Robert E. London

Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart.

Changes in cytosolic free calcium concentration during myocardial ischemia were measured by 19F NMR in 5FBAPTA-loaded perfused rat hearts. The hearts were perfused with Krebs-Henseleit buffer containing 5 μM of the acetoxymethyl ester of 5FBAPTA, which was hydrolyzed by cytosolic esterases to achieve cytosolic concentrations of 5FBAPTA of 0.12 to 0.65 mM. Cytosolic free calcium concentrations were calculated as the product of the ratio of peak areas for bound and free 5FBAPTA in the NMR spectra and the dissociation constant (708 nM). The basal cytosolic calcium concentration, measured in potassium or magnesium arrested hearts, was 252 nM, and the time-average calcium concentration in beating hearts was 630 nM. Following the onset of total ischemia, there was no immediate substantial change in cytosolic calcium despite a rapid decline in creatine phosphate and ATP and a marked increase in inorganic phosphate as monitored by 31P NMR, but by 10 minutes, there was a substantial increase in free calcium concentration. The ratio of peak areas of bound and free 5FBAPTA returned to the preischemic value during reperfusion, and there was no detectable loss of 5FBAPTA from the heart. Creatine phosphate was also restored to its preischemic level during reperfusion. These results indicate that cytosolic free calcium increases during ischemia and is not immediately associated with lethal injury. This increase in cytosolic calcium may activate degradative enzymes that eventually could compromise myocyte viability.

The structure of the dust mite allergen Der p 7 reveals similarities to innate immune proteins.

BACKGROUND Sensitization to house dust mite allergens is strongly correlated with asthma. Der p 7 elicits strong IgE antibody and T-cell responses in patients with mite allergy. However, the structure and biological function of this important allergen are unknown. Allergen function might contribute to allergenicity, as shown for the protease activity of group 1 mite allergens and the interaction with the innate immune system by group 2 mite allergens. OBJECTIVE We sought to determine the crystal structure of Der p 7 and to investigate its biological function. METHODS X-ray crystallography was used to determine the Der p 7 structure. Nuclear magnetic resonance analysis and biochemical assays were used to examine the binding of Der p 7 to predicted ligands. RESULTS Der p 7 has an elongated structure, with two 4-stranded antiparallel beta-sheets that wrap around a long C-terminal helix. The fold of Der p 7 is similar to that of LPS-binding protein (LBP), which interacts with Toll-like receptors after binding LPS and other bacterially derived lipid ligands. Nuclear magnetic resonance and biochemical assays indicate that Der p 7 does not bind LPS but binds with weak affinity to the bacterial lipopeptide polymyxin B in the predicted binding site of Der p 7. CONCLUSIONS Der p 7 binds a bacterially derived lipid product, a common feature of some allergens. The finding that the group 7, as well as the group 2, mite allergens are structurally similar to different proteins in the Toll-like receptor pathway further strengthens the connections between dust mites, innate immunity, and allergy.

Targeted Deletion of Thioredoxin-Interacting Protein Regulates Cardiac Dysfunction in Response to Pressure Overload

Biomechanical overload induces cardiac hypertrophy and heart failure, and reactive oxygen species (ROS) play a role in both processes. Thioredoxin-Interacting Protein (Txnip) is encoded by a mechanically-regulated gene that controls cell growth and apoptosis in part through interaction with the endogenous dithiol antioxidant thioredoxin. Here we show that Txnip is a critical regulator of the cardiac response to pressure overload. We generated inducible cardiomyocyte-specific and systemic Txnip-null mice (Txnip-KO) using Flp/frt and Cre/loxP technologies. Compared with littermate controls, Txnip-KO hearts had attenuated cardiac hypertrophy and preserved left ventricular (LV) contractile reserve through 4 weeks of pressure overload; however, the beneficial effects were not sustained and Txnip deletion ultimately led to maladaptive LV remodeling at 8 weeks of pressure overload. Interestingly, these effects of Txnip deletion on cardiac performance were not accompanied by global changes in thioredoxin activity or ROS; instead, Txnip-KO hearts had a robust increase in myocardial glucose uptake. Thus, deletion of Txnip plays an unanticipated role in myocardial energy homeostasis rather than redox regulation. These results support the emerging concept that the function of Txnip is not as a simple thioredoxin inhibitor but as a metabolic control protein.

Magnetic resonance imaging studies of the brains of anesthetized rats treated with manganese chloride

An understanding of the distribution of manganese ions in the brain is of interest in connection with the development of an understanding of the neurotoxicity of this element. Information about the time dependent biodistribution of manganese ions in the brains of intact rats subsequent to single IP injections of MnCl2 has been obtained from magnetic resonance imaging (MRI) studies. The enhanced MRI contrast is based on the reduction in the spin lattice relaxation time (T1) of water protons which exchange into the coordination sphere of the manganese ions. These studies indicate rapid and significant accumulations of water accessible manganese in the ventricles, the pineal gland, and the pituitary gland. The rapid appearance of high levels of manganese in the ventricular cerebrospinal fluid indicates that manganese readily crosses the filtration barrier of the choroid plexus and is thereafter apparently absorbed by the ependymal surfaces of the ventricles and transported to the subarachnoid space.

Relaxation-matrix analysis of the transferred nuclear Overhauser effect for finite exchange rates

Abstract A matrix formalism is developed for calculations of time-dependent nuclear Overhauser effects in systems undergoing chemical exchange, and the analysis is applied to the interpretation of data obtained in transferred-NOE experiments. Simulations have been performed for a wide variety of nuclear geometries, and preliminary data for the reversible binding of the inhibitor tubercidin to bacterial purine nucleoside phosphorylase have also been obtained. Although theoretically the initial buildup rate of the NOE interaction is independent of the exchange-rate constant k, this independence persists for such a short period of time that it is experimentally unobservable unless the exchange rate is extremely high. For the parameters used in the models, calculated buildup rates corresponding to mixing times of 10–100 ms exhibit a strong dependence on k. Of greatest interest is the observation that a lag in the development of the transferred NOE, generally believed to characterize indirect relaxation pathways, frequently is observable only at very high rates of chemical exchange and is so minimal as to be experimentally undetectable. Alternatively, a pronounced lag phase is predicted if the exchange rate is sufficiently slow that the observed NOE corresponds to only the free species. An apparent lag can also be predicted to result from the small negative NOEs of the uncomplexed species for some exchange parameters. The analysis has also been extended to include macromolecule (enzyme) protons, and several models for the effects of enzyme-mediated relaxation have been evaluated. These calculations, combined with the limited ability of the researcher to control most of the parameters involved in this type of study, suggest significant problems with interpretations based on initial rate measurements and strongly support the need for an independent determination of the rate of chemical exchange and for the use of model calculations such as those presented here in the interpretation of transferred-NOE studies.

Dependence of Amino Acid Side Chain 13C Shifts on Dihedral Angle: Application to Conformational Analysis

Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of side chain (13)C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cgamma resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (nonproton) gamma-substituents and with gauche conformational orientations of gamma-substituents. The (13)C shift/conformation correlations are most apparent for Cgamma carbons but also can be observed at positions further from the backbone. Intraresidue steric conflict leads to a correlation between upfield-shifted side chain (13)C resonances and statistically lower probabilities in surveys of protein side chain conformation. Illustrative applications to the DNA pol lambda lyase domain and to dihydrofolate reductase are discussed. In the latter case, (13)C shift analysis indicates that the conformation of the remote residue V119 on the betaF-betaG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. It is anticipated that (13)C shift data for protein sidechains can provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, deltaCdelta1-deltaCdelta2, on both chi1 and chi2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available.

Lactate Dehydrogenase C and Energy Metabolism in Mouse Sperm

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD+ cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC. Lack of LDHC impairs mouse sperm glycolysis but not pyruvate to lactate conversion.

Regulation of the Ca2+ Gradient Across the Sarcoplasmic Reticulum in Perfused Rabbit Heart A 19F Nuclear Magnetic Resonance Study

Myocardial contractility depends on Ca2+ release from and uptake into the sarcoplasmic reticulum (SR). The Ca2+ gradient between the SR matrix and the cytosol (SR Ca2+ gradient) is maintained by the SR Ca2+-ATPase using the free energy available from hydrolysis of ATP. The activity of the SR Ca2+-ATPase is not only dependent on the energy state of the cell but is also kinetically regulated by SR proteins such as phospholamban. To evaluate the importance of thermodynamic and kinetic regulation of the SR Ca2+ gradient, we examined the relationship between the energy available from ATP hydrolysis (DeltaGATP) and the energy required for maintenance of the SR Ca2+ gradient (DeltaGCa2+SR) during physiological and pathological manipulations that alter DeltaGATP and the phosphorylation state of phospholamban. We used our previously developed 19F nuclear magnetic resonance method to measure the ionized [Ca2+] in the SR of Langendorff-perfused rabbit hearts. We found that addition of either pyruvate or isoproterenol resulted in an increase in left ventricular developed pressure and an increase in [Ca2+]SR. Pyruvate increased DeltaGATP, and the increase in the SR Ca2+ gradient was matched to the increase in DeltaGATP; DeltaGATP increased from 58.3+/-0.5 to 60.4+/-1.0 kJ/mol (P<0.05), and DeltaGCa2+SR increased from 47.1+/-0.3 to 48.5+/-0.1 kJ/mol (P<0.05). In contrast, the increase in the SR Ca2+ gradient in the presence of isoproterenol occurred despite a decline in DeltaGATP from 58. 3+/-0.5 to 55.8+/-0.6 kJ/mol. Thus, the data indicate that the SR Ca2+ gradient can be increased by an increase in DeltaGATP, and that the positive inotropic effect of pyruvate can be explained by improved energy-linked SR Ca2+ handling, whereas the results with isoproterenol are consistent with removal of the kinetic limitation of phospholamban on the activity of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, which allows the SR Ca2+ gradient to move closer to its thermodynamic limit. Ischemia decreases DeltaGATP, and this should also have an effect on SR Ca2+ handling. During 30 minutes of ischemia, DeltaGATP decreased by 12 kJ/mol, but the decrease in DeltaGCa2+SR was 16 kJ/mol, greater than would be predicted by the fall in DeltaGATP and consistent with increased SR Ca2+ release and increased SR Ca2+ cycling. Because ischemic preconditioning is reported to decrease SR Ca2+ cycling during a subsequent sustained period of ischemia, we examined whether ischemic preconditioning affects the relationship between the fall in DeltaGATP and the fall in DeltaGCa2+SR during ischemia. We found that preconditioning attenuated the fall in DeltaGCa2+SR during ischemia; the fall in DeltaGCa2+SR was of comparable magnitude to the fall in DeltaGATP, and this was associated with a significant improvement in functional recovery during reperfusion. The data suggest that there is both thermodynamic regulation of the SR Ca2+ gradient by DeltaGATP and kinetic regulation, which can alter the relationship between DeltaGATP and DeltaGCa2+SR.

Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity

To cite this article: Mueller GA, Gosavi RA, Pomés A, Wünschmann S, Moon AF, London RE, Pedersen LC. Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity. Allergy 2011; 66: 878–885.

Reaction mechanism of the ε subunit of E. coli DNA polymerase III: Insights into active site metal coordination and catalytically significant residues

The 28 kDa epsilon subunit of Escherichia coli DNA polymerase III is the exonucleotidic proofreader responsible for editing polymerase insertion errors. Here, we study the mechanism by which epsilon carries out the exonuclease activity. We performed quantum mechanics/molecular mechanics calculations on the N-terminal domain containing the exonuclease activity. Both the free-epsilon and a complex epsilon bound to a theta homologue (HOT) were studied. For the epsilon-HOT complex Mg(2+) or Mn(2+) were investigated as the essential divalent metal cofactors, while only Mg(2+) was used for free-epsilon. In all calculations a water molecule bound to the catalytic metal acts as the nucleophile for hydrolysis of the phosphate bond. Initially, a direct proton transfer to H162 is observed. Subsequently, the nucleophilic attack takes place followed by a second proton transfer to E14. Our results show that the reaction catalyzed with Mn(2+) is faster than that with Mg(2+), in agreement with experiment. In addition, the epsilon-HOT complex shows a slightly lower energy barrier compared to free-epsilon. In all cases the catalytic metal is observed to be pentacoordinated. Charge and frontier orbital analyses suggest that charge transfer may stabilize the pentacoordination. Energy decomposition analysis to study the contribution of each residue to catalysis suggests that there are several important residues. Among these, H98, D103, D129, and D146 have been implicated in catalysis by mutagenesis studies. Some of these residues were found to be structurally conserved on human TREX1, the exonuclease domains from E. coli DNA-Pol I, and the DNA polymerase of bacteriophage RB69.