1985 |
Krafft, Grant Arthur |
R23Activity Code Description: Undocumented code - click on the grant title for more information. |
Cytochalasin/Probes of Cytoskeletal Function @ Syracuse University At Syracuse
The cytochalasins are a group of cytotoxic fungal metabolites which exhibit a complex array of biological activities. These molecules have been used extensively as probes of cell biological processes. The specific objectives of our investigation are: (1)\the design and synthesis of simplified cytochalasin analogs into which [unreadable]13[unreadable]C-labels, fluorophores, affinity labeling functionality, and/or modified functional groups have been incorporated; (2)\the determination of specific structure-activity correlations of simplified cytochalasins; (3)\determination of the specific molecular aspects of actin polymerization; and (4)\identification and characterization of cellular cytochalasin receptors. Efficient synthetic strategies have been developed to prepare functionally versatile perhydroisoindolones from which simplified cytochalasin derivatives are being prepared. One of these simplified derivatives, lacking the C-7 OH and the large ring retains ca. 50 percent of the actin assembly inhibition activity of cytochalasin D. It is anticipated that subsequently synthesized analogs will exhibit a more well-defined spectrum of biological activities, enabling delineation of specific molecular events involved with membrane and cytoskeletal processes. (L)
|
0.943 |
1985 — 1986 |
Krafft, Grant Arthur |
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. |
Photoactive Fluorophores @ Syracuse University At Syracuse
The design, synthesis and application of a new type of fluorescent biological probe is proposed. Photoactivable fluorophores (PAFs) will possess latent fluorescent properties that can be revealed by photochemical activation. Activation of PAFs attached to cellular components or antibody complexes will selectively switch "on" fluorescence at any arbitrarily designated time and in a spatially well-defined cellular compartment or location. PAF's will enable studies of cellular dynamics, motility and translocation phenomena, now difficult or impossible using conventional fluorescence techniques or other tracer methodology. Several fundamentally different types of PAFs will be prepared, each based on a different photoinitiated chemical sequence to convert the non-fluorescent PAFs to ultimate fluorophores. Deactivating or quenching protecting groups, removable by photochemical reaction, will be integral components of one type of PAF. Photostimulated nucleophilic aromatic substitution, photoinduced stilbene type cyclizations, and photo chemically generated nitrenes and carbenes, which can readily cyclize by intramolecular C-H bond insertions, will provide the mechanistic bases for other types of PAFs. Studies will be conducted to determine kinetics, efficiency and wavelength dependence of the photoactivation process and to evaluate fluorescence characteristics of the PAFs and photogenerated fluorescent molecules. Purified actin will be labeled with a suitably functionalized PAF, and employed in the development of new fluorescence technique. The new "fluorescence dissipation" technique (to be developed in collaboration with Professor Ben Ware) will be a highly sensitive replacement for fluorescence photobleaching recovery (FPR) methods in studies of actin assembly. This new technique will be a generally applicable replacement for FPR methods in many other types of investigations. Photoactivable fluorophores will represent an innovative technology, capable of revolutionizing existing fluorescence-based studies and breaking new ground in emerging biophysical and biochemical investigations of significant relevance to fundamental biomedical research.
|
0.943 |
1996 — 2000 |
Krafft, Grant Arthur |
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. |
Structure/Function of Alzheimers Amyloid-B Aggregates @ Northwestern University |
1 |
1997 — 2001 |
Krafft, Grant Arthur |
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. |
Preparation and Characterization of Ab-Derived Active Ligands
This Program Project seeks to test the hypothese that non-fibrillar aggregated assemblies of amyloid beta (AB), which we have term Abeta-derived active ligands (ADALs), are responsible for triggering processes leading to the glial activation, neuronal plasticity malfunction, synaptic degeneration and cell death in Alzheimer's disease. Extending this hypothesis, it is proposed that particular glial proteins modulate competing Abeta assembly process-to enhance ADAL formation. In this program project we will prepare and characterize ADALs, definign conditions that promote their formation, and we will identify the specific responses activated by ADALs in neurons, glia, mixed cultures and organotypic brain slices. These studies will be coordinated with histochemical studies to establish the relevance of ADALs to AD pathology. The focus of project 1 is the preparation and characterization of ADALs and investigation of conditions reponsible for their formation. We will define optimal in vitro conditions for reproducible formation of ADALs,preparing sufficient quantities for structural, biochemical and biophysical characterization studies, and for investigations of their biological responses in collaboration with Projects 2 and 3. The mechanisms governing ADAL formation will be investigated, with an emphasis on the role played by selected glial co-factor proteins associated wit Abeta in AD. We will extend our studies involving atomic force microscopy (AFM) to provide more detailed analysis of ADAL morphology and distribution, and we will use techniques such as field flow fractionation and native gel electrophoresis for ADAL purification. We will employ laser light scattering, analytical ultracentrifugation and fluroescence polarization and energy transfer techniques to investigate ADAL assembly mechanisms. The major questions to be addressed in this project are: What experimental conditions favor the formation of ADALs and what techniques enable the isolation of homogeneous preparation of ADAL structures? What factors govern the formation of different ADAL structures, and inactive fibrillar or soluble Abeta assemblies? What are the biochemical and biophysical characteristics of ADALs? What specific features of the Abeta 1-42 primary structure are important for assembly into different ADAL and fibrillar structures? What amino acid residues are important for the biological activity of ADALs in neurons? in glia? The integration of these studies with the extensive biological capabilities within this Program Project will define the molecular and cellular processes that lead to the inflammatory pathology, synaptic degeneration and neuronal cell death that occurs in Alzheimer's disease.
|
0.91 |
1997 — 2001 |
Krafft, Grant Arthur |
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. |
Supramolecular Abeta Structure--Glial/Neuronal Response
The overall hypothesis addressed by this program is that non- fibrillar aggregated assemblies of amyloid beta (Abeta) which we refer to as Abeta-derived active ligands (ADALs) are active in triggering Alzheimer's disease (AD)-specific cellular responses leading to glial activation, neuronal plasticity malfunction, degeneration and ultimately cell death. We further propose that the generation of these active supramolecular structures of Abeta aggregates can be influenced by the presence of other plaque components, particularly those components derived film glia, and that responses of glial cells contribute to an environment that facilitates and enhances the formation of these bioactive ADALs. It is the goal of this program to isolate and characterize the ADALs examine the conditions that promote their formation, determine the glial and neuronal responses elicited by specific ADALs, and elucidate the interplay of ADAL-glial-neuronal responses. A longer term goal is correlation of bioactive Abeta structures and cellular responses that we characterize in vitro with evidence that these same Abeta structures and responses are associated with pathology in AD brain. Towards these goals, three highly integrated and interactive projects are proposed. Project l will prepare and characterize ADALs structurally, examine the kinetics of ADAL formation (with or without added glial proteins provided by project 2), and determine what factors influence the formation of ADAl-s. The ADAL preps will be provided to Projects 2 and 3 for evaluation of bioactivity on glia and neurons, respectively. Immunohistochemical studies on AD brain tissue will also be done to document whether manifestations of specific molecular events induced by Abeta in glia or neuronal cultures can be detected in AD brain. These cooperative and synergistic interactions among a group of investigators with complementary expertises provide a broad-based, yet focused approach to addressing fundamental mechanistic questions about the processes and pathways involved in generation of AD neuropathology. In addition, our collaborations have led to significant new discoveries about the genesis of neurotoxic amyloid, the influence of glial-derived proteins on amyloid toxicity, and the specific molecular signaling pathways that mediate amyloid Abeta- induced neurodegeneration. These extensive feasibility data suggest a high probability for successful accomplishment of our proposed goals.
|
0.91 |
1998 — 2002 |
Krafft, Grant Arthur |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Structure &Function of Alzheimers Amyloid Beta Aggregates
Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disease characterized by cognitive decline, and at autopsy, its distinct brain pathologies, neurofibrillary tangles and neuritic plaques. The major protein component of neuritic plaques is a peptide known as amyloid _ (A_), mechanistically linked to AD by virtue of mutations in its precursor protein (APP). The objective of this research is to define the structure of A_ aggregates and precisely determine the molecular interactions responsible for AD-relevant neuronal responses elicited by A_. A further objective is to characterize structural aspects of A_ interactions with two other plaque components, apolipoprotein E (apoE) and apoJ, which are elevated in AD brain tissue. Definition of molecular interactions linked to AD-relevant neuronal responses is essential for identification of drug targets to slow or block the progression of disease pathology. Mass spectrometric techniques are crucial t o t hesemechanistic studies.
|
0.952 |