Use of gated perfusion to study early effects of anoxia on cardiac energy metabolism: a new 31P NMR method

A novel 31P NMR method is described that is capable of determining rapid changes in the intracellular levels of various phosphorus-containing compounds in an isolated, perfused working rat heart. This technique involves the gating of 31P NMR measurements to a heart that is alternately perfused with a modified Krebs-Henseleit medium containing 10 mM pyruvate and equilibrated with either 95% O2/5% CO2 or 95% N2/5% CO2. The experimental design allows up to three NMR measurements to be made during a single O2/N2 perfusion cycle. When these measurements are repeated at different intervals during the cycle, rapid changes in metabolite levels can be determined. Preliminary studies have shown that hearts remain hemodynamically stable to the aerobic/anoxic perfusion cycle as judged by heart rate, peak systolic pressure, aortic output, and coronary flow for at least 80 min in the magnet when subjected to cycle times of 4.5-s O2 and 1.5-s N2 perfusions. NMR measurements made under these conditions showed that a transition from full aerobic perfusion to this cycle revealed a new steady state, with an increased inorganic phosphate level from 6% total observable phosphorus to 10% and a possibly significant decreased measurement of creatine phosphate level (from 35 to 31%). Comparison of individual NMR measurements made during this perfusion cycle shows apparent rapid cyclical variations in intracellular pH and the levels of Pi, ATP, and NAD(H). These changes, expressed as variations above and below mean values measured during the cycle, showed that (a) intracellular pH, as measured by the chemical shift of Pi, reversibly decreases by more than 0.1 pH unit within 0.5-1 s following maximal anoxic perfusion and (b) coincident with a decrease in intracellular pH, Pi levels increased by a maximum of 30-40% whereas ATP levels decreased by a maximum of 15-20%. The amount of total observable phosphorous detected during the cycle is essentially constant. Unexpectedly, creatine phosphate levels are most stable, indicating that their levels are being maintained at the expense of ATP. Also unexpected is the finding that NAD(H) levels varied from maximal to undetectable levels during the perfusion cycle. The current method of aerobic/anoxic perfusion is capable of resolving metabolic events much faster than previous NMR methods and yielding information that is unobtainable by any other technique.     

Evolutionary Psychology, Human Universals, and the Standard Social Science Model

Proponents of evolutionary psychology take the existence of humanuniversals to constitute decisive evidence in favor of their view. Ifthe same social norms are found in culture after culture, we have goodreason to believe that they are innate, they argue. In this paper Ipropose an alternative explanation for the existence of humanuniversals, which does not depend on them being the product of inbuiltpsychological adaptations. Following the work of Brian Skyrms, I suggestthat if a particular convention possesses even a very small advantageover competitors, whatever the reason for that advantage, we shouldexpect it to become the norm almost everywhere. Tiny advantages aretranslated into very large basins of attraction, in the language of gametheory. If this is so, universal norms are not evidence for innatepsychological adaptations at all. Having shown that the existence ofuniversals is consistent with the so-called Standard Social ScienceModel, I turn to a consideration of the evidence, to show that thisstyle of explanation is preferable to the evolutionary explanation, atleast with regard to patterns of gender inequality.     

Integrating Air Pollution,Climate Change,and Economics in a Risk-Based Life-Cycle Analysis: A Case Study of Residential Insulation

One of the ways in which risk assessment can inform life-cycle analysis (LCA) is by providing a mechanism to translate midpoint categories into common endpoints. Although this analytical step is complex and often highly uncertain, it can allow for prioritization among disparate midpoints and subsequent analytical refinements focused on the endpoints that dominate policy decisions. In this article, we present an approach to address three widely differing impact categories—particulate matter air pollution, greenhouse gas emissions, and personal income. We use the case of increased residential insulation as a measure to reduce energy consumption, which implies economic and public health tradeoffs across all three categories. We apply previously developed models that combined input-output LCA and risk assessment to address public health impacts from particulate matter, and extend the framework to address greenhouse gases and the public health consequences of changes in income. For a hypothetical loan program applied to both new and existing single-family homes, we find a payback period of approximately one year for the particulate matter and greenhouse gas–related midpoints and endpoints, with the structure of the loan implying that no economic payback is required. Our central estimates for avoided disability adjusted life years (DALYs) for a 50-year period are approximately 200,000 for particulate matter, 900,000 for greenhouse gases, and 300,000 for income changes, although values are highly dependent on discount rates and other model assumptions. We conclude that all three impact categories are potentially significant in this case, indicating that analytical refinements should be considered for all three impact categories to reduce model uncertainties. Our study demonstrates how LCA and risk assessment can work together in a framework that includes multiple impact categories, aiding in the evaluation of the net impacts of an energy policy change on society.     

The mechanics of atherosclerotic plaque rupture by inclusion/matrix interfacial decohesion

Histological investigation along with finite element analysis of arterial wall/atherosclerotic plaque geometries indicates the paradoxical result that ruptures often occur at sites with predicted stresses of half the plaque cap strength. Recent experiments have revealed calcified cells within the cap suggesting that these inclusions, situated close to the cap/luminal blood surface, precipitate rupture at low nominal loads by concentrating stress. In this paper, we investigate the proposition that rupture at low nominal loads occurs by (possibly brittle) decohesion of the calcification/cap interface followed by tearing of cap tissue. A novel boundary value problem is analyzed consisting of a remotely loaded linear elastic layer (extracellular matrix cap) containing a rigid spherical inclusion (calcified cell) that interacts with it through a nonlinear structural interface which models the binding of the calcified cell to the extracellular matrix via integrin receptor proteins. Equilibrium solutions are obtained from equations derived from the Boussinesq potentials for spherical domains. Results indicate a brittle character to the rupture process with the size of the domains between the inclusion center and the matrix surfaces determining the concentration of stress. For an inclusion close to a surface the abrupt unloading of the interface during brittle decohesion produces a sharp spike in circumferential stress. We conjecture that when this dynamic stress exceeds the cap strength, tearing occurs followed by thrombus formation and possibly infarction.     

Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains

The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.     

Production of myeloid cell cytosols functionally and immunochemically deficient in the 47 kDa or 67 kDa NADPH oxidase cytosolic factors

Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.     

Functional Characterization of a WWP1/Tiul1 Tumor-derived Mutant Reveals a Paradigm of Its Constitutive Activation in Human Cancer

Although E3 ubiquitin ligases are deemed to play key roles in normal cell function and homeostasis, whether their alterations contribute to cancer pathogenesis remains unclear. In this study, we sought to investigate potential mechanisms that govern WWP1/Tiul1 (WWP1) ubiquitin ligase activity, focusing on its ability to trigger degradation of TGFβ type I receptor (TβRI) in conjunction with Smad7. Our data reveal that the WWP1 protein is very stable at steady states because its autopolyubiquitination activity is silenced due to an intra-interaction between the C2 and/or WW and Hect domains that favors WWP1 monoubiquitination at the expense of its polyubiquitination or polyubiquitination of TβRI. Upon binding of WWP1 to Smad7, this functional interplay is disabled, switching its monoubiquitination activity toward a polyubiquitination activity, thereby driving its own degradation and that of TβRI as well. Intriguingly, a WWP1 point mutation found in human prostate cancer disrupts this regulatory mechanism by relieving the inhibitory effects of C2 and WW on Hect and thereby causing WWP1 hyperactivation. That cancer-driven alteration of WWP1 culminates in excessive TβRI degradation and attenuated TGFβ cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1.