Imaging of Myocardial Fatty Acid Oxidation

Myocardial fuel selection is a key feature of the health and function of the heart, with clear links between myocardial function and fuel selection and important impacts of fuel selection on ischemia tolerance. Radiopharmaceuticals provide uniquely valuable tools for in vivo, non-invasive assessment of these aspects of cardiac function and metabolism. Here we review the landscape of imaging probes developed to provide noninvasive assessment of myocardial fatty acid oxidation (MFAO). Also, we review the state of current knowledge that myocardial fatty acid imaging has helped establish of static and dynamic fuel selection that characterizes cardiac and cardiometabolic disease and the interplay between fuel selection and various aspects of cardiac function

Acid activation mechanism of the influenza A M2 proton channel

The homotetrameric influenza A M2 channel (AM2) is an acid-activated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.United States. National Institutes of Health (R01-GM088204

Computational Design and Elaboration of a De Novo Heterotetrameric α-Helical Protein that Selectively Binds an Emissive Abiological (Porphinato)zinc Chromophore

The first example of a computationally de novo designed protein that binds an emissive abiological chromophore is presented, in which a sophisticated level of cofactor discrimination is pre-engineered. This heterotetrameric, C(2)-symmetric bundle, A(His):B(Thr), uniquely binds (5,15-di[(4-carboxymethyleneoxy)phenyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The A(2)B(2) heterotetrameric protein reflects ligand-directed elements of both positive and negative design, including hydrogen bonds to second-shell ligands. Experimental support for the appropriate formulation of [(DPP)Zn:A(His):B(Thr)](2) is provided by UV/visible and circular dichroism spectroscopies, size exclusion chromatography, and analytical ultracentrifugation. Time-resolved transient absorption and fluorescence spectroscopic data reveal classic excited-state singlet and triplet PZn photophysics for the A(His):B(Thr):(DPP)Zn protein (k(fluorescence) = 4 x 10(8) s(-1); tau(triplet) = 5 ms). The A(2)B(2) apoprotein has immeasurably low binding affinities for related [porphinato]metal chromophores that include a (DPP)Fe(III) cofactor and the zinc metal ion hemin derivative [(PPIX)Zn], underscoring the exquisite active-site binding discrimination realized in this computationally designed protein. Importantly, elements of design in the A(His):B(Thr) protein ensure that interactions within the tetra-alpha-helical bundle are such that only the heterotetramer is stable in solution; corresponding homomeric bundles present unfavorable ligand-binding environments and thus preclude protein structural rearrangements that could lead to binding of (porphinato)iron cofactors

Imaging of cardiac neuronal function after cocaine exposure using Carbon-11 hydroxyephedrine and positron emission tomography

AbstractObjectives. The aim of the study was to define the effect of cocaine on the myocardial uptake and retention of C-11 hydroxyephedrine in the anesthetized dog model.Background. Cardiac toxcity of cocaine has been linked to its inhibitory effect on norepinephrine reuptake by the sympathetic nerve terminals of the heart. Carbon-11 hydroxyephedrine is a C-11-labled norepinephrine analog that has high specific affinity for uptake-1 and thus makes possible the assessment of the effect of cocaine on norepinephrine reuptake by cardiac sympathetic nerve terminals.Methods. The cardiac kinetics of C-11 hydroxyephedrine as assessed by dynamic positron emission tomographic imaging were used to characterize norepinephrine reuptake by the sympathetic nerve terminals. Carbon-11 hydroxyephedrine was injected intravenously before, as well as at 5 min and 2.5 h after, intravenous administration of 2 mg/kg body weight of cocaine in anesthetized dogs. Hemodynamic variables and microsphere-determined cardiac blood flow were also measured before and after cocaine exposure.Results. Intravenous injection of cocaine did not significantly affect hemodynamic variables and myocardial blood flow in the anesthetized animals. Compared with baseline, myocardial retention of C-11 hydroxyephedrine was significantly reduced by 78 ± 3% (mean ± SD) at 5 min and remained significantly reduced (28 ± 17%) at 2.5 h after cocaine injection. Cocaine administration after C-11 hydroxyephedrine injection (39 min) resulted in rapid biexponential clearance of C-11 hydroxyephedrine from myocardium.Conclusions. These results suggest prolonged effects of cocaine on the sympathetic nerve terminals of the heart. Positron emission tomography provides a noninvasive and sensitive means to objectively assess the cardiac pharmacokinetics of drugs such as cocaine

Adhesion of MC3T3-E1 cells to RGD peptides of different flanking residues: Detachment strength and correlation with long-term cellular function

We synthesized a series of RGD peptides and immobilized them to an amine-functional self-assembled monolayer using a modified maleimide-based conjugate technique that minimizes nonspecific interactions. Using a spinning disc apparatus, a trend in the detachment strength (τ50) of RGD peptides of different flanking residues was found: RGDSPK ≻ RGDSVVYGLR ≈ RGDS ≻ RGES. Using blocking monoclonal antibodies, cellular adhesion to the peptides was shown to be primarily α√-integrin-mediated. In contrast, the τ50 value of the cells on fibronectin (Fn)-coated substrates of similar surface density was 6-7 times higher and involved both α5β1 and ανβ3 integrins. Cellular spreading was enhanced on RGD peptides after 1 h when compared to RGE and unmodified substrates. However, no significant differences were observed between the different RGD peptides. Long-term function of MC3T3-E1 cells was also evaluated by measuring alkaline phosphatase (ALP) activity and mineral deposition. Among the four peptides, RGDSPK exhibited the highest level of ALP activity after 11 days and mineralization after 15 days and reached comparable levels as Fn substrates after 15 and 24 days, respectively. These findings collectively illustrate both the advantages and limitations of enhancing cellular adhesion and function by the design of RGD peptides

Spectral analysis on infinite Sierpinski fractafolds

A fractafold, a space that is locally modeled on a specified fractal, is the fractal equivalent of a manifold. For compact fractafolds based on the Sierpinski gasket, it was shown by the first author how to compute the discrete spectrum of the Laplacian in terms of the spectrum of a finite graph Laplacian. A similar problem was solved by the second author for the case of infinite blowups of a Sierpinski gasket, where spectrum is pure point of infinite multiplicity. Both works used the method of spectral decimations to obtain explicit description of the eigenvalues and eigenfunctions. In this paper we combine the ideas from these earlier works to obtain a description of the spectral resolution of the Laplacian for noncompact fractafolds. Our main abstract results enable us to obtain a completely explicit description of the spectral resolution of the fractafold Laplacian. For some specific examples we turn the spectral resolution into a "Plancherel formula". We also present such a formula for the graph Laplacian on the 3-regular tree, which appears to be a new result of independent interest. In the end we discuss periodic fractafolds and fractal fields

Microwave Assisted Synthesis of Py-Im Polyamides

Microwave synthesis was utilized to rapidly build Py-Im polyamides in high yields and purity using Boc-protection chemistry on Kaiser oxime resin. A representative polyamide targeting the 5′-WGWWCW-3′ (W = A or T) subset of the consensus Androgen and Glucocorticoid Response Elements was synthesized in 56% yield after 20 linear steps and HPLC purification. It was confirmed by Mosher amide derivatization of the polyamide that a chiral α-amino acid does not racemize after several additional coupling steps

De Novo Design of a Single Chain Diphenylporphyrin Metalloprotein

We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes

Computational De Novo Design and Characterization of a Protein That Selectively Binds a Highly Hyperpolarizable Abiological Chromophore

This work reports the first example of a single-chain protein computationally designed to contain four α-helical segments and fold to form a four-helix bundle encapsulating a supramolecular abiological chromophore that possesses exceptional nonlinear optical properties. The 109-residue protein, designated SCRPZ-1, binds and disperses an insoluble hyperpolarizable chromophore, ruthenium(II) [5-(4\u27-ethynyl-(2,2\u27;6\u27,2″-terpyridinyl))-10,20-bis(phenyl)porphinato]zinc(II)-(2,2\u27;6\u27,2″-terpyridine)(2+) (RuPZn) in aqueous buffer solution at a 1:1 stoichiometry. A 1:1 binding stoichiometry of the holoprotein is supported by electronic absorption and circular dichroism spectra, as well as equilibrium analytical ultracentrifugation and size exclusion chromatography. SCRPZ-1 readily dimerizes at micromolar concentrations, and an empirical redesign of the protein exterior produced a stable monomeric protein, SCRPZ-2, that also displayed a 1:1 protein:cofactor stoichiometry. For both proteins in aqueous buffer, the encapsulated cofactor displays photophysical properties resembling those exhibited by the dilute RuPZn cofactor in organic solvent: femtosecond, nanosecond, and microsecond time scale pump-probe transient absorption spectroscopic data evince intensely absorbing holoprotein excited states having large spectral bandwidth that penetrate deep in the near-infrared energy regime; the holoprotein electronically excited triplet state exhibits a microsecond time scale lifetime characteristic of the RuPZn chromophore. Hyper-Rayleigh light scattering measurements carried out at an incident irradiation wavelength of 1340 nm for these holoproteins demonstrate an exceptional dynamic hyperpolarizabilty (β1340 = 3100 × 10(-30) esu). X-ray reflectivity measurements establish that this de novo-designed hyperpolarizable protein can be covalently attached with high surface density to a silicon surface without loss of the cofactor, indicating that these assemblies provide a new approach to bioinspired materials that have unique electro-optic functionality

Increased Brain Fatty Acid Uptake in Metabolic Syndrome

OBJECTIVE: To test whether brain fatty acid uptake is enhanced in obese subjects with metabolic syndrome (MS) and whether weight reduction modifies it. RESEARCH DESIGN AND METHODS: We measured brain fatty acid uptake in a group of 23 patients with MS and 7 age-matched healthy control subjects during fasting conditions using positron emission tomography (PET) with [(11)C]-palmitate and [(18)F]fluoro-6-thia-heptadecanoic acid ([(18)F]-FTHA). Sixteen MS subjects were restudied after 6 weeks of very low calorie diet intervention. RESULTS: At baseline, brain global fatty acid uptake derived from [(18)F]-FTHA was 50% higher in patients with MS compared with control subjects. The mean percentage increment was 130% in the white matter, 47% in the gray matter, and uniform across brain regions. In the MS group, the nonoxidized fraction measured using [(11)C]-palmitate was 86% higher. Brain fatty acid uptake measured with [(18)F]-FTHA-PET was associated with age, fasting serum insulin, and homeostasis model assessment (HOMA) index. Both total and nonoxidized fractions of fatty acid uptake were associated with BMI. Rapid weight reduction decreased brain fatty acid uptake by 17%. CONCLUSIONS: To our knowledge, this is the first study on humans to observe enhanced brain fatty acid uptake in patients with MS. Both fatty acid uptake and accumulation appear to be increased in MS patients and reversed by weight reduction