Alexandra L. Klinger

Confirmation of a unique intra-dimer cooperativity in the human hemoglobin ?1?1half-oxygenated intermediate supports the symmetry rule model of allosteric regulation

The contribution of the α1β1half‐oxygenated tetramer [αβ:αO2βO2] (species 21) to human hemoglobin cooperativity was evaluated using cryogenic isoelectric focusing. The cooperative free energy of binding, reflecting O2‐driven protein structure changes, was measured as 21ΔGc = 5.1 ± 0.3 kcal for the Zn/FeO2 analog. For the Fe/FeCN analog, 21ΔGc was estimated as 4.0 kcal after correction for a CN ligand rearrangement artifact, demonstrating that ligand rearrangement does not invalidate previous conclusions regarding this species. In the context of the entire Hb cooperativity cascade, which includes eight intermediate species, the 21 tetramer is highly abundant relative to the other doubly‐ligated species, providing strong support for the previously determined consensus partition function of O2 binding and for the Symmetry Rule model of hemoglobin cooperativity (Ackers et al., Science 1992;255:54–63). Cooperativity of normal human hemoglobin is shown to depend on site‐configuration, and not solely the number of O2 bound, nor the occupancy of α vs. β subunits. Verification of a unique contribution from the α1β1doubly‐oxygenated species to the equilibrium O2 binding curve strongly reinforces the Symmetry Rule interpretation that the α1β1dimer acts both as a structural and functional element in cooperative O2 binding. Proteins 2000;41:23–43.

Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores

Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50-150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway.

A synchrotron-based hydroxyl radical footprinting analysis of amyloid fibrils and prefibrillar intermediates with residue-specific resolution.

Structural models of the fibrils formed by the 40-residue amyloid-β (Aβ40) peptide in Alzheimer’s disease typically consist of linear polypeptide segments, oriented approximately perpendicular to the long axis of the fibril, and joined together as parallel in-register β-sheets to form filaments. However, various models differ in the number of filaments that run the length of a fibril, and in the topological arrangement of these filaments. In addition to questions about the structure of Aβ40 monomers in fibrils, there are important unanswered questions about their structure in prefibrillar intermediates, which are of interest because they may represent the most neurotoxic form of Aβ40. To assess different models of fibril structure and to gain insight into the structure of prefibrillar intermediates, the relative solvent accessibility of amino acid residue side chains in fibrillar and prefibrillar Aβ40 preparations was characterized in solution by hydroxyl radical footprinting and structural mass spectrometry. A key to the application of this technology was the development of hydroxyl radical reactivity measures for individual side chains of Aβ40. Combined with mass-per-length measurements performed by dark-field electron microscopy, the results of this study are consistent with the core filament structure represented by two- and three-filament solid state nuclear magnetic resonance-based models of the Aβ40 fibril (such as 2LMN, 2LMO, 2LMP, and 2LMQ), with minor refinements, but they are inconsistent with the more recently proposed 2M4J model. The results also demonstrate that individual Aβ40 fibrils exhibit structural heterogeneity or polymorphism, where regions of two-filament structure alternate with regions of three-filament structure. The footprinting approach utilized in this study will be valuable for characterizing various fibrillar and nonfibrillar forms of the Aβ peptide.

Mechanotransductional basis of endothelial cell response to intravascular bubbles

Vascular air embolism resulting from too rapid decompression is a well-known risk in deep-sea diving, aviation and space travel. It is also a common complication during surgery or other medical procedures when air or other endogenously administered gas is entrained in the circulation. Preventive and post-event treatment options are extremely limited for this dangerous condition, and none of them address the poorly understood pathophysiology of endothelial response to intravascular bubble presence. Using a novel apparatus allowing precise manipulation of microbubbles in real time fluorescence microscopy studies, we directly measure human umbilical vein endothelial cell responses to bubble contact. Strong intracellular calcium transients requiring extracellular calcium are observed upon cell-bubble interaction. The transient is eliminated both by the presence of the stretch activated channel inhibitor, gadolinium, and the transient receptor potential vanilliod family inhibitor, ruthenium red. No bubble induced calcium upsurge occurs if the cells are pretreated with an inhibitor of actin polymerization, cytochalasin-D. This study explores the biomechanical mechanisms at play in bubble interfacial interactions with endothelial surface layer (ESL) macromolecules, reassessing cell response after selective digestion of glycocalyx glycosoaminoglycans, hyaluran (HA) and heparin sulfate (HS). HA digestion causes reduction of cell-bubble adherence and a more rapid induction of calcium influx after contact. HS depletion significantly decreases calcium transient amplitudes, as does pharmacologically induced sydencan ectodomain shedding. The surfactant perfluorocarbon Oxycyte abolishes any bubble induced calcium transient, presumably through direct competition with ESL macromolecules for interfacial occupancy, thus attenuating the interactions that trigger potentially deleterious biochemical pathways.

Analysis of spectra from multiwavelength oxygen-binding studies of mixed metal hybrid hemoglobins.

Abstract Computational models of proton binding can range from the chemically complex and statistically simple (as in the quantum calculations) to the chemically simple and statistically complex. Much progress has been made in the multiple-site titration problem. Calculations have improved with the inclusion of more flexibility in regard to both the geometry of the proton binding and the larger scale protein motions associated with titration. This article concentrated on the principles of current calculations, but did not attempt to survey their quantitative performance. This is (1) because such comparisons are given in the cited papers and (2) because continued developments in understanding conformational flexibility and interaction energies will be needed to develop robust methods with strong predictive power. Nevertheless, the advances achieved over the past few years should not be underestimated: serious calculations of protonation behavior and its coupling to conformational change can now be confidently pursued against a backdrop of increasing understanding of the strengths and limitations of such models. It is hoped that such theoretical advances will also spur renewed experimental interest in measuring both overall titration curves and individual p K a values or p K a shifts. Exploration of the shapes of individual titration curves (as measured by Hill coefficients and other parameters) would also be useful in assessing the accuracy of computations and in drawing connections to functional behavior.

Pefluorocarbon inhibition of bubble induced Ca2+ transients in an in vitro model of vascular gas embolism

Endothelial injury resulting from deleterious interaction of gas microbubbles occurs in many surgical procedures and other medical interventions. The symptoms of vascular air embolism (VAE), while serious, are often difficult to detect, and there are essentially no pharmaceutical preventative or post-event treatments currently available. Perfluorocarbons (PFCs), however, have shown particular promise as a therapeutic option in reducing endothelial injury both in- and ex-vivo. Recently, we demonstrated the effectiveness of Oxycyte, a third-generation PFC formulated in a phosphotidylcholine emulsion, using an in vitro model of VAE developed in our laboratory. This apparatus allows live cell imaging concurrent with precise manipulation of physiologically sized microbubbles so that they may be brought into individual contact with human umbilical vein endothelial cells dye-loaded with the Ca2+ sensitive Fluo-4. Herein, we expand use of this fluorescence microscopy-based cell culture model. Specifically, we examined the concentration dependence of Oxycyte in reducing both the amplitude and frequency of large intracellular Ca2+ currents that are both a hallmark of bubble contact and a quantifiable indication that abnormal intracellular signaling has been triggered. We measured dose dependence curves and fit the resultant data using a modified Black and Leff operational model of agonism. The half maximal inhibitory concentrations of Oxycyte for (i) inhibition of occurrence and (ii) amplitude reduction were 229 ± 49 µM and 226 ± 167 µM, respectively. This investigation shows the preferential gas/liquid interface occupancy of the PFC component of Oxycyte over that of mechanosensing glycocalyx components and validates Oxycyte’s specific surfactant mechanism of action. Further, no lethality was observed for any concentration of this bioinert PFC, as it acts as a competitive allosteric inhibitor of syndecan activation to ameliorate cell response to bubble contact.

Understanding the Mechanotransductional Basis of Intravascular Bubble Injury

Intravascular bubbles, commonly introduced during surgery or precipitated from dissolved gases in decompression sickness, can occlude vessels, diminish perfusion, and initiate thrombotic and inflammatory pathways. The specific interactions between bubbles and the endothelium that lead to cell injury and death are poorly understood. We report live single cell time-lapse imaging of human umbilical vein endothelial cells (HUVECs) perturbed by microbubbles produced by injecting air through a pulled glass capillary ground so that its bevel opposes the buoyant force of the bubble. The bubble is moved with a three-stage micromanipulator into contact with a cell containing Fluo-4, a calcium sensitive dye, and imaged using phase-contrast and fluorescence microscopy. A significant transient elevation in intracellular calcium is observed in response to bubble impact. Membrane depolarization (external K+, 130 mM) does not block the calcium response, indicating that voltage-dependent calcium channels are not involved. Extracellular calcium and an intact actin cytoskeleton are required for the elevation of intracellular calcium upon bubble impact, which is reduced with the addition of gadolinium, a general inhibitor of mechanosensitive channels. These results suggest that a combination of the mechanical deformation and the air-water interface of the bubble activate a plasmalemma ion channel triggering further release of internal calcium stores. The calcium response is ameliorated with addition of surfactant, likely competing for occupancy of the air-liquid bubble interface. Supported by ONR N00014-08-1-0436 and NIH R01 HL67986.