The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Amide proton transfer (APT) imaging may potentially detect mobile proteins/peptides non-invasively in vivo, but its specificity may be reduced by contamination from other confounding effects such as asymmetry of non-specific magnetization transfer (MT) effects and spin-lattice relaxation with rate R1 (=1/T1). Previously reported spillover, MT and R1 correction methods were based on a two-pool model, in which the existence of multiple water compartments with heterogeneous relaxation properties in real tissues was ignored. Such simple models may not adequately represent real tissues, and thus such corrections may be unreliable. The current study investigated the effectiveness and accuracy of correcting for R1 in APT imaging via simulations and in vivo experiments using tumor-bearing rats subjected to serial injections of Gd-DTPA that produced different tissue R1 values in regions of blood-brain-barrier breakdown. The results suggest that conventional measurements of APT contrast (such as APT* and MTRasym ) may be significantly contaminated by R1 variations, while the R1 -corrected metric AREX* was found to be relatively unaffected by R1 changes over a broad range (0.4-1 Hz). Our results confirm the importance of correcting for spin-lattice relaxation effects in quantitative APT imaging, and demonstrate the reliability of using the observed tissue R1 for corrections to obtain more specific and accurate measurements of APT contrast in vivo. The results also indicate that, due to relatively fast transcytolemmal water exchange, the influence of intra- and extracellular water compartments on CEST measurements with seconds long saturation time may be ignored in tumors.
Copyright © 2015 John Wiley & Sons, Ltd.
Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10.
Copyright © 2014 the authors 0270-6474/14/3414079-17$15.00/0.
RATIONALE - Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear.
OBJECTIVE - To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering and to determine the resulting arrhythmogenic changes in Ca homeostasis in the intact mouse heart.
METHODS AND RESULTS - Using cardiomyocytes isolated from mice expressing troponin T (TnT) mutants (TnT-I79N, TnT-F110I, TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca binding. The underlying cause was an increase in the cytosolic Ca binding affinity, whereas maximal Ca binding capacity was unchanged. The effect was sufficiently large to alter Ca handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca content and release after pauses. Accordingly, action potential (AP) regulation was altered, with postpause action potential prolongation, afterdepolarizations, and triggered activity. Acute Ca sensitization with EMD 57033 mimicked the effects of Ca-sensitizing TnT mutants and produced pause-dependent ventricular ectopy and sustained ventricular tachycardia after acute myocardial infarction.
CONCLUSIONS - Myofilament Ca sensitization increases cytosolic Ca binding affinity. A major proarrhythmic consequence is a pause-dependent potentiation of Ca release, action potential prolongation, and triggered activity. Increased cytosolic Ca binding represents a novel mechanism of pause-dependent arrhythmia that may be relevant for inherited and acquired cardiomyopathies.
Muscle functional magnetic resonance imaging (MRI) refers to changes in the contrast properties of certain MR images that occur in exercising muscles. In part, these changes result indirectly from increased rates of cellular energy metabolism, which alter the image contrast properties by increasing the water content and by decreasing the intracellular pH. Also, increases in blood oxygen extraction cause a rapidly evolving, small, and negative contribution to signal. Together, these changes produce a complex time course of contrast changes during exercise. Analysis of this time course may provide insight into the physiology of exercising muscles. These contrast changes also provide a non-invasive method for determining the spatial pattern of muscle activation.
Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Galpha(i) and Galpha(q) proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.
Peripheral nerve section promotes regenerative, elongated neuritic growth of adult sensory neurons. Although the role of chloride homeostasis, through the regulation of ionotropic GABA receptors, in the growth status of immature neurons in the CNS begins to emerge, nothing is known of its role in the regenerative growth of injured adult neurons. To analyze the intracellular Cl- variation after a sciatic nerve section in vivo, gramicidin perforated-patch recordings were used to study muscimol-induced currents in mice dorsal root ganglion neurons isolated from control and axotomized neurons. We show that the reversal potential of muscimol-induced current, E(GABA-A), was shifted toward depolarized potentials in axotomized neurons. This was attributable to Cl- influx because removal of extracellular Cl- prevented this shift. Application of bumetanide, an inhibitor of NKCC1 cotransporter and E(GABA-A) recordings in sensory neurons from NKCC1-/- mice, identified NKCC1 as being responsible for the increase in intracellular Cl- in axotomized neurons. In addition, we demonstrate with a phospho-NKCC1 antibody that nerve injury induces an increase in the phosphorylated form of NKCC1 in dorsal root ganglia that could account for intracellular Cl- accumulation. Time-lapse recordings of the neuritic growth of axotomized neurons show a faster growth velocity compared with control. Bumetanide, the intrathecal injection of NKCC1 small interfering RNA, and the use of NKCC1-/- mice demonstrated that NKCC1 is involved in determining the velocity of elongated growth of axotomized neurons. Our results clearly show that NKCC1-induced increase in intracellular chloride concentration is a major event accompanying peripheral nerve regeneration.
The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Delta6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Delta6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Delta6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Delta6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Delta6-27p33) did not exhibit detectable inhibitory activity. The Delta6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Delta6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Delta6-27p33 or Delta(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.
Endothelial cells preserve vascular integrity in part by synthesizing type IV collagen for the basement membrane of blood vessels. Vitamin C, which at physiologic pH is largely the ascorbate mono-anion, both protects these cells from oxidant stress and is required for collagen synthesis. Therefore, cultured endothelial cells were used to correlate intracellular concentrations of ascorbate with its uptake and ability to stimulate collagen release into the culture medium. The kinetics and inhibitor specificity of ascorbate transport into EA.hy926 endothelial cells were similar to those observed in other cell types, indicative of a specific high affinity transport process. Further, transport of the vitamin generated intracellular ascorbate concentrations that were 80-100-fold higher than concentrations in the medium following overnight culture, and transport inhibition with sulfinpyrazone and phloretin partially prevented such ascorbate accumulation. On the other hand, low millimolar intracellular concentrations of ascorbate impaired its transport measured after overnight culture. Synthesis and release of type IV collagen into the culture medium was markedly stimulated by ascorbate in a time-dependent manner, and was saturable with increasing medium concentrations of the vitamin. Optimal rates of collagen synthesis required intracellular concentrations of the vitamin up to 2 mM. Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium.
Etomidate, an intravenous imidazole general anaesthetic, is thought to produce anaesthesia by modulating or activating ionotropic Cl(-)-permeable GABA(A) receptors. Chromaffin cells are known to express functional GABA(A) receptors with properties similar to their neuronal counterparts. We have shown that activation of the GABA(A) receptors, with specific GABA(A) agonists, leads to cellular excitation. Our goal was to determine whether etomidate mimicked this response and to explore the functional consequences of this activation. Imaging experiments with the Ca(2+)-indicator dye fura-2 were used to assay [Ca(2+)](i). Bovine adrenal chromaffin cells were superfused with a variety of GABA(A)-selective drugs to determine their effects on [Ca(2+)](i). Amperometric measurements were used to assay catecholamine release in real-time. We show that bovine adrenal chromaffin cells were excited by etomidate at clinically relevant concentrations. Etomidate directly activated GABA(A) receptors found in chromaffin cells thereby elevating [Ca(2+)](i). The effects of etomidate were mimicked by the specific GABA(A) agonist muscimol and blocked by the specific antagonist bicuculline. Our data show that low concentrations of etomidate modulated GABA(A) receptor activation by muscimol. Blockade of voltage-dependent Ca(2+) channels prevented the elevation of [Ca(2+)](i) by GABA. Application of etomidate directly to the chromaffin cells elicited robust catecholamine secretion from these cells. The data indicate that clinically relevant concentrations of etomidate can directly activate GABA(A) receptors, which, due to the positive anion equilibrium potential, depolarizes chromaffin cells. This depolarization activates voltage-dependent Ca(2+) channels thereby stimulating catecholamine release. Our data suggest that circulating catecholamine levels may be elevated after etomidate application.
Recently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure. We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of H(2)O(2), peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells (BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 microM menadione), and polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues.