Biomedical subjects

Alan Jasanoff

Publications and source records attributed to Alan Jasanoff.

7 recordsLinked to original sources

PubMed2006

Rational design of new binding specificity by simultaneous mutagenesis of calmodulin and a target peptide.

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies the role of CaM as a shared participant in calcium-dependent signal transduction pathways but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/target pairs that retain high affinity for one another but lack affinity for wild-type CaM and its natural interaction partners would therefore be useful as sensor components and possibly also as elements of "synthetic" cellular-signaling networks. Here, we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal-muscle light-chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained an affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple-linked binding equilibria.

Amino Acid Sequence↗

PubMed2006

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

Animals↗

PubMed2006

Dynamic imaging with MRI contrast agents: quantitative considerations.

Time-resolved MRI has had enormous impact in cognitive science and may become a significant tool in basic biological research with the application of new molecular imaging agents. In this paper, we examine the temporal characteristics of MRI contrast agents that could be used in dynamic studies. We consider "smart" T1 contrast agents, T2 agents based on reversible aggregation of superparamagnetic nanoparticles and sensors that produce changes in saturation transfer effects (chemical exchange saturation transfer, CEST). We discuss response properties of several agents with reference to available experimental data, and we develop a new theoretical model that predicts the response rates and relaxivity changes of aggregation-based sensors. We also perform calculations to define the extent to which constraints on temporal resolution are imposed by the imaging methods themselves. Our analysis confirms that some small T1 agents may be compatible with MRI temporal resolution on the order of 100 ms. Nanoparticle aggregation T2 sensors are applicable at much lower concentrations, but are likely to respond on a single second or slower timescale. CEST agents work at high concentrations and temporal resolutions of 1-10 s, limited by a requirement for long presaturation periods in the MRI pulse sequence.

Brain Mapping↗

PubMed2005

Phosphine oxide polymer for water-soluble nanoparticles.

A phosphine oxide polymer was developed using bis(dichlorophosphino)ethane and poly(ethylene glycol). This polymer system was used to transfer various nanoparticles from organic solvents to water, retaining their physical properties and reactivities.

Gels↗

PubMed2005

Functional MRI using molecular imaging agents.

Contrast agents for magnetic resonance imaging (MRI) have recently been used as cellular-level probes of neural function. New in vivo labeling strategies now enable researchers to follow plasticity of brain activation patterns and cellular structure over time. On the horizon is the prospect that molecular imaging agents specifically designed for functional imaging (fMRI) on a relatively fast timescale could offer an alternative to conventional hemodynamics-based approaches. Development of several MRI sensors has defined principles by which imaging agents for "molecular fMRI" can be constructed; application of engineered sensors for cellular-level correlates of neuronal activity would allow researchers to combine the noninvasiveness of MRI with spatial resolution of tens of microns and temporal resolution of 100ms or less. Facilitated by advances in imaging-agent delivery methods and model systems appropriate for high-resolution neuroimaging, novel molecular imaging strategies continue to potentiate MRI as a tool for mechanistic investigation of neural systems.

Animals↗

PubMed2003

In vivo oxygen detection using exogenous hemoglobin as a contrast agent in magnetic resonance microscopy.

In this work we show that exogenous molecular hemoglobin (Hb) is an effective indicator of relative local oxygen tension in magnetic resonance (MR) microscopy studies in vivo. This approach is more sensitive than other MRI oximetry methods; it can be used at higher resolutions and in specimens with no blood oxygen level-dependent (BOLD) effects. Using injection studies in flies, we show that Hb can permeate through relatively dense neural tissue, and that it is not obviously disruptive to physiology. Hb-injected flies show large changes in signal intensity (40-50%) when external O(2) levels are manipulated artificially from 0% to 21%. Oxygen-dependent contrast changes produced by exogenous Hb are detected in T(2)-weighted imaging experiments, and can be roughly calibrated if necessary. These studies demonstrate the feasibility of a contrast agent technique that may be useful for functional MRI (fMRI) studies of metabolism at tens of microns resolution.

Animals↗

PubMedDate not supplied

In vivo magnetic resonance microscopy of brain structure in unanesthetized flies.

We present near-cellular-resolution magnetic resonance (MR) images of an unanesthetized animal, the blowfly Sarcophaga bullata. Immobilized flies were inserted into a home-built gradient probe in a 14.1-T magnet, and images of voxel size (20-40 microm)(3)--comparable to the diameter of many neuronal cell bodies in the fly's brain--were obtained in several hours. Use of applied field gradients on the order of 60 G/cm allowed minimally distorted images to be produced, despite significant susceptibility differences across the specimen. The images we obtained have exceptional contrast-to-noise levels; comparison with histology-based anatomical information shows that the MR microscopy faithfully represents patterns of nervous tissue and allows distinct brain regions to be clearly identified. Even at the highest resolutions we explored, morphological detail was pronounced in the apparent absence of instabilities or movement-related artifacts frequently observed during imaging of live animal specimens. This work demonstrates that the challenges of noninvasive in vivo MR microscopy can be overcome in a system amenable to studies of brain structure and physiology.

Animals↗