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1.  Inhibition of erythrocyte sickling in vitro by pyridoxal. 
Journal of Clinical Investigation  1978;62(4):888-891.
To test the antisickling activity of pyridoxal, we compared the oxygen affinity and the percent sickling at low PO2 of untreated erythrocytes with values for cells from the same blood sample incubated with pyridoxal, glyceraldehyde, or pyridoxine. Pyridoxal increased oxygen affinity much more than glyceraldehyde. 20 mM pyridoxal and glyceraldehyde had equivalent antisickling activity. At PO2 levels above 20 mm Hg, both agents reduced sickling to less than 2%. In samples examined by electron microscopy, pyridoxal reduced the percent sickled cells and the percent cells that contain hemoglobin S fibers by the same amount (from 74 to 3%). Pyridoxine had no effect on oxygen affinity or sockling. Pyridoxal reacts with intracellular hemoglobin to increase oxygen affinity, which inhibits hemoglobin S polymerization and sickling.
Images
PMCID: PMC371842  PMID: 701485
2.  Effects of o-vanillin on K+ transport of red blood cells from patients with sickle cell disease 
Blood Cells, Molecules & Diseases  2014;53(1-2):21-26.
Aromatic aldehydes like o-vanillin were designed to reduce the complications of sickle cell disease (SCD) by interaction with HbS, to reduce polymerisation and RBC sickling. Present results show that o-vanillin also directly affects RBC membrane permeability. Both the K+–Cl− cotransporter (KCC) and the Ca2 +-activated K+ channel (or Gardos channel) were inhibited with IC50 of about 0.3 and 1 mM, respectively, with activities almost completely abolished by 5 mM. Similar effects were observed in RBCs treated with the thiol reacting reagent N-ethylmaleimide or with the Ca2 + ionophore A23187, to circumvent any action via HbS polymerisation. The deoxygenation-induced cation conductance (sometimes termed Psickle) was partially inhibited, whilst deoxygenation-induced exposure of phosphatidylserine was completely abrogated. Na+/K+ pump activity was also reduced. Notwithstanding, o-vanillin stimulated K+ efflux through an unidentified pathway and resulted in reduction in cell volume (as measured by wet weight − dry weight). These actions are relevant to understanding how aromatic aldehydes may affect RBC membrane permeability per se as well as HbS polymerisation and thereby inform design of compounds most efficacious in ameliorating the complications of SCD.
doi:10.1016/j.bcmd.2014.02.004
PMCID: PMC4039999  PMID: 24594314
Sickle; Red blood cells; Aromatic aldehydes; o-Vanillin; Potassium permeability
3.  Solenostemon monostachyus, Ipomoea involucrata and Carica papaya seed oil versus Glutathione, or Vernonia amygdalina: Methanolic extracts of novel plants for the management of sickle cell anemia disease 
Background
Sickle cell disease (SCD) is a genetic disease caused by an individual inheriting an allele for sickle cell hemoglobin from both parents and is associated with unusually large numbers of immature blood cells, containing many long, thin, crescent-shaped erythrocytes. It is a disease prevalent throughout many populations. The use of medicinal plants and nutrition in managing SCD is gaining increasing attention.
Methods
The antisickling effects of Solenostemon monostachyus (SolMon), Carica papaya seed oil (Cari-oil) and Ipomoea involucrata (Ipocrata) in male (HbSSM) and female (HbSSF) human sickle cell blood was examined in vitro and compared with controls, or cells treated with glutathione or an antisickling plant (Vernonia amygdalina; VerMyg).
Results
Levels of sickle blood cells were significantly reduced (P < 0.05) in all the plant-extract treated SCD patients’ blood compared with that of untreated SCD patients. RBCs in SolMon, Ipocrata, and Cari-oil treated samples were significantly higher (P < 0.05) compared with VerMyg-treated samples. The Fe2+/Fe3+ ratio was significantly reduced (P < 0.05) in all plant extract-treated HbSSM samples compared with controls. Hemoglobin concentration was significantly increased (P < 0.05) by SolMon treatment in HbSSF compared with VerMyg. Sickle cell polymerization inhibition exhibited by SolMon was significantly higher (P < 0.05) compared with that of VerMyg in HbSSF blood. Sickle cell polymerization inhibition in SolMon and Ipocrata were significantly higher (P < 0.05) compared with VerMyg in HbSSM blood. All plant extracts significantly reduced (P < 0.05) lactate dehydrogenase activity in both HbSSM and HbSSF-treated blood. Catalase activity was significantly increased (P < 0.05) in HbSSF blood treated with Ipocrata compared with glutathione. Cari-oil treated HbSSM and HbSSF blood had significantly increased (P < 0.05) peroxidase activity compared with controls.
Conclusions
Methanolic extracts from S. monostachyus, C. papaya seed oil and I. involucrata exhibited particular antisickling properties coupled with the potential to reduce stress in sickle cell patients. Each plant individually or in combination may be useful for the management of sickle cell disease.
doi:10.1186/1472-6882-12-262
PMCID: PMC3553046  PMID: 23259718
Sickle cell disease; Health; Management; Antisickling; Underutilized; Plants
4.  The effect of deoxygenation on whole-cell conductance of red blood cells from healthy individuals and patients with sickle cell disease 
Blood  2006;109(6):2622-2629.
Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed Psickle, contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na+ and K+, but was less permeable to NMDG+ and Cl−. Conductance to Ca2+ was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 μM), Zn2+ (100 μM), and by Gd3+ (IC50 of approximately 2 μM). It therefore shares some properties with Psickle. These findings represent the first electrical recordings of single HbS cells and will facilitate progress in understanding altered red blood cell cation transport characteristics of SCD.
doi:10.1182/blood-2006-03-001404
PMCID: PMC2951509  PMID: 17138828
5.  Laccase Catalyzed Synthesis of Iodinated Phenolic Compounds with Antifungal Activity 
PLoS ONE  2014;9(3):e89924.
Iodine is a well known antimicrobial compound. Laccase, an oxidoreductase which couples the one electron oxidation of diverse phenolic and non-phenolic substrates to the reduction of oxygen to water, is capable of oxidizing unreactive iodide to reactive iodine. We have shown previously that laccase-iodide treatment of spruce wood results in a wash-out resistant antimicrobial surface. In this study, we investigated whether phenolic compounds such as vanillin, which resembles sub-structures of softwood lignin, can be directly iodinated by reacting with laccase and iodide, resulting in compounds with antifungal activity. HPLC-MS analysis showed that vanillin was converted to iodovanillin by laccase catalysis at an excess of potassium iodide. No conversion of vanillin occurred in the absence of enzyme. The addition of redox mediators in catalytic concentrations increased the rate of iodide oxidation ten-fold and the yield of iodovanillin by 50%. Iodinated phenolic products were also detected when o-vanillin, ethyl vanillin, acetovanillone and methyl vanillate were incubated with laccase and iodide. At an increased educt concentration of 0.1 M an almost one to one molar ratio of iodide to vanillin could be used without compromising conversion rate, and the insoluble iodovanillin product could be recovered by simple centrifugation. The novel enzymatic synthesis procedure fulfills key criteria of green chemistry. Biocatalytically produced iodovanillin and iodo-ethyl vanillin had significant growth inhibitory effects on several wood degrading fungal species. For Trametes versicolor, a species causing white rot of wood, almost complete growth inhibition and a partial biocidal effect was observed on agar plates. Enzymatic tests indicated that the iodinated compounds acted as enzyme responsive, antimicrobial materials.
doi:10.1371/journal.pone.0089924
PMCID: PMC3940670  PMID: 24594755
6.  Hypoxia Activates a Ca2+-Permeable Cation Conductance Sensitive to Carbon Monoxide and to GsMTx-4 in Human and Mouse Sickle Erythrocytes 
PLoS ONE  2010;5(1):e8732.
Background
Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca2+] ([Ca2+]i) and subsequent activation of KCa 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.
Methodology/Principal Findings
We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca2+- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca2+]i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca2+]i in mouse sickle erythrocytes did not require KCa3.1 activity.
Conclusions/Significance
The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca2+-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca2+ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.
doi:10.1371/journal.pone.0008732
PMCID: PMC2806905  PMID: 20090940
7.  Abnormal rheology of oxygenated blood in sickle cell anemia 
Journal of Clinical Investigation  1970;49(4):623-634.
The viscosity of oxygenated blood from patients with sickle cell anemia (Hb SS disease) was found to be abnormally increased, a property which contrasts with the well recognized viscous aberration produced by deoxygenation of Hb SS blood. Experiments designed to explain this finding led to considerations of deformation and aggregation, primary determinants of the rheologic behavior of erythrocytes as they traverse the microcirculation. Deformability of erythrocytes is in turn dependent upon internal viscosity (i.e. the state and concentration of hemoglobin in solution) and membrane flexibility. Definition of the contribution made by each of these properties to the abnormal viscosity of oxygenated Hb SS blood was made possible by analysis of viscosity measurements, made over a wide range of shear rates and cell concentrations, on Hb SS erythrocytes and normal erythrocytes suspended in Ringer's solution (where aggregation does not occur) and in plasma. Similar measurements were made on the two cell types separated by ultracentrifugation of Hb SS erythrocytes: high density erythrocytes composed of 50 to 70% irreversibly “sickled” cells (ISC) and low density erythrocytes composed of over 95% non-ISC.
Under all experimental conditions (hematocrit, shear rate, and suspending medium) the viscosity of ISC exceeds that of normal erythrocytes. The viscosity of non-ISC is elevated only in the absence of aggregation and over intermediate ranges of hematocrit. Analyses of the data reveal (a) an elevated internal viscosity of ISC: (b) a reduced membrane flexibility of both ISC and non-ISC, particularly at low shear rates; and (c) a reduced tendency for aggregation displayed by both cell types.
The abnormal viscosity of oxygenated Hb SS blood can be attributed to the altered rheology of ISC and, to a lesser extent, of non-ISC. These studies assign a role to the abnormal rheology of Hb SS erythrocytes in the pathogenesis of sickle cell anemia, even under conditions of complete oxygenation.
Images
PMCID: PMC322516  PMID: 5443167
8.  A Vanillin Derivative Causes Mitochondrial Dysfunction and Triggers Oxidative Stress in Cryptococcus neoformans 
PLoS ONE  2014;9(2):e89122.
Vanillin is a well-known food and cosmetic additive and has antioxidant and antimutagenic properties. It has also been suggested to have antifungal activity against major human pathogenic fungi, although it is not very effective. In this study, the antifungal activities of vanillin and 33 vanillin derivatives against the human fungal pathogen Cryptococcus neoformans, the main pathogen of cryptococcal meningitis in immunocompromised patients, were investigated. We found a structural correlation between the vanillin derivatives and antifungal activity, showing that the hydroxyl or alkoxy group is more advantageous than the halogenated or nitrated group in benzaldehyde. Among the vanillin derivatives with a hydroxyl or alkoxy group, o-vanillin and o-ethyl vanillin showed the highest antifungal activity against C. neoformans. o-Vanillin was further studied to understand the mechanism of antifungal action. We compared the transcriptome of C. neoformans cells untreated or treated with o-vanillin by using RNA sequencing and found that the compound caused mitochondrial dysfunction and triggered oxidative stress. These antifungal mechanisms of o-vanillin were experimentally confirmed by the significantly reduced growth of the mutants lacking the genes involved in mitochondrial functions and oxidative stress response.
doi:10.1371/journal.pone.0089122
PMCID: PMC3930674  PMID: 24586538
9.  Effect of fetal hemoglobin on microvascular regulation in sickle transgenic-knockout mice 
Journal of Clinical Investigation  2004;114(8):1136-1145.
In sickle cell disease, intravascular sickling and attendant flow abnormalities underlie the chronic inflammation and vascular endothelial abnormalities. However, the relationship between sickling and vascular tone is not well understood. We hypothesized that sickling-induced vaso-occlusive events and attendant oxidative stress will affect microvascular regulatory mechanisms. In the present studies, we have examined whether microvascular abnormalities expressed in sickle transgenic-knockout Berkeley (BERK) mice (which express exclusively human α- and βS-globins with <1% γ-globin levels) are amenable to correction with increased levels of antisickling fetal hemoglobin (HbF). In BERK mice, sickling, increased oxidative stress, and hemolytic anemia are accompanied by vasodilation, compensatory increases in eNOS and COX-2, and attenuated vascular responses to NO-mediated vasoactive stimuli and norepinephrine. The hypotension and vasodilation (required for adequate oxygen delivery in the face of chronic anemia) are mediated by non-NO vasodilators (i.e., prostacyclin) as evidenced by induction of COX-2. In BERK mice, the resistance to NO-mediated vasodilators is associated with increased oxidative stress and hemolytic rate, and in BERK + γ mice (expressing 20% HbF), an improved response to these stimuli is associated with reduced oxidative stress and hemolytic rate. Furthermore, BERK + γ mice show normalization of vessel diameters, and eNOS and COX-2 expression. These results demonstrate a strong relationship between sickling and microvascular function in sickle cell disease.
doi:10.1172/JCI200421633
PMCID: PMC522244  PMID: 15489961
10.  Low concentrations of nitric oxide increase oxygen affinity of sickle erythrocytes in vitro and in vivo. 
Journal of Clinical Investigation  1997;100(5):1193-1198.
The hallmark of sickle cell disease (SCD) is the polymerization of deoxygenated sickle hemoglobin (HbS). In SCD patients, one strategy to reduce red blood cell (RBC) sickling is to increase HbS oxygen affinity. Our objective was to determine if low concentrations of nitric oxide (NO) gas would augment the oxygen affinity of RBCs containing homozygous HbS (SS). Blood containing normal adult hemoglobin (AA) or SS RBCs was incubated in vitro in the presence of varying concentrations of NO up to 80 ppm, and oxygen dissociation curves (ODCs) were measured. In addition, blood was obtained from three AA and nine SS volunteers, before and after breathing 80 ppm NO in air for 45 min, and the ODCs were measured. Exposure of SS RBCs to 80 ppm NO in vitro for 5 min or longer decreased the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen (P50), an average of 15% (4.8+/-1.7 mmHg mean+/-SE; P < 0.001). The increase in SS RBC oxygen affinity correlated with the NO concentration. The P50 of AA RBCs was unchanged (P > 0.1) by 80 ppm NO. In SS volunteers breathing 80 ppm NO for 45 min, the P50 decreased (P < 0.001) by 4.6+/-2.0 mmHg. 60 min after NO breathing was discontinued, the RBC P50 remained decreased in five of seven volunteers in whom the ODC was measured. There was no RBC P50 change (P > 0.1) in AA volunteers breathing NO. Methemoglobin (Mhb) remained low in all subjects breathing NO (SS Mhb 1.4+/-0.5%), and there was no correlation (r = 0.02) between the reduction in P50 and the change in Mhb. Thus, low concentrations of NO augment the oxygen affinity of sickle erythrocytes in vitro and in vivo without significant Mhb production. These results suggest that low concentrations of NO gas may offer an attractive new therapeutic model for the treatment of SCD.
PMCID: PMC508295  PMID: 9276736
11.  Pathophisiology of Sickle Cell Disease and New Drugs for the Treatment 
A homozygous mutation in the gene for β globin, a subunit of adult hemoglobin A (HbA), is the proximate cause of sickle cell disease (SCD). Sickle hemoglobin (HbS) shows peculiar biochemical properties, which lead to polymerizing when deoxygenated. HbS polymerization is associated with a reduction in cell ion and water content (cell dehydration), increased red cell density which further accelerate HbS polymerization. Dense, dehydrated erythrocytes are likely to undergo instant polymerization in conditions of mild hypoxia due to their high HbS concentration, and HbS polymers may be formed under normal oxygen pressure. Pathophysiological studies have shown that the dense, dehydrated red cells may play a central role in acute and chronic clinical manifestations of sickle cell disease, in which intravascular sickling in capillaries and small vessels leads to vaso-occlusion and impaired blood flow in a variety of organs and tissue. The persistent membrane damage associated with HbS polymerization also favors the generation of distorted rigid cells and further contributes to vaso-occlusive crisis (VOCs) and cell destruction in the peripheral circulation. These damaged, dense sickle red cells also show a loss of phospholipid asymmetry with externalization of phosphatidylserine (PS), which is believed to play a significant role in promoting macrophage recognition with removal of erythrocytes (erythrophagocytosis). Vaso-occlusive events in the microcirculation result from a complex scenario involving the interactions between different cell types, including dense, dehydrated sickle cells, reticulocytes, abnormally activated endothelial cells, leukocytes, platelets and plasma factors such as cytokine and oxidized pro-inflammatory lipids. Hydroxycarbamide (hydroxyurea) is currently the only drug approved for chronic administration in adult patients with sickle cell disease to prevent acute painful crises and reduce the incidence of transfusion and acute chest crises. Here, we will focus on consolidated and experimental therapeutic strategies for the treatment of sickle cell disease, including: agents which reduce or prevent sickle cell dehydrationagents which reduce sickle cell-endothelial adhesive eventsnitric oxide (NO) or NO-related compoundsanti-oxidant agents
Correction of the abnormalities ranging from membrane cation transport pathways to red cell-endothelial adhesive events, might constitute new pharmacological targets for treating sickle cell disease.
doi:10.4084/MJHID.2009.024
PMCID: PMC3033152  PMID: 21415994
12.  Nitric oxide reduces sickle hemoglobin polymerization: Potential role of nitric oxide-induced charge alteration in depolymerization 
We previously demonstrated that inhaling nitric oxide (NO) increases the oxygen affinity of sickle red blood cells (RBCs) in patients with sickle cell disease (SCD). Our recent studies found that NO lowered the P50 values of sickle hemoglobin (HbS) hemolysates but did not increase methemoglobin (metHb) levels, supporting the role of NO, but not metHb, in the oxygen affinity of HbS. Here we examine the mechanism by which NO increases HbS oxygen affinity. Because anti-sickling agents increase sickle RBC oxygen affinity, we first determined whether NO exhibits anti-sickling properties. The viscosity of HbS hemolysates, measured by falling ball assays, increased upon deoxygenation; NO treatment reduced the increment. Multiphoton microscopic analyses showed smaller HbS polymers in deoxygenated sickle RBCs and HbS hemolysates exposed to NO. These results suggest that NO inhibits HbS polymer formation and has anti-sickling properties. Furthermore, we found that HbS treated with NO exhibits an isoelectric point similar to that of HbA, suggesting that NO alters the electric charge of HbS. NO–HbS adducts had the same elution time as HbA upon high performance liquid chromatography analysis. This study demonstrates that NO may disrupt HbS polymers by abolishing the excess positive charge of HbS, resulting in increased oxygen affinity.
doi:10.1016/j.abb.2011.03.013
PMCID: PMC3889650  PMID: 21457702
Sickle cell disease; Nitric oxide; Oxygen affinity; Anti-sickling; Polymer formation
13.  Reduction of Inflammatory Responses and Enhancement of Extracellular Matrix Formation by Vanillin-Incorporated Poly(Lactic-co-Glycolic Acid) Scaffolds 
Tissue Engineering. Part A  2012;18(19-20):1967-1978.
Vanillin is one of the major components of vanilla, a commonly used flavoring agent and preservative and is known to exert potent antioxidant and anti-inflammatory activities. In this work, vanillin-incorporated poly(lactic-co-glycolic acid) (PLGA) films and scaffolds were fabricated to evaluate the effects of vanillin on the inflammatory responses and extracellular matrix (ECM) formation in vitro and in vivo. The incorporation of vanillin to PLGA films induced hydrophilic nature, resulting in the higher cell attachment and proliferation than the pure PLGA film. Vanillin also reduced the generation of reactive oxygen species (ROS) in cells cultured on the pure PLGA film and significantly inhibited the PLGA-induced inflammatory responses in vivo, evidenced by the reduced accumulation of inflammatory cells and thinner fibrous capsules. The effects of vanillin on the ECM formation were evaluated using annulus fibrous (AF) cell-seeded porous PLGA/vanillin scaffolds. PLGA/vanillin scaffolds elicited the more production of glycosaminoglycan and collagen than the pure PLGA scaffold, in a concentration-dependent manner. Based on the low level of inflammatory responses and enhanced ECM formation, vanillin-incorporated PLGA constructs make them promising candidates in the future biomedical applications.
doi:10.1089/ten.tea.2012.0001
PMCID: PMC3463284  PMID: 22551555
14.  Use of Citrate Agar Electrophoresis in Evaluation of Antisickling Agents 
Hemoglobin S (Hb S), treated with antisickling agents which interfere with gel formation, has been found to migrate on citrate agar electrophoresis in a manner similar to Hb A or Hb F. This finding suggests a rapid and inexpensive method for screening potential antisickling compounds of the types which interfere with gelation. This investigation has also led to an improved understanding of the molecular basis of citrate agar electrophoresis and to the realization that agar itself may contain a natural antisickling agent.
Images
PMCID: PMC2552633  PMID: 7205973
15.  Differences in Response to Fetal Hemoglobin Induction Therapy in β-Thalassemia and Sickle Cell Disease 
Inducers of fetal hemoglobin (HbF) have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia (β-Thal). To understand the basis of these differences in clinical effectiveness, we compared the effects of butyrate and hemin on the expression of the different globin genes in progenitors-derived erythroid cells from patients with β-Thal intermedia and SCD. Exposure to butyrate resulted in an augmentation of γ-globin mRNA levels in both SCD and β-Thal. Interestingly, butyrate exposure increased α-globin expression in β-Thal, while α-globin mRNA levels decreased in SCD in response to butyrate. As a result, the favorable effects of the butyrate-induced increase in γ-globin expression on α:β-like globin mRNA imbalance in β-Thal were reduced as a result of the associated increase in α-globin expression. Hemin had similar but less profound effects on all three globin genes in both categories of patients. Although the majority of patients with β-Thal did not correct their globin imbalance in response to butyrate or hemin induction of HbF in a minority of patients resulted in marked reduction in globin imbalance. Thus, we believe that the poor clinical response in a majority of patients with β-Thal to inducers of γ-globin expression may be a reflection of unfavorable effects of these agents on the other globin genes.
doi:10.1016/j.bcmd.2009.02.006
PMCID: PMC2707824  PMID: 19346141
Thalassemia; Sickle Cells; Hemoglobin Expression; Butyrate; Hemin
16.  THE PHYSICAL STATE OF HEMOGLOBIN IN SICKLE-CELL ANEMIA ERYTHROCYTES IN VIVO 
Venous blood removed anaerobically from patients with sickle-cell anemia was transferred immediately into fixative, thus precluding significant loss or gain of oxygen by the cells. Electron microscopy demonstrated an intraerythrocytic fibrillar fine structure similar to that described in prior studies on erythrocytes sickled by deoxygenation in vitro. Observations reported here lead to these conclusions: (a) explanations of the sickling process derived from in vitro experimentation may with validity be applied to sickling in vivo; and (b) the term "sickled" must be used with caution: a sickle-shaped membrane does not necessarily endose Hb S in filamentous form.
PMCID: PMC2138473  PMID: 5642466
17.  Cation depletion by the sodium pump in red cells with pathologic cation leaks. Sickle cells and xerocytes. 
Journal of Clinical Investigation  1986;78(6):1487-1496.
The mechanism by which sickle cells and xerocytic red cells become depleted of cations in vivo has not been identified previously. Both types of cells exhibit elevated permeabilities to sodium and potassium, in the case of sickle cells, when deoxygenated. The ouabain-insensitive fluxes of sodium and potassium were equivalent, however, in both cell types under these conditions. When incubated 18 hours in vitro, sickle cells lost cations but only when deoxygenated. This cation depletion was blocked by ouabain, removal of external potassium, or pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonate, which blocks the increase in cation permeability induced by deoxygenation. The loss of cation exhibited by oxygenated xerocytes similarly incubated was also blocked by ouabain. These data support the hypothesis that the elevated "passive" cation fluxes of xerocytes and deoxygenated sickle cells are not directly responsible for cation depletion of these cells; rather, these pathologic leaks interact with the sodium pump to produce a net loss of cellular cation.
PMCID: PMC423903  PMID: 2430999
18.  Antisickling activity of butyl stearate isolated from Ocimum basilicum (Lamiaceae) 
Objective
To perform phytochemical analyses on the leaves of Ocimum basilicum L. (O. basilicum), to elucidate the structure of isolate and then perform the antisickling activity on the crude extract and on the isolate.
Methods
The Emmel test performed on the acidified methanolic extract of this plant was used to evaluate the antisickling activity. The structure characterization of the active compound was performed using chromatographic techniques for the separation and the spectroscopic ones for structure elucidation (1H-NMR, 13C-NMR, COSY, HMBC).
Results
The chemical screening on the crude extract revealed the presence of polyphenols (flavonoids, anthocyanins, leucoanthocyanins, tannins, quinones) alkaloids, saponins, triterpenoids and steroids. The obtained extract after evaporation yielded 34.50 g (11.5%) out of 300 g of powdered leaves of O. basilicum. The acidified methanolic extract and butyl stearate showed an interesting antisickling activity.
Conclusions
The acidified methanolic extract and butyl stearate from O. basilicum displayed a good antisickling activity. To the best of our knowledge, this is the first time to report the antisickling activity of this compound in this plant. The synthesized compound presented the same spectroscopic characteristics than the natural one and the antisickling activities of its derivatives are understudying.
doi:10.12980/APJTB.4.2014C1329
PMCID: PMC3985055
Sickle cell disease; Antisickling plant; Ocimum basilicum; Butyl stearate
19.  Metastable Polymerization of Sickle Hemoglobin in Droplets 
Journal of molecular biology  2007;369(5):1170-1174.
Sickle cell disease arises from a genetic mutation of one amino acid in each of the two hemoglobin β chains, leading to the polymerization of hemoglobin in the red cell upon deoxygenation, and is characterized by vascular crises and tissue damage due to the obstruction of small vessels by sickled cells. It has been an untested assumption that, in red cells that sickle, the growing polymer mass would consume monomers until the thermodynamically well-described monomer solubility was reached. By photolyzing droplets of sickle hemoglobin suspended in oil we find that polymerization does not exhaust the available store of monomers, but stops prematurely, leaving the solutions in a supersaturated, metastable state typically 20% above solubility at 37°C, though the particular values depend on the details of the experiment. We propose that polymer growth stops because the growing ends reach the droplet edge, whereas new polymer formation is thwarted by long nucleation times, since the hemoglobin concentration is lowered by depletion of monomers into the polymers that have formed. This finding suggests a new aspect to the pathophysiology of sickle cell disease, namely, that cells deoxygenated in the microcirculation are not merely undeformable, but will actively wedge themselves tightly against the walls of the microvasculature by a ratchet-like mechanism driven by the supersaturated solution.
doi:10.1016/j.jmb.2007.04.030
PMCID: PMC1950749  PMID: 17493634
20.  Effect of Intracellular Magnesium and Oxygen Tension on K+-Cl− Cotransport in Normal and Sickle Human Red Cells 
In red cells from normal individuals (HbA cells), the K+-Cl− cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb polymerisation, but not on [Mg2+]i.
doi:10.1159/000092073
PMCID: PMC1475928  PMID: 16543728
KCl cotransport; Oxygen; Magnesium; Sickle cell
21.  Sodium-potassium pump, ion fluxes, and cellular dehydration in sickle cell anemia. 
Journal of Clinical Investigation  1987;79(6):1621-1628.
We studied the role of the sodium-potassium pump in erythrocytes of 12 patients with sickle cell anemia (SS). Ouabain-binding sites per cell and pump-mediated Rb/K uptake were significantly higher in SS patients than in white or black controls. Ouabain-resistant Rb/K influx was also greater than in normal controls or patients with sickle cell trait. Deoxygenation of SS erythrocytes increased ouabain-sensitive Rb/K influx without altering ouabain binding, presumably as the consequence of an increase in the passive influx of sodium. Deoxygenation increased mean corpuscular hemoglobin concentration (MCHC) by 5.5%, and studies of the density distribution of SS cells indicated an increase in highly dense fractions known to contain sickled erythrocytes. Ouabain prevented the rise in MCHC and reduced the percentage of dense cells. These findings indicate a magnified role for the sodium-potassium pump in the pathophysiology of SS erythrocytes and suggest that its inhibition might prove useful in therapy.
PMCID: PMC424484  PMID: 3034977
22.  Effects of deoxygenation on active and passive Ca2+ transport and on the cytoplasmic Ca2+ levels of sickle cell anemia red cells. 
Journal of Clinical Investigation  1993;92(5):2489-2498.
Elevated [Ca2+]i in deoxygenated sickle cell anemia (SS) red cells (RBCs) could trigger a major dehydration pathway via the Ca(2+)-sensitive K+ channel. But apart from an increase in calcium permeability, the effects of deoxygenation on the Ca2+ metabolism of sickle cells have not been previously documented. With the application of 45Ca(2+)-tracer flux methods and the combined use of the ionophore A23187, Co2+ ions, and intracellular incorporation of the Ca2+ chelator benz-2, in density-fractionated SS RBCs, we show here for the first time that upon deoxygenation, the mean [Ca2+]i level of SS discocytes was significantly increased, two- to threefold, from a normal range of 9.4 to 11.4 nM in the oxygenated cells, to a range of 21.8 to 31.7 nM in the deoxygenated cells, closer to K+ channel activatory levels. Unlike normal RBCs, deoxygenated SS RBCs showed a two- to fourfold increase in pump-leak Ca2+ turnover. Deoxygenation of the SS RBCs reduced their Ca2+ pump Vmax, more so in reticulocyte- and discocyte-rich than in dense cell fractions, and decreased their cytoplasmic Ca2+ buffering. Analysis of these results suggests that both increased Ca2+ influx and reduced Ca2+ pump extrusion contribute to the [Ca2+]i elevation.
PMCID: PMC288434  PMID: 8227363
23.  Pyridoxal phosphate as an antisickling agent in vitro. 
Journal of Clinical Investigation  1983;71(5):1224-1229.
Although pyridoxal phosphate is known to inhibit gelation of purified hemoglobin S, antisickling activity has never been demonstrated for intact erythrocytes. We incubated washed erythrocytes at 37 degrees C either in buffer alone, or with added pyridoxal phosphate or pyridoxal, washed these cells, suspended them in untreated buffer, and compared the percent modified hemoglobin, the oxygen affinity, and the extent of sickling under hypoxia. Pyridoxal phosphate modified intracellular hemoglobin more slowly than pyridoxal. Pyridoxal phosphate lowered the oxygen affinity of normal cells, but had no effect on oxygen binding by sickle cells. Pyridoxal increased the oxygen affinity of normal and sickle erythrocytes equally. Pyridoxal phosphate significantly inhibited sickling of sickle or sickle trait erythrocytes (P less than 0.001). Inhibition of sickling by pyridoxal phosphate was largely independent of oxygen binding; whereas inhibition of sickling by pyridoxal was almost entirely dependent on increased oxygen binding. Although pyridoxal phosphate and pyridoxal both inhibit sickling by modification of hemoglobin S, they differ in the kinetics of whole cell modification, the effect on oxygen affinity of intact cells, and the mechanism of action of the antisickling activity.
PMCID: PMC436982  PMID: 6853710
24.  Identification of the Ca2+ entry pathway involved in deoxygenation-induced phosphatidylserine exposure in red blood cells from patients with sickle cell disease 
Pflugers Archiv  2013;465:1651-1660.
Phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients is increased compared to levels in normal individuals and may participate in the anaemic and ischaemic complications of SCD. Exposure is increased by deoxygenation and occurs with elevation of intracellular Ca2+ to low micromolar levels. The Ca2+ entry step has not been defined but a role for the deoxygenation-induced pathway, Psickle, is postulated. Partial Psickle inhibitors 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS), 4,4′-dithiocyano-2,2′-stilbene-disulphonic acid (DIDS) and dipyridamole inhibited deoxygenation-induced PS exposure (DIDS IC50, 118 nM). Inhibitors and activators of other pathways (including these stimulated by depolarisation, benzodiazepines, glutamate and stretch) were without effect. Zn2+ and Gd3+ stimulated PS exposure to high levels. In the case of Zn2+, this effect was independent of oxygen (and hence HbS polymerisation and RBC sickling) but required extracellular Ca2+. The effect was completely abolished when Zn2+ (100 μM) was added to RBCs suspended in autologous plasma, implying a requirement of high levels of free Zn2+.
doi:10.1007/s00424-013-1308-y
PMCID: PMC3825215  PMID: 23775402
Sickle cell disease; Red blood cell; Phosphatidylserine; Deoxygenation; Calcium; Cation channel
25.  Effects of Cyanate and 2,3-Diphosphoglycerate on Sickling RELATIONSHIP TO OXYGENATION 
Journal of Clinical Investigation  1973;52(10):2542-2547.
Cyanate and 2,3-diphosphoglycerate (2,3-DPG) both influence the oxygen affinity of hemoglobin. The studies presented here concern the effects of these compounds on the sickling phenomenon. The inhibitory effect of cyanate on sickling is largely due to the fact that it increases the percentage of oxyhemoglobin S at a given oxygen tension. In addition, cyanate inhibits sickling by a mechanism that is independent of oxygenation. In this paper, we have demonstrated that the viscosity of carbamylated sickle blood was lower than that of non-carbamylated controls at the same oxygen saturation. Furthermore, carbamylation resulted in an increase in the minimum concentration of deoxy-sickle hemoglobin required for gelation.
Like cyanate, 2,3-DPG affected sickling of intact erythrocytes by two mechanisms. Since 2,3-DPG decreases the percentage of oxyhemoglobin S at a given oxygen tension, sickling is enhanced. In addition, 2,3-DPG had a direct effect. When the intracellular 2,3-DPG concentration was increased in vitro, a greater percentage of cells were sickled at a given oxygen saturation. Conversely, sickling was inhibited in cells in which 2,3-DPG was artificially lowered. These data indicate that the enhancement of sickling by 2,3-DPG is in part independent of its influence on oxygen affinity.
PMCID: PMC302513  PMID: 4729047

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