23 research outputs found
2-Triazole-substituted adenosines: a new class of selective Aā adenosine receptor agonists, partial agonists, and antagonists
''Click chemistry" was explored to synthesize two series of 2-(1,2,3-triazolyl) adenosine derivatives (1-14). Binding affinity at the human A(1), A(2A), and A(3)ARs (adenosine receptors) and relative efficacy at the A(3)AR were determined. Some triazol-1-yl analogues showed A(3)AR affinity in the low nanomolar range, a high ratio of A(3)/A(2A) selectivity, and a moderate-to-high A(3)/A(1) ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A(3)AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A(3)AR efficacy. Thus, several 5'-OH derivatives appeared to be selective A(3)AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A(1)AR and displaying a characteristic docking mode in an A(3)AR model. The corresponding 5'-ethyluronamide analogues generally showed increased A(3)AR affinity and behaved as full antagonists, i.e., 17, with 910-fold A(3)/A(1) selectivity. Thus, N-6-substituted 2-( 1,2,3-triazolyl)-adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A(3)AR antagonists, partial agonists, and agonists
A Chemical Glycoproteomics Platform Reveals O-GlcNAcylation of Mitochondrial Voltage-Dependent Anion Channel 2
SummaryProtein modification by O-linked Ī²-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2ā/ā cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating aĀ functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts
Isotopic Signature Transfer and Mass Pattern Prediction (IsoStamp): An Enabling Technique for Chemically-Directed Proteomics
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New Tools for Chemically Directed Glycoproteomics and Xe-based MRI Contrast Agents
In recent years, technological advancements in the fields of mass spectrometry (MS) and molecular imaging have enabled scientists to ask and answer questions that once seemed impossibly challenging. For example, advancements in mass spectrometry have made it possible to provide a quantitative description of a human cell proteome, where both the identities and absolute abundances of thousands of proteins are simultaneously and reproducibly calculated. Equivalently, advancements in molecular imaging have made it possible to monitor the dynamics of posttranslational modifications, such as glycosylation, in living organisms. Inspired by these advancements, this dissertation is divided into two sections that describe chemical approaches for both identifying and imaging biomolecules in living systems. In the first section, the biomolecules of interest are glycoproteins, and I describe chemical tools and computational strategies for identifying glycoproteins through MS. After a brief discussion of the importance of protein glycosylation in Chapter 1, I survey chemical methods that facilitate the study of glycoproteins through MS in Chapter 2. A major theme that emerges from this discussion is that although glycoproteins are highly abundant biomolecules, they exist as a complex mixture of enormously diverse structures, which has made identifying any one member of the population a great analytical challenge. In Chapter 3, I introduce the concept of chemically directed proteomics. This method is used to direct MS analysis to specific species of interest (regardless of abundance) by chemically tagging them with an identifiable isotopic signature. Towards extending chemically directed proteomics beyond chemical labeling, in Chapter 4 I explore metabolic strategies to incorporate an isotopic signature directly into glycans using a specific mixture of monosaccharide isotopologs. In the second section, the focus is molecular imaging, and I describe how viral nanoparticles can be used as molecular scaffolds to develop high sensitivity contrast agents for magnetic resonance imaging (MRI). After a brief discussion of the advantageous role that viral nanoparticles have played in the development of gadolinium-based contrast agents in Chapter 5, I then focus on a new class of contrast agents known as xenon biosensors. In Chapter 6, I describe the utility of xenon-based MRI for in vivo image and illustrate that through the combination of hyperpolarization and chemical exchange saturation transfer detection, xenon biosensors can achieve low detection thresholds.Towards improving the detection sensitivity of xenon MRI further, biosensors can be assembled onto supramolecular scaffolds such as viral nanoparticles. In Chapter 7, I discuss the development of a bacteriophage MS2-based xenon biosensor-the first viral capsid functionalized as a 129Xe-based MRI contrast agent. Subsequently, in Chapter 8, I extend the application of viral capsids from spherical bacteriophage to filamentous bacteriophage by generating an M13 bacteriophage-based xenon biosensor. Bacteriophages where chosen because they are routinely used in phage display techniques for identifying new epitope-targeting groups such as peptides and antibody fragments. Accordingly, in Chapter 9, I describe the development of a phage-based xenon biosensor that possesses antibody fragments for targeted imaging of cancer cells in vitro.In summary, this dissertation describes a number of chemical approaches for both identifying and imaging biomolecules within biologically relevant environments. In the future, it will be exciting to watch as these tools are further refined and improved upon to address outstanding questions in disease biology. In particular, there are a number of research projects underway to identify cancer-related cell surface glycoproteins that can serve as biomarkers for disease states. Additionally, efforts are underway to apply phage-based xenon biosensors to lung cancer detection and imaging in vivo
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A New Algorithm for Computational Image Analysis of Deformable Motion at High Spatial and Temporal Resolution Applied to Root Growth: Roughly Uniform Elongation in the Meristem and also, After an Abrupt Acceleration, in the Elongation Zone
A requirement for understanding morphogenesis is being able to quantify expansion at the cellular scale. Here, we present new software (RootflowRT) for measuring the expansion profile of a growing root at high spatial and temporal resolution. The software implements an image processing algorithm using a novel combination of optical flow methods for deformable motion. The algorithm operates on a stack of nine images with a given time interval between each (usually 10 s) and quantifies velocity confidently at most pixels of the image. The root does not need to be marked. The software calculates components of motion parallel and perpendicular to the local tangent of the root\u27s midline. A variation of the software has been developed that reports the overall root growth rate versus time. Using this software, we find that the growth zone of the root can be divided into two distinct regions, an apical region where the rate of motion, i.e. velocity, rises gradually with position and a subapical region where velocity rises steeply with position. In both zones, velocity increases almost linearly with position, and the transition between zones is abrupt. We observed this pattern for roots of Arabidopsis, tomato (Lycopersicon lycopersicum), lettuce (Lactuca sativa), alyssum (Aurinia saxatilis), and timothy (Phleum pratense). These velocity profiles imply that relative elongation rate is regulated in a step-wise fashion, being low but roughly uniform within the meristem and then becoming high, but again roughly uniform, within the zone of elongation. The executable code for RootflowRT is available from the corresponding author on request
Direct detection of nitrotyrosine-containing proteins using an aniline-based oxidative coupling strategy
A convenient two-step method is described for the detection of nitrotyrosine-containing proteins. First, nitrotyrosines are reduced to aminophenols using sodium dithionite. Following this, an oxidative coupling reaction is used to attach anilines bearing fluorescence reporters or affinity probes. Features of this approach include fast reaction times, pmol-level sensitivity, and excellent chemoselectivity
A Chemical Method for Labeling Lysine Methyltransferase Substrates
LuxCorp was exhibited at Canberra Contemporary Art Space in May as part of the Metis 2004 arts and science festival.
CD 1/28/2008Materials:
Bioluminescent bacteriainstallation view, Luxcorp installatio