Rhoda Elison Hirsch

Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.

Liquid–liquid separation in solutions of normal and sickle cell hemoglobin

We show that in solutions of human hemoglobin (Hb)—oxy- and deoxy-Hb A or S—of near-physiological pH, ionic strength, and Hb concentration, liquid–liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40°C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid–liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.

Intrinsic fluorescence emission of intact oxy hemoglobins

Abstract Fluorescence has not been previously detected in intact hemoproteins. We have been able to measure significant fluorescence emission in purified oxy HbA using front-face fluorometry. The excitation maximum (293 nm), the emission maximum (325 nm) and the fluorescence spectra of Hb Rothschild (β 37 Trp → Arg) allows us to conclude that β 37 Trp is primarily responsible for the fluorescence signal of HbA. We propose that this intrinsic fluorescence of hemoglobin may be used as a probe to study conformational changes in hemoglobin and possibly other heme-containing proteins.

A FIRST EVALUATION OF THE NATURAL HIGH MOLECULAR WEIGHT POLYMERIC Lumbricus terrestris HEMOGLOBIN AS AN OXYGEN CARRIER.

Lumbricus terrestris hemoglobin (LtHb), an unusually stable Hb (MW approximately 4x10(6) Da) with respect to dissociation and oxidation, circulates extracellularly in the earthworm and at neutral pH exhibits oxygen affinity and cooperativity similar to that of human HbA. Results suggest that LtHb may serve as a model for a high molecular weight extracellular oxygen carrier. Mice and a rat model partially exchanged with LtHb showed no apparent behavioral and physical changes. 31P NMR spectroscopy of perfused guinea pig hearts, used to assess phosphocreatine levels as an indication of the ability of LtHb to serve as an oxygen carrier to the heart, demonstrated that LtHb provides oxygen to the tissue and maintains the energy metabolism significantly better than the control non-Hb perfusion media. One day after infusion, video enhanced microscopy imaging of the mice cremaster muscle vasculature reveals temporal adhesion of leukocytes to the endothelial walls with temporal infiltration of leukocytes to the surrounding tissue, correlated with dosage. Exchanged mice rechallenged with LtHb show no overt allergic response or death. Further evaluation of this natural extracellular Hb as a potential polymeric Hb blood substitute/perfusion agent is warranted.

Liquid-Liquid Phase Separation in Hemoglobins: Distinct Aggregation Mechanisms of the β6 Mutants

Reversible liquid-liquid (L-L) phase separation in the form of high concentration hemoglobin (Hb) solution droplets is favored in an equilibrium with a low-concentration Hb solution when induced by inositol-hexaphosphate in the presence of polyethylene glycol 4000 at pH 6.35 HEPES (50 mM). The L-L phase separation of Hb serves as a model to elucidate intermolecular interactions that may give rise to accelerated nucleation kinetics of liganded HbC (beta6 Lys) compared to HbS (beta6 Val) and HbA (beta6 Glu). Under conditions of low pH (pH 6.35) in the presence of inositol-hexaphosphate, COHb assumes an altered R-state. The phase lines for the three Hb variants in concentration and temperature coordinates indicate that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS and HbA. Over time, L-L phase separation gives rise to amorphous aggregation and subsequent formation of crystals of different kinetics and habits, unique to the individual Hb. The composite of R- and T-like solution aggregation behavior indicates that this is a conformationally driven event. These results indicate that specific contact sites, thermodynamics, and kinetics all play a role in L-L phase separation and differ for the beta6 mutant hemoglobins compared to HbA. In addition, the dense liquid droplet interface or aggregate interface noticeably participates in crystal nucleation.

ROLE OF REDOX POTENTIAL OF HEMOGLOBIN-BASED OXYGEN CARRIERS ON METHEMOGLOBIN REDUCTION BY PLASMA COMPONENTS

A functional requirement for all hemoglobin-based oxygen carriers (HBOCs) is the maintenance of the heme-iron in the reduced state. This is necessary for the reversible binding/release of molecular oxygen and minimization of methemoglobin (Fe+3) formation. Acellular hemoglobins are especially susceptible to oxidation and denaturation. In the absence of the intrinsic reducing systems of the red blood cell, the reduced heme-Fe+2 can be oxidized to form increasing levels of methemoglobin that can give rise to free radicals and oxidative cellular damage. If acellular HBOCs are to be utilized as red cell substitutes for oxygen delivery, these carriers must be stabilized in the plasma, the carrier medium. Normal plasma contains reducing components, such as ascorbic acid and glutathione, that can afford protection to these acellular HBOCs through electron-transfer mediated processes. For these components to provide effective reduction to an HBOCs, a favorable reduction potential difference must exist between the reducing agent and the HBOCs. Using a modified thin-layer spectroelectrochemical method, a determination of the formal reduction potential (vs. Ag/AgCl) of several oxygen carriers, including monomeric myoglobin, tetrameric HbA and HbS, chemically cross-linked HbXL99α, polymerized oxyglobin (FDA approved for canine anemia), and the natural cross-linked polymeric Lumbricus hemoglobin, have been determined. In contrast to the negative formal reduction potentials (−155 to −50 mV) obtained for Mb, HbA, HbS, HbXL99α, and oxyglobin, Lumbricus hemoglobin exhibited a positive formal reduction potential (~100 mV). These results may help explain the greater effectiveness of the tested reducing agents to reduce met Lumbricus hemoglobin, compared to the other HBOCs, back to the required reduced form necessary for physiological binding/release of oxygen. Each reducing agent was capable of reducing met Lumbricus hemoglobin to the fully reduced state, although the kinetics of these reactions were different. HbA, HbXL99α, and oxyglobin were only partially reduced (10 to 37%) by glutathione, β-NADH, and ascorbic acid under similar conditions.

Acellular Invertebrate Hemoglobins as Model Therapeutic Oxygen Carriers: Unique Redox Potentials

Natural acellular polymeric hemoglobins (Hb) provide oxygen transport and delivery within many terrestrial and marine invertebrate organisms. It has been our premise that these natural acellular Hbs may serve as models of therapeutic hemoglobin-based oxygen carriers (HBOC). Our attention has focused on the acellular Hb from the terrestrial invertebrate, Lumbricus terrestris (Lt), which possesses a unique hierarchical structure and a unique ability to function extracellularly without oxidative damage. Lumbricus Hb and Arenicola Hb are resistant to autoxidation, chemical oxidation by potassium ferricyanide, and have little or no capacity to transfer electrons to Fe+3-complexes at 37°C. An understanding of how these invertebrate acellular oxygen carriers maintain their structural integrity and redox stability in vivo is vital for the design of a safe and effective red cell substitute. We report here a positive redox potential for these giant hemoglobins that may lie at the basis for its resistance to oxidation.

Miniaturized Scintillation Technique for Protein Solubility Determinations

We have developed a miniaturized (volume of crystallizing solution ∼100 μl) technique for the determination of protein solubility as a function of temperature. After nucleation, crystals are detected by the light they scatter. Then the temperature at which a solution with the initial concentration is in equilibrium with the crystals is sought by stepwise, equilibrium dissolution of the crystals. The approach to solubility from the side of dissolution provides for higher accuracy of the determinations. The method was used to determine the temperature dependence of the solubility of human hemoglobin (Hb) C, for which high-resolution x-ray crystallography data are needed to understand the structural basis for the drastically different in vivo aggregation/crystallization behavior of β6 Hb mutants.

Solution-active Structural Alterations in Liganded Hemoglobins C (β6 Glu → Lys) and S (β6 Glu → Val)

Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the α and β chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC. We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the α1β2interface (β37), and in the A-helix (α14/β15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC ∼ HbS > HbA.

Generating S-nitrosothiols from hemoglobin: Mechanisms, conformational dependence, and physiological relevance

Background: The mechanism for production of N2O3 from MetHb, nitrite, and NO is controversial. Results: An Hb intermediate attributed to heme-bound N2O3 is characterized. Conclusion: Partially met-R state Hb can function as a generator of long lived forms of bioactive NO. Significance: The results provide insight into how Hb reactivity with nitrite can be harnessed physiologically and therapeutically. In vitro, ferrous deoxy-hemes in hemoglobin (Hb) react with nitrite to generate nitric oxide (NO) through a nitrite reductase reaction. In vivo studies indicate Hb with nitrite can be a source of NO bioactivity. The nitrite reductase reaction does not appear to account fully for this activity because free NO is short lived especially within the red blood cell. Thus, the exporting of NO bioactivity both out of the RBC and over a large distance requires an additional mechanism. A nitrite anhydrase (NA) reaction in which N2O3, a potent S-nitrosating agent, is produced through the reaction of NO with ferric heme-bound nitrite has been proposed (Basu, S., Grubina, R., Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang, A., He, X., Azarov, I., Seibert, R., Mehta, A., Patel, R., King, S. B., Hogg, N., Ghosh, A., Gladwin, M. T., and Kim-Shapiro, D. B. (2007) Nat. Chem. Biol. 3, 785–794) as a possible mechanism. Legitimate concerns, including physiological relevance and the nature of the mechanism, have been raised concerning the NA reaction. This study addresses these concerns demonstrating NO and nitrite with ferric hemes under near physiological conditions yield an intermediate having the properties of the purported NA heme-bound N2O3 intermediate. The results indicate that ferric heme sites, traditionally viewed as a source of potential toxicity, can be functionally significant, especially for partially oxygenated/partially met-R state Hb that arises from the NO dioxygenation reaction. In the presence of low levels of nitrite and either NO or a suitable reductant such as l-cysteine, these ferric heme sites can function as a generator for the formation of S-nitrosothiols such as S-nitrosoglutathione and, as such, should be considered as a source of RBC-derived and exportable bioactive NO.