %D 2007 %V 99 %N 9 %P 1012-20 %@ 1524-4571 %X The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI(1-192)) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 mum, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic beta-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI(1-192) in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca(2+) sensitivity of the contractile apparatus was increased in preparations containing cTnI(1-192) (pCa(50): 5.73+/-0.03 versus 5.52+/-0.03 for cTnI(1-192) and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI(1-192)-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved. %0 Journal Article %~ Pubmed %A Nicolau, Dan V %A Solana, Gerardin %A Kekic, Murat %A Fulga, Florin %A Mahanivong, Chitladda %A Wright, Jonathan %A dos Remedios, Cristobal G %T Surface hydrophobicity modulates the operation of actomyosin-based dynamic nanodevices. %B Langmuir : the ACS journal of surfaces and colloids %D 2007 %V 23 %N 21 %P 10846-54 %@ 0743-7463 %X We studied the impact of surface hydrophobicity on the motility of actin filaments moving on heavy-meromyosin (HMM)-coated surfaces. Apart from nitrocellulose (NC), which is the current standard for motility assays, all materials tested are good candidates for microfabrication: hydrophilic and hydrophobic glass, poly(methyl methacrylate) (PMMA), poly(tert-butyl methacrylate) (PtBuMA), and a copolymer of O-acryloyl acetophenone oxime with a 4-acryloyloxybenzophenone (AAPO). The most hydrophilic (hydrophilic glass, contact angle 35 degrees) and the most hydrophobic (PtBuMA, contact angle 78 degrees) surfaces do not maintain the motility of actin filaments, presumably because of the low density of adsorbed HMM protein or its high levels of denaturation, respectively. The velocity of actin filaments presents higher values in the middle of this "surface hydrophobicity motility window" (NC, PMMA), and a bimodal distribution, which is more apparent at the edges of this motility window (hydrophobic glass and AAPO). A molecular surface analysis of HMM and its S1 units suggests that the two very different, temporally separated conformations of the HMM heads could exacerbate the surface-modulated protein behavior, which is common to all microdevices using surface-immobilized proteins. An explanation for the above behavior proposes that the motility of actin filaments on HMM-functionalized surfaces is the result of the action of three populations of motors, each in a different surface-protein conformation, that is, HMM with both heads working (high velocities), working with one head (low velocities), and fully denatured HMM (no motility). It is also proposed that the molecularly dynamic nature of polymer surfaces amplifies the impact of surface hydrophobicity on protein behavior. The study demonstrates that PMMA is a good candidate for the fabrication of future actomyosin-driven dynamic nanodevices because it induces the smoothest motility of individual nano-objects with velocities comparable with those obtained on NC. %0 Journal Article %~ Pubmed %A Dedova, Irina V %A Nikolaeva, Olga P %A Safer, Daniel %A De La Cruz, Enrique M %A dos Remedios, Cris G %T Thymosin beta4 induces a conformational change in actin monomers. %B Biophysical Journal %D 2006 %V 90 %N 3 %P 985-92 %@ 0006-3495 %X Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin beta(4) (tbeta(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tbeta(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 A and decreases the distance between the purine base of bound ATP (epsilonATP) and Lys-61 by 1.9 A, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tbeta(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tbeta(4) binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tbeta(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tbeta(4) and cofilin compete for actin binding. Tbeta(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tbeta(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tbeta(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding. %0 Book %A Zhang, Shu Qin %A Yao, Cai Shi %A dos Remedios, Cristobal %T The Amazing Human Body: the anatomical display of real human bodies %B %D 2006 %C Australia %I Redtail %V %N %P %@ 9780977521104 %X %Z FOR Codes: 110399 %0 Journal Article %~ Pubmed %A Steel, Bradley C %A McKenzie, David R %A Bilek, Marcela Mm %A Nosworthy, Neil J %A Dos Remedios, Cristobal G %T Nanosecond responses of proteins to ultra-high temperature pulses. %B Biophysical Journal %D 2006 %V 91 %N 6 %P L66-8 %@ 0006-3495 %X Observations of fast unfolding events in proteins are typically restricted to <100 degrees C. We use a novel apparatus to heat and cool enzymes within tens of nanoseconds to temperatures well in excess of the boiling point. The nanosecond temperature spikes are too fast to allow water to boil but can affect protein function. Spikes of 174 degrees C for catalase and approximately 290 degrees C for horseradish peroxidase are required to produce irreversible loss of enzyme activity. Similar temperature spikes have no effect when restricted to 100 degrees C or below. These results indicate that the "speed limit" for the thermal unfolding of large proteins is shorter than 10(-8) s. The unfolding rate at high temperature is consistent with extrapolation of low temperature rates over 12 orders of magnitude using the Arrhenius relation. %Z FOR Codes: 110106 %0 Journal Article %~ Isi %A Gorbatyuk, V. Y. %A Nosworthy, N. J. %A Robson, S. A. %A Bains, N. P. S. %A Maciejewski, M. W. %A dos Remedios, C. G. %A King, G. F. %T Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction. %B Molecular Cell %D 2006 %C United States %I Cell Press %V 24 %N 4 %P 511-522 %@ 1097-2765 %X %Z FOR Codes: 111799 %0 Journal Article %~ Pubmed %A Chhabra, Deepak %A Nosworthy, Neil J %A dos Remedios, Cristobal G %T The N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin complex. %B Proteomics %D 2005 %V 5 %N 12 %P 3131-6 %@ 1615-9853 %X Apoptosis is essential in embryonic development, clonal selection of cells of the immune system and in the prevention of cancer. Apoptotic cells display characteristic changes in morphology that precede the eventual fragmentation of nuclear DNA resulting in cell death. Current evidence implicates DNase I as responsible for hydrolysis of DNA during apoptosis. In vivo, it is likely that cytoplasmic actin binds and inhibits the enzymatic activity and nuclear translocation of DNase I and that disruption of the actin-DNase I complex results in activation of DNase I. In this report we demonstrate that the N-terminal fragment of gelsolin (N-gelsolin) disrupts the actin-DNase I interaction. This provides a molecular mechanism for the role of the N-gelsolin in regulating DNase I activity. We also show that cofilin stabilises the actin-DNase I complex by forming a ternary complex that prevents N-gelsolin from releasing DNase I from actin. We suggest that both cofilin and gelsolin are essential in modulating the release of DNase I from actin. %0 Journal Article %A Chhabra, Deepak %A dos Remedios, Cristobal %T Intracellular fluorescence resonance energy transfer %B Biophysical Journal %D 2005 %C 9650 Rockville Pike, Bethesda, Md, 20814-3998 %I Biophysical Society %V 89 %N %P 1902-1908 %@ 1542-0086 %X %Z FOR Codes: 110199 %0 Journal Article %~ Pubmed %A Jiang, Lele %A Bardini, Michelle %A Keogh, Anne %A dos Remedios, Cristobal G %A Burnstock, Geoffrey %T P2X1 receptors are closely associated with connexin 43 in human ventricular myocardium. %B International Journal of Cardiology %D 2005 %V 98 %N 2 %P 291-7 %@ 0167-5273 %X BACKGROUND: It has been suggested that gap-junctional conductance between cardiomyocytes is regulated through a specific ligand-receptor interaction between ATP and connexins. In this study we examined the localization of P2X1 ionotropic receptors and their relation to connexin43 in gap junctions in human left ventricles. METHODS AND RESULTS: Using immunohistochemistry, we detected P2X1 expression predominantly in the intercalated discs. Labelling of the P2X1 receptor and the gap junction protein connexin43 showed close association in some gap junctions, while in others the two proteins often appeared to be spatially discrete. Western blotting detected four major bands at 45, 60, 95 and 120 kDa in the protein extracts from human left ventricles corresponding to equivalent bands from rat vas deferens. The most prominent band in human left ventricles was at 95 kDa, possibly a dimer of the native P2X1 receptor, whereas in rat vas deferens it was at 60 kDa. After preincubation of the antibody with its epitope peptide, the 45 and 60 kDa bands almost disappeared and the 95 and 120 kDa bands were significantly attenuated. CONCLUSIONS: P2X1 receptors in human myocardium are densely localized in gap junctions at intercalated discs between muscle cells. Close association of P2X1 receptors and connexin 43 occurred in some regions of some gap junctions, but in others they were spatially separate. Little difference in the pattern of distribution of P2X1 receptors was found in failing left ventricles of patients with dilated cardiomyopathy, although Western blots showed an enhancement of P2X1 receptor protein. %Z FOR Codes: 110106 110201 %0 Journal Article %~ Pubmed %A Martinez-Neira, R %A Kekic, M %A Nicolau, D %A dos Remedios, C G %T A novel biosensor for mercuric ions based on motor proteins. %B Biosensors & bioelectronics %D 2005 %V 20 %N 7 %P 1428-32 %@ 0956-5663 %X 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. %Z FOR Codes: 100499 %0 Journal Article %~ Isi %A Lui, R. %A Tsubakihara, M. %A Macdonald, P. %A Hayward, C. %A dos Remedios, C. G. %T Analysing inflammation in heart failure using microarray platforms. %B Journal of Molecular and Cellular Cardiology %D 2005 %C United Kingdom %I Academic Press %V 38 %N 5 %P 854-855 %@ 0022-2828 %X %0 Conference Proceedings %A Biek, Marcela %A McKenzie, David R %A Nosworthy, Neil J %A DAVIES, Kerrie %A MORROW, RICHARD %A THORDARSON, PALLI %A GAN, BEE K. %A dos Remedios, Cristobal %T Functional attachment of horse radish peroxidase to plasma-treated surfaces %B Smart Materials III %D 2005 %C Univ New South Wales Dec 13 %I SPIE proceedings, unknown %V 5648 %N %P 62-67 %@ %E Wilson, Alan R. %X %0 Conference Proceedings %A Ramdutt, Devin %A Lui, Rodney %A Boswell, Roderick %A dos Remedios, Cristobal %A Charles, Christine %A Bilek, Marcela %A McKenzie, David %T Development of the nanotiter plate for use in antibody and cell array technologies. %B SPIE %D 2005 %C UNSW %I SPIE Proceedings, unknown %V 5651 %N %P 409-417 %@ %E Nicolau, Dan %X %0 Journal Article %~ Pubmed %A Chhabra, D %A dos Remedios, C G %T Cofilin, actin and their complex observed in vivo using fluorescence resonance energy transfer. %B Biophysical Journal %D 2005 %V 89 %N 3 %P 1902-8 %@ 0006-3495 %X 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. %Z FOR Codes: 110106 %0 Journal Article %~ Isi %A Kekic, M. %A Martinez, R. %A dos Remedios, C. %A Nicolau, D. %T An acto/myosin in vitro motility assay as a motion based sensor. %B Biophysical Journal %D 2005 %C United States %I Biophysical Society %V 88 %N 1 %P 503A-503A %@ 0006-3495 %X %0 Journal Article %A dos Remedios, Cristobal %T AES is reborn as AEPS %B Proteomics %D 2004 %C Po Box 10 11 61, Weinheim, Germany, D-69451 %I Wiley-V C H Verlag Gmbh %V 4 %N %P 1857 %@ 1615-9861 %X %Z FOR Codes: 110106 %0 Journal Article %~ Pubmed %A Borovikov, Yu S %A Dedova, I V %A dos Remedios, C G %A Vikhoreva, N N %A Vikhorev, P G %A Avrova, S V %A Hazlett, T L %A Van Der Meer, B W %T Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin. %B Biophysical Journal %D 2004 %V 86 %N 5 %P 3020-9 %@ 0006-3495 %X 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. %0 Journal Article %~ Pubmed %A Dedova, I V %A Avrova, S V %A Vikhoreva, N N %A Vikhorev, R G %A Hazlett, T L %A Van der Meer, W %A Dos Remedios, C G %A Borovikov, Iu S %T [Conformational changes of actin induced by strong or weak myosin subfragment-1 binding] %B Tsitologiia %D 2004 %V 46 %N 8 %P 719-34 %@ 0041-3771 %X Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction. %Z FOR Codes: 110199 %0 Journal Article %~ Pubmed %A Dedova, Irina V %A Nikolaeva, Olga P %A Mikhailova, Valeria V %A dos Remedios, Cris G %A Levitsky, Dmitrii I %T Two opposite effects of cofilin on the thermal unfolding of F-actin: a differential scanning calorimetric study. %B Biophysical chemistry %D 2004 %V 110 %N 1-2 %P 119-28 %@ 0301-4622 %X Differential scanning calorimetry was used to examine the effects of cofilin on the thermal unfolding of actin. Stoichiometric binding increases the thermal stability of both G- and F-actin but at sub-saturating concentrations cofilin destabilizes F-actin. At actin:cofilin molar ratios of 1.5-6 the peaks corresponding to stabilized (66-67 degrees C) and destabilized (56-57 degrees C) F-actin are observed simultaneously in the same thermogram. Destabilizing effects of sub-saturating cofilin are highly cooperative and are observed at actin:cofilin molar ratios as low as 100:1. These effects are abolished by the addition of phalloidin or aluminum fluoride. Conversely, at saturating concentrations, cofilin prevents the stabilizing effects of phalloidin and aluminum fluoride on the F-actin thermal unfolding. These results suggest that cofilin stabilizes those actin subunits to which it directly binds, but destabilizes F-actin with a high cooperativity in neighboring cofilin-free regions. %0 Journal Article %~ Pubmed %A dos Remedios, Cristobal G %T The 32nd European Muscle Conference. %B Journal of muscle research and cell motility %D 2004 %V 25 %N 1 %P 3-5 %@ 0142-4319 %X %0 Journal Article %~ Pubmed %A Tsubakihara, Masako %A Williams, Neal K %A Keogh, Anne %A dos Remedios, Cristobal G %T Comparison of gene expression between left atria and left ventricles from non-diseased humans. %B Proteomics %D 2004 %V 4 %N 1 %P 261-70 %@ 1615-9853 %X We examine the reliability and accuracy of gene array technology in analyzing differences in gene expression between human non-diseased left atrium and left ventricle. We have used cDNA gene arrays and validated those data by carefully designed quantitative real-time polymerase chain reaction (PCR). We have identified pitfalls using cDNA gene array technology based on comparisons with other gene array studies and with changes reported for the levels of expression of the genes corresponding to these cDNAs. The high error rate reported here underscores the cautionary comments reported by others in this field. %0 Journal Article %~ Pubmed %A Steel, B %A Bilek, M M %A dos Remedios, C G %A McKenzie, D R %T Apparatus for exposing cell membranes to rapid temperature transients. %B European biophysics journal : EBJ %D 2004 %V 33 %N 2 %P 117-20 %@ 0175-7571 %X 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. %0 Journal Article %~ Pubmed %A Lal, Sean %A Lui, Rodney %A Nguyen, Lisa %A Macdonald, Peter %A Denyer, Gareth %A dos Remedios, Cristobal %T Increases in leukocyte cluster of differentiation antigen expression during cardiopulmonary bypass in patients undergoing heart transplantation. %B Proteomics %D 2004 %V 4 %N 7 %P 1918-26 %@ 1615-9853 %X Cardiopulmonary bypass (CPB) is essential in heart transplantation surgery. It is also used in coronary artery grafting surgery where it has been associated with a postoperative inflammatory response. This manifests as an increase in pro-inflammatory proteins like interleukin-8 and tumour necrosis factor alpha accompanied by increases in neutrophil populations and increased expression of cluster of differentiation (CD) antigens. The latter have been investigated using flow cytometry, which is limited to the measurement of three to four CD antigens simultaneously. We have developed a novel antibody array that can simultaneously measure the expression of 72 different CD antigens. The purpose of this study was to use this technology to measure CD antigen expression in patients undergoing CPB during heart transplantation. Twelve patients undergoing this operation were studied. A preoperative sample acted as an internal control while a second sample taken during CPB was the comparator. No previous studies have examined changes in CD antigen expression during heart transplantation. We report an increase in the expression of 10 different CD antigens across all patients between the two time points. %0 Journal Article %A Ng, DCH %A Court, NW %A dos Remedios, CG %A Bogoyevitch, MA %T Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts %B Cardiovascular Research %D 2003 %C PO BOX 211, AMSTERDAM, NETHERLANDS, 1000 A %I Elsevier Science Bv %V 57 %N %P 333-346 %@ 0008-6363 %X %0 Journal Article %~ Pubmed %A dos Remedios, C G %A Chhabra, D %A Kekic, M %A Dedova, I V %A Tsubakihara, M %A Berry, D A %A Nosworthy, N J %T Actin binding proteins: regulation of cytoskeletal microfilaments. %B Physiological reviews %D 2003 %V 83 %N 2 %P 433-73 %@ 0031-9333 %X 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. %Z FOR Codes: 110199 %0 Journal Article %A Jiang, L %A Huang, Y %A Hunyor, S %A dos Remedios, CG %T Cardiomyocyte apoptosis is associated with increased wall stress in chronic failing left ventricle %B European Heart Journal %D 2003 %C 32 JAMESTOWN RD, LONDON, ENGLAND, NW1 7B %I W B Saunders Co Ltd %V 24 %N %P 742-751 %@ 0195-668x %X %0 Journal Article %A Steel, BC %A Bilek, M %A dos Remedios, CG %A McKenzie, DR %T Apparatus for exposing cell membranes to rapid temperature transients %B European Biophysics Journal With Biophysics Letters %D 2003 %C 175 FIFTH AVE, NEW YORK, USA, NY, 1001 %I Springer-Verlag %V 33 %N %P 117-120 %@ 0175-7571 %X %0 Journal Article %~ Pubmed %A Dunn, Tirith %A Heller, C Ann %A McCarthy, Stanley W %A Dos Remedios, Cristobal %T Anatomical study of the "trochanteric bursa". %B Clinical anatomy (New York, N.Y.) %D 2003 %V 16 %N 3 %P 233-40 %@ 0897-3806 %X To resolve ambiguity in the literature about the anatomy of the "trochanteric bursa" or trochanteric subgluteus maximus bursa, this study examines the constancy, structure, and relationships of this bursa in a series of anatomical dissections of the hip. Sixteen embalmed hip specimens, from subjects aged 63-91 years, were examined. Subgluteus maximus bursae were demonstrated in 13 hips. In each of these a bursa, the deep bursa, was seen immediately superficial to the common attachment of the gluteus medius, minimus, and vastus lateralis muscles onto the greater trochanter. In five hips a smaller second bursa, the superficial bursa, was reflected with the gluteus maximus muscle. In two hips, four bursae were identified. The additional bursae were associated with either the deep or the superficial bursa. Examination of histological samples from the bursal walls confirmed the presence of a synovial lining in varying stages of development in seven of the eight bursae examined. Branches of the inferior gluteal nerve were seen to supply deep and superficial bursae in two dissections. The study data indicate that subgluteus maximus bursae at the level of the greater trochanter are an expected finding in the older age group and that they vary in number, position, and histological appearance. These features give rise to the hypothesis that these bursae are acquired as a consequence of excessive friction between the greater trochanter and the gluteus maximus as it inserts into the fascia lata. %Z FOR Codes: 110399 %0 Journal Article %A Mahanivong, C %A Wright, JP %A Kekic, M %A Pham, DK %A dos Remedios, CG %A Nicolau, DV %T Manipulation of the motility of protein molecular motors on microfabricated substrates %B Biomedical Microdevices %D 2002 %C Van Godewijckstraat 30, Dordrecht, Netherlands, 3311 Gz %I Kluwer Academic Publ %V 4:02 %N %P 111-116 %@ 1387-2176 %X %0 Journal Article %~ Pubmed %A Lal, Sean P %A Christopherson, Richard I %A dos Remedios, Cristobal G %T Antibody arrays: an embryonic but rapidly growing technology. %B Drug discovery today %D 2002 %V 7 %N 18 Suppl %P S143-9 %@ 1359-6446 %X Protein arrays are now an attractive proposition as they can measure a diverse range of protein interactions not possible with traditional DNA arrays. Antibody arrays are a specific subset of this technology. Originally conceived as multi-analyte detectors, antibody arrays are now used in a wide variety of applications. For instance, the potential of this technology to diagnose human diseases, such as leukemia, breast cancer and, potentially, heart failure, has stimulated much interest. Furthermore, identification of new protein targets in particular disease states will prove to be an invaluable tool in drug discovery and development. Patient prognosis and treatment are also potential applications of the technology. Antibody arrays have proved to be dynamic in response to these broad range of possibilities. This review examines variations in antibody array design and discusses current and potential applications of this novel and interesting technology. %Z FOR Codes: 100404 %0 Book %A Thomas, DT %A dos Remedios, CG %T Molecular Interactions of Actin; Actin-Myosin Interaction & Actin-Based Regulation %B %D 2002 %C Germany %I Springer-Verlag %V %N %P %@ 3540671110 %X %0 Journal Article %A Bains, NPS %A Gorbatyuk, VY %A Nosworthy, NJ %A Robson, SA %A Maciejewski, MW %A dos Remedios, CG %A King, GF %T Letter to the Editor: Backbone & side-chain 1H, 15N, and 13C assignments for chick cofilin %B Journal Of Biomolecular Nmr %D 2002 %C Van Godewijckstraat 30, Dordrecht, Netherlands, 3311 Gz %I Kluwer Academic Publ %V 22 %N %P 193-194 %@ 0925-2738 %X %0 Journal Article %A Court, NW %A dos Remedios, CG %A Cordell, J %A Bogoyevitch, MA %T Cardiac expression & subcellular localization of the p38 Mitogen-activated protein kinase member, stress-activated protein kinases-3 (SAPK3) %B Journal Of Molecular And Cellular Cardiology %D 2002 %C 24-28 Oval Rd, London, England, Nw1 7D %I Academic Press Ltd Elsevier Science Ltd %V 34 %N %P 413-426 %@ 0022-2828 %X %0 Journal Article %A Heller, CA %A Marucci, DD %A Dunn, T %A Barr, EM %A Houang, MTW %A dos Remedios, CG %T Inguinal canal 'Lipoma' %B Clinical Anatomy %D 2002 %C Div John Wiley & Sons Inc,605 Third Ave, New York, Ny, 10158-0012 %I Wiley-Liss %V 15 %N %P 280-285 %@ 0897-3806 %X %0 Journal Article %A Chhabra, D %A Bao, SS %A dos Remedios, CG %T The distribution of cofilin and DNase I in vivo %B Cell Research %D 2002 %C 16 Donghuangchenggen North St, Beijing, Peoples R China, 100717 %I Science China Press %V 12(3-4) %N %P 207-214 %@ 1001-0602 %X %0 Journal Article %A Steel, BC %A Bilda, Z %A McKenzie, DR %A dos Remedios, CG %T A technique for microsecond heating & cooling of a thin (submicron) biological sample %B European Biophysics Journal With Biophysics Letters %D 2002 %C 175 Fifth Ave, New York, Ny, 10010 %I Springer-Verlag %V 31 %N %P 378-382 %@ 0175-7571 %X %0 Journal Article %A Moens, PDJ %A dos Remedios, CG %T Analysis of models of F-Actin using fluorescence resonance energy transfer spectroscopy %B Molecular Interactions of Actin: Actin structure and actin-binding proteins %D 2001 %C 1114 First Ave, 4Th Fl, New York, Ny, 10021 %I Rockefeller Univ Press %V 194 %N %P 189-203 %@ 0022-1007 %X %0 Journal Article %A Heinke, MY %A Yao, M %A Chang, D %A Einstein, R %A dos Remedios, CG %T Apoptosis of Ventricular and Atrial Myocytes from Pacing-Induced Canine Heart Failure %B Cardiovascular Research %D 2001 %C Hudson Rd, Leeds, England, Ls9 7Dl %I W S Maney & Sons Ltd %V 6 %N %P 389-392 %@ 1351-0002 %X %0 Journal Article %A Jiang, L %A Tsubakihara, M %A Heinke, MY %A Phillips, WD %A dos Remedios, CG %A Nosworthy, NJ %A Yao, M %T Heart failure and apoptosis: Electrophoretic methods support data from micro- and macro-arrays. A critical review of genomics and proteomics %B Proteomics %D 2001 %C Oxford %I Pergamon-Elsevier Science Ltd %V 52 %N %P 863-870 %@ 0277-9535 %X %0 Book Section %A Belov, L %A de la Vega, O %A dos Remedios, CG %A Mulligan, SP %A Christopherson, RI %T Immunophenotyping of leukemias using a cluster of differentiation antibody microarray1 %B Cancer Research %D 2001 %C Usa %I Wb Saunders Company %V %N %P 558-570 %@ 721682979 %X %0 Journal Article %A dos Remedios, CG %A Thomas, DD %T An overview of actin structure and actin-binding proteins %B Molecular Interactions of Actin; Actin structure and actin-binding proteins %D 2001 %C The Boulevard, Langford Lane,Kidlington, Oxford, England, Ox5 1Gb %I Pergamon-Elsevier Science Ltd %V 19 %N %P 415-425 %@ 0736-5748 %X %0 Journal Article %A Berry, DA %A Keogh, A %A dos Remedios, CG %T Nuclear membrane proteins in failing human dilated cardiomyopathy %B Proteomics %D 2001 %C 4350 East West Highway, Suite 500, Bethesda, Md, 20814-4110 %I Endocrine Soc %V 142 %N %P 2147-2150 %@ 0013-7227 %X %0 Journal Article %~ Pubmed %A Belov, L %A de la Vega, O %A dos Remedios, C G %A Mulligan, S P %A Christopherson, R I %T Immunophenotyping of leukemias using a cluster of differentiation antibody microarray. %B Cancer Research %D 2001 %V 61 %N 11 %P 4483-9 %@ 0008-5472 %X 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. %Z FOR Codes: 110704 %0 Journal Article %A Heinke, MY %A Yao, M %A Chang, D %A Einstein, R %A dos Remedios, CG %T Apoptosis of ventricular and atrial myocytes from pacinmg-induced canine heart failure %B Cardiovascular Research %D 2001 %C Hudson Rd, Leeds, England, Ls9 7Dl %I W S Maney & Sons Ltd %V 6 %N %P 200-203 %@ 1351-0002 %X %0 Journal Article %~ Pubmed %A Kekic, M %A Nosworthy, N J %A Dedova, I %A Collyer, C A %A dos Remedios, C G %T Regulation of the cytoskeleton assembly: a role for a ternary complex of actin with two actin-binding proteins. %B Results and problems in cell differentiation %D 2001 %V 32 %N %P 165-79 %@ 0080-1844 %X %Z FOR Codes: 60199 %0 Journal Article %A Berry, DA %A Balcar, VJ %A Barden, JA %A Keogh, A %A dos Remedios, CG %T Determination of P2X1 (alpha)-sarcoglycan (adhalin) expression levels in failing human dilated cardiomyopathic left ventricles %B Electrophoresis %D 2000 %C %I Wiley-Vch %V 21 (170 %N %P 3857-3862 %@ 0173-0835 %X %0 Journal Article %A Hambly, BD %A Walsh, J %A dos Remedios, CG %T Electrophoresis - a multidisciplinary and unifying technology %B Electrophoresis %D 2000 %C %I Wiley-V CH Verlag GMBH %V 21 %N %P 3781-3783 %@ 0173-0835 %X %0 Journal Article %A Chhabra, D %A Nosworthy, NJ %A dos Remedios, CG %T The role of ATP, ADP and divalent cations in the formation of binary and ternary complexes of actin, cofilin and DNase I %B Electrophoresis %D 2000 %C %I Wiley-Vch verlag GmbH %V 21 (17) %N %P 3863-3869 %@ 0173-0835 %X