Basic Examination of Blood and Bone Marrow

Vajpayee, Neerja; Graham, Susan S.; Bem, Sylva

doi:10.1016/b978-1-4377-0974-2.00030-0

Cited by 42 publications

(26 citation statements)

References 65 publications

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“…The triple cysteine substitutions to the H64Q Ngb reduce intramolecular cross-linking and increase protein solubility, allowing for the preparation of stable >12 mM stock solutions. High concentrations of Ngb could allow for “unit” infusions of saline containing 50 – 100 g of Ngb in the field by paramedics, necessary to increase the plasma levels to 1 – 2 g/dL and stoichiometrically scavenge CO from CO-Hb complexes (56), which would reduce the CO-Hb level by an absolute percentage of approximately 10–15% (e.g. from 30% CO-Hb to 15–20%).…”

Section: Discussionmentioning

confidence: 99%

Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning

et al. 2016

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Carbon monoxide (CO) is a leading cause of poisoning deaths worldwide, with no available antidotal therapy. We introduce a potential treatment paradigm for CO poisoning, based on near-irreversible binding of CO by an engineered human neuroglobin (Ngb). Ngb is a six-coordinate hemoprotein, with the heme iron coordinated by two histidine residues. We mutated the distal histidine to glutamine (H64Q) and substituted three surface cysteines with less reactive amino acids to form a five-coordinate heme protein (Ngb-H64Q-CCC). This molecule exhibited an unusually high affinity for gaseous ligands, with a P50 value for oxygen of 0.015 mmHg. Ngb-H64Q-CCC bound CO about 500 times more strongly than did hemoglobin. Incubation of Ngb-H64Q-CCC with 100% CO-saturated hemoglobin, either cell-free or encapsulated in human red blood cells, reduced the half-life of carboxy-hemoglobin to 0.11 and 0.41 minutes, respectively, from a value that is ≥ 200 minutes when the hemoglobin or cells are only exposed to air. Infusions of Ngb-H64Q-CCC to CO-poisoned mice enhanced CO removal from red blood cells, restored heart rate and blood pressure, increased survival, and were followed by rapid renal elimination of CO-bound Ngb-H64Q-CCC. Heme-based scavenger molecules with very high CO binding affinity such as our mutant five-coordinate Ngb are potential antidotes for CO poisoning by virtue of their ability to bind and eliminate CO.

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Section: Discussionmentioning

confidence: 99%

Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning

et al. 2016

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“…The 7-day K562 induction time course (Figure 4B) produced a mean maximum MCH value of 7.31 pg/cell in induced samples compared to only 0.983 pg/ cell in the uninduced controls (Day 7). The induced value is significantly lower than the normal red blood cell level of 2733 pg/cell (31), which has only been approached previously in K562 cells with glutamine starvation combined with hemin treatment (32). The 5-day MEL induction time course (Figure 4C) resulted in a mean maximum MCH of 4.47 pg/cell in induced cultures and 0.583 pg/cell in uninduced cultures (Day 5).…”

Section: Resultsmentioning

confidence: 94%

Rapid and Sensitive Quantitation of Heme in Hemoglobinized Cells

et al. 2016

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Rapid and accurate heme quantitation in the research lab has become more desirable as the crucial role that intracellular hemoproteins play in metabolism continues to emerge. Here, the time-honored approaches of pyridine hemochromogen and fluorescence heme assays are compared with direct absorbance-based technologies using the CLARiTY spectrophotometer. All samples tested with these methods were rich in hemoglobin-associated heme, including buffered hemoglobin standards, whole blood from mice, and murine erythroleukemia (MEL) and K562 cells. While the pyridine hemochromogen assay demonstrated the greatest linear range of heme detection, all 3 methods demonstrated similar analytical sensitivities and normalized limits of quantitation of ∼1 µM. Surprisingly, the fluorescence assay was only shown to be distinct in its ability to quantitate extremely small samples. Using the CLARiTY system in combination with pyridine hemochromogen and cell count data, a common hemoglobin extinction coefficient for blood and differentiating MEL and K562 cells of 0.46 µM-1 cm-1 was derived. This value was applied to supplemental experiments designed to measure MEL cell hemoglobinization in response to the addition or removal of factors previously shown to affect heme biosynthesis (e.g., L-glutamine, iron).

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“…Val and Leu were used for this analysis because they are effluxed at high rates and the parasite lacks biosynthetic pathways for these amino acids. Using an adult human mean corpuscular hemoglobin value of 30 pg/cell [26] and the efflux rates for Val and Leu in Table 2, and assuming that these rates are sustained for a third of the 44 hour asexual cycle during which 70% of erythrocyte hemoglobin is degraded [1], we calculate that 41% of Val and 40% of Leu generated from hemoglobin catabolism is effluxed. This exercise suggests that a large fraction of the amino acids generated through hemoglobin catabolism is lost from the parasite and is consistent with a previous report that asexual P. falciparum uses only one-fifth of the amino acids generated from hemoglobin catabolism for protein synthesis [2].…”

Section: Resultsmentioning

confidence: 99%

Amino acid efflux by asexual blood-stage Plasmodium falciparum and its utility in interrogating the kinetics of hemoglobin endocytosis and catabolism in vivo

Dalal

Klemba

2015

Molecular and Biochemical Parasitology

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The endocytosis and catabolism of large quantities of host cell hemoglobin is a hallmark of the intraerythrocytic asexual stage of the human malaria parasite Plasmodium falciparum. It is known that the parasite’s production of amino acids from hemoglobin far exceeds its metabolic needs. Here, we show that P. falciparum effluxes large quantities of certain non-polar (Ala, Leu, Val, Pro, Phe, Gly) and polar (Ser, Thr, His) amino acids to the external medium. That these amino acids originate from hemoglobin catabolism is indicated by the strong correlation between individual amino acid efflux rates and their abundances in hemoglobin, and the ability of the food vacuole falcipain inhibitor E-64d to greatly suppress efflux rates. We then developed a rapid, sensitive and precise method for quantifying flux through the hemoglobin endocytic-catabolic pathway that is based on leucine efflux. Optimization of the method involved the generation of a novel amino acid-restricted RPMI formulation as well as the validation of D-norvaline as an internal standard. The utility of this method was demonstrated by characterizing the effects of the phosphatidylinositol-3-kinase inhibitors wortmannin and dihydroartemisinin on the kinetics of Leu efflux. Both compounds rapidly inhibited Leu efflux, which is consistent with a role for phosphtidylinositol-3-phosphate production in the delivery of hemoglobin to the food vacuole; however, wortmannin inhibition was transient, which was likely due to the instability of this compound in culture medium. The simplicity, convenience and non-invasive nature of the Leu efflux assay described here makes it ideal for characterizing the in vivo kinetics of hemoglobin endocytosis and catabolism, for inhibitor target validation studies, and for medium-throughput screens to identify novel inhibitors of cytostomal endocytosis.

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Basic Examination of Blood and Bone Marrow

Cited by 42 publications

References 65 publications

Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning

Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning

Rapid and Sensitive Quantitation of Heme in Hemoglobinized Cells

Amino acid efflux by asexual blood-stage Plasmodium falciparum and its utility in interrogating the kinetics of hemoglobin endocytosis and catabolism in vivo

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