1985 — 2001 |
Inesi, Giuseppe |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Calcium Ion Control in Cardiac Function @ University of Maryland Baltimore
The myofilaments of cardiac and skeletal cells are critically dependent on the intracellular concentration of Ca2+ for regulation of their contractile activity, In turn, the intracellular concentration of Ca2+ is controlled by membrane structures which are endowed with specific transport systems for the divalent cation. It is the aim of this proposal to combine ultrastructural, biochemical and physiological methods of investigation on order to clarify the mechanisms of Ca2+ uptake and release by these membranes. The proposed research will be carried out with a range of experimental systems including dissociated cells, isolated sarcoplasmic reticulum vesicles, solubilized and purified proteins (Ca2+ ATPase) and reconstituted membranes. Specific ultrastructural and/or conformational features of these systems will be related to functional activities with specific reference to active Ca2+ transport and coupled enzyme activity. Sequential steps of the enzyme cycle will be studied by rapid mixing techniques in the forward and reverse directions, leading to active transport and ATP synthesis respectively. Passive Ca2+ fluxes will be also studied, and their relevance to contractile regulation will be evaluated by monitoring sarcomere activation in dissociated cell preparations. Comparative studies will be performed under the influence of pharmacological agents. These studies will lead to clarification of basic biological mechanisms: ion transport, energy transduction and contractile control.
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1 |
1989 — 1994 |
Inesi, Giuseppe Demeis, Leopoldo |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
U.S.-Brazil Cooperative Research: Calcium Transport Across Biological Membranes @ University of Maryland At Baltimore
This award supports cooperative research in biophysics to be conducted by Giuseppe Inesi of the University of Maryland at Baltimore and Leopoldo de Meis of the Federal University in Rio de Janeiro, Brazil. The topic is calcium transport across biological membranes. The specific aim is to clarify the mechanisms of calcium uptake in membrane vesicles obtained from muscle tissue. The biological significance of the research is related to the contraction and relaxation of muscle fibers. Chemical significance lies in understanding enzyme kinetics and catalysis, the coupling of catalytic and tranport activities, channels and transmembrane flow of electrolytes and the structure of proteins. There has been a long-standing, productive collaboration between Dr. Inesi and several Brazilian scientists who, as postdoctoral associates, have spent extended periods of time in his laboratory. This grant will allow for the continuation of this cooperation and enhance Dr. Inesi's participation in the work in Brazil. Both investigators are well qualified and will carry out work on an important problem in a vital area of research.
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1 |
1997 — 2001 |
Inesi, Giuseppe |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Sarcoplasmic Reticulum Calcium Atpase @ University of Maryland Baltimore
The sarcoplasmic reticulum ATPase concentrates cytosolic Ca2+ into intracellular compartments, thereby permitting relaxation and subsequent tension development of cardiac and skeletal muscle. We will use ATPase proteins and cDNA clones to study catalytic, transport and inhibitory mechanisms, as well as ATPase expression following gene transfer, as follows: 1-Characterization of the mechanism of Ca2+ binding, ATP utilization, intermediate enzyme phosphorylation and Ca2+ transport, as they relate to ATPase topology and structure. Following previous information obtained by indirect measurement, we will extend our mutational analysis by expressing mutated ATPase in large quantities for direct measurements of stoichiometry, equilibrium and kinetic constants, and the effects of specific amino acid mutations of the partial reactions of the catalytic and transport cycle. ATPase mutants will be also used for spectroscopic studies to relate functional steps to structural features, an explain intermolecular linkages of separate function domains. 2-Topology of the specific ATPase inhibition by thapsigargin. We will synthesize high affinity thapsigargin analogs, including a radioactive azido derivative for (covalent) photo labeling of the native ATPase, proteolytic digestion, proteolytic digestion, proteolytic digestion, sequencing and determination of labeled amino acid(s) as the specific binding site. Furthermore guided by previous findings on the effects of large chimerical exchanges, we will test the effects of specific amino acid mutations on the sensitivity of the Ca2+ ATPase to thapsigargin. The specific aim is to define the topology of the thapsigargin inhibitory site and characterize the inhibitory mechanism. 3-Gene transfer and ATPase expression in foreign cells and cardiac muscle. A variety of cultured cells including cardiac myocytes will be transfected with SERCA1 or SERCA2A cDNA using various methods to obtain maximal efficiency and expression of Ca2+ ATPase. A variety of DNA constructs will be used, including dual gene constructs for expression under selective pressure, high yield promoters for overexpression, and tissue specific promoters for cardiac cells. Expression and targeting will be monitored by immunofluorescence in permeabilized cells and in isolated subcellular fractions. The specific aims are to: (a) recover large amounts of expressed ATPase for mutational studies of transport and catalytic function by the isolated enzyme, and (b) used gene transfer to established functional intracellular organelles and to influence cytosolic Ca2+ homeostasis.
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1 |
2002 — 2010 |
Inesi, Giuseppe |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular and Cellular Studies of Ca2+ Transport Atpase @ California Pacific Med Ctr Res Institute
DESCRIPTION (provided by applicant): The Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA) is the operator of active Ca2+ transport into intracellular stores. The stored Ca2+ is in turn released to trigger cytosolic Ca2"1" signaling. In cardiac and skeletal muscle Ca2+ transport and Ca2+ signaling play prominent roles in control of relaxation and contraction, as well as other functions such as transcriptional activation. These functions are altered in cardiac failure. The aims of this project are: (1) clarification of the molecular mechanism whereby ATP is utilized to move Ca2+ against a concentration gradient;(2) establishment of strategies for gene transfer into cardiac myocytes, and definition of the consequences of overexpression or silencing SERCA and other genes encoding Ca2+ signaling proteins. The research related to aim (1) will produce specific modifications in native and recombinant ATPase by protein chemistry and site directed mutagenesis, and will define the effects of these modifications on the sequential ATPase reactions that are coupled to Ca2+ transport. The findings will be related to crystallographic data and diffraction analysis, to indicate how various ATPase protein domains and specific amino acid residues are involved in energy transduction. Binding sites and specific effects of inhibitors will be defined. It is expected that the mechanism of this enzyme, as a prototype of active transport and energy transduction, will be solved at the molecular and atomic level. The research related to aim (2) will be mostly based on exogenous cDNA delivery to cardiac myocytes by means of recombinant adenovirus vectors under control of specific promoters, thereby optimizing gene transfer and silencing strategies for basic studies of cardiac cell physiology in culture. The functional consequences of SERCA up- or downregulation on Ca2+ signaling, contraction/relaxation cycle and cellular homeostasis will be defined. In addition the effects of silencing specifically SERCA or other genes (i.e., calcineurin) on transcription and expression of other proteins and remodeling of the Ca2+ signaling pathways will be studied. This work will allow us to explore and clarify a new and important concept indicating that in addition to short term functional modulation (i.e., adrenergic), long term changes in copy number, diversity and profile of Ca2+ signaling proteins are important factors in cardiac remodeling, hypertrophy, failure, and possible treatment.
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1 |
2003 |
Inesi, Giuseppe |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Faseb Summer Research Conference: Transport Atpases @ Federation of Amer Soc For Exper Biology
DESCRIPTION (provided by applicant): This is an application for partial funding for a FASEB summer research conference titled "Transport ATPases: Genomics, Mechanisms and Relevance to Diseases". The conference will be held in the Vermont Academy, Saxton River, Vermont, on July 12-17, 2003. The Conference was reviewed and approved by the appropriate FASEB Committee, and will be the fourth meeting held every two years on this subject. The major goal of the meeting is to bring together about 150 participants, including the top scientists in this field, but also younger investigators and postdoctoral fellows. The conference schedule include two keynote lectures, nine sessions with 4 talks each, poster viewing and discussion, and a postdoc forum. The subjects of discussion will be: Genomic survey of transport ATPases, crystal structure and mechanism of the Ca 2+ATPase, crystal structure and mechanism of the ABC transporters, transport ATPases in yeast systems, crystal structure and mechanisms of the ATP synthase, comparative ATPase structure and mechanisms, mechanism and modulation of plasma Na+,K+ and Ca 2+ATPases, Ca 2+ATPase in cardiac function and disease, and relevance of transport ATPases to human diseases. In addition to lectures, there will be poster presentations and a post-doc forum to foster communication among more established and younger scientists. It is expected that the conference will be highly significant with regard to both basic and medical sciences. The astounding progress in definition of crystal structures facilitates understanding of mechanisms and open up avenues for modeling and drug development. Furthermore, genomic surveys reveal an increasing number of genetically linked diseases of transport ATPases. Finally, it is clear that pathogenetic features of common disease states, such as heart failure and drug resistance, are related to altered expression and/or function of transport ATPases. The conference is related to the mission of the NHBLI (Ca 2+ and Na+,K+ATPase; ABC transporters), NIAMS (Ca 2+ATPases), NIGMS (all transport ATPases), NIDDK (CFTR, copper ATPase), NCI (MDR/Pglycoprotein and MRP1, and the NEI (ABCR). There is a good representation of established and junior scientists at the meeting, both male and female.
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0.915 |