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1.  A non-electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease 
The Journal of Physiology  2013;591(6):1463-1474.
Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.
doi:10.1113/jphysiol.2012.246579
PMCID: PMC3607166  PMID: 23297308
2.  A non-electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease 
The Journal of Physiology  2013;591(Pt 6):1463-1474.
Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.
doi:10.1113/jphysiol.2012.246579
PMCID: PMC3607166  PMID: 23297308
3.  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
4.  Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease 
American journal of hematology  2014;89(6):598-603.
In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient’s disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged “sickled” and “normal” erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = −0.558, P = 0.027), negatively with pH (R = −0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = −0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.
doi:10.1002/ajh.23699
PMCID: PMC4180063  PMID: 24585634
5.  The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress 
Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin.
doi:10.3389/fmicb.2015.01390
PMCID: PMC4676198  PMID: 26696995
Saccharomyces cerevisae; ADH6; ADH7; vanillin; translational repression; lignocellulosic biomass; bioethanol production
6.  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.
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PMCID: PMC371842  PMID: 701485
7.  Structure of fully liganded Hb ζ2β2 s trapped in a tense conformation 
The crystallographic analysis of fully liganded Hb ζ2β2 s trapped in a tense conformation is reported.
A variant Hb ζ2β2 s that is formed from sickle hemoglobin (Hb S; α2β2 s) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2β2 s inhibits the polymerization of deoxy­genated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2β2), Hb ζ2β2 s exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-­diphosphoglycerate allostery. Here, the 1.95 Å resolution crystal structure of human Hb ζ2β2 s that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2β2 s displays a central water cavity, a ζ1–βs2 (or ζ2–βs1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal βHis146 salt-bridge interactions, and a β-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1βs1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2β2 s and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.
doi:10.1107/S0907444913019197
PMCID: PMC3792644  PMID: 24100324
hemoglobin; tense state; relaxed state; Bohr effect; 2,3-disphosphoglycerate; cooperativity; allostery
8.  Hypoxia-induced in vivo sickling of transgenic mouse red cells. 
Journal of Clinical Investigation  1991;87(2):639-647.
To develop an animal model for sickle cell anemia, we have created transgenic mice that express a severe naturally occurring human sickling hemoglobin, Hb S Antilles. Due to its low solubility and oxygen affinity, Hb S Antilles has a greater propensity to cause red cell sickling than Hb S. To make transgenic animals that express a high level of Hb S Antilles, the erythroid-specific DNAse I hypersensitive site II from the human beta-globin cluster was linked independently to the human alpha 2-globin gene and to the beta S Antilles gene. Embryos were injected with both constructs simultaneously and seven transgenic mice were obtained, three of which contained both the human alpha and the human beta S Antilles transgene. After crossing the human transgenes into the mouse beta-thalassemic background a transgenic mouse line was derived in which approximately half the beta-globin chains in the murine red cells were human beta S Antilles. Deoxygenation of the transgenic red cells in vitro resulted in extensive sickling. An increase of in vivo sickling was achieved by placing these transgenic mice in a low oxygen environment. This murine model for red cell sickling should help to advance our understanding of sickle cell disease and may provide a model to test therapeutic interventions.
Images
PMCID: PMC296354  PMID: 1991848
9.  Effect of cetiedil, an in vitro antisickling agent, on erythrocyte membrane cation permeability. 
Journal of Clinical Investigation  1981;68(5):1215-1220.
Cetiedil has been reported to relieve painful crises in sickle cell anemia and to have antisickling properties in vitro. The drug alters neither oxygen affinity nor the solubility of deoxyhemoglobin S. Because the viscosity of the erythrocyte interior and the kinetics of gelation are dependent on the concentration of hemoglobin, we postulated that cetiedil might inhibit sickling by modifying erythrocyte sodium or potassium movements in a manner that would increase cell water content and thus dilute the cell hemoglobin. The drug has two such effects: it inhibits the specific increase in potassium permeability that follows a rise in cytoplasmic calcium concentration and it causes a rise in passive sodium movements. These effects are further evidence that cell ion and water movements may be important in the process of sickling and suggest a mechanism for the results reported with cetiedil.
PMCID: PMC370916  PMID: 7298848
10.  Phytomedicines and Nutraceuticals: Alternative Therapeutics for Sickle Cell Anemia 
The Scientific World Journal  2013;2013:269659.
Sickle cell anemia is a genetically inherited disease in which the “SS” individual possesses an abnormal beta globin gene. A single base substitution in the gene encoding the human β-globin subunit results in replacement of β6 glutamic acid by valine, leading to the devastating clinical manifestations of sickle cell disease. This substitution causes drastic reduction in the solubility of sickle cell hemoglobin (HbS) when deoxygenated. Under these conditions, the HbS molecules polymerize to form long crystalline intracellular mass of fibers which are responsible for the deformation of the biconcave disc shaped erythrocyte into a sickle shape. First-line clinical management of sickle cell anemia include, use of hydroxyurea, folic acid, amino acids supplementation, penicillinprophylaxis, and antimalarial prophylaxis to manage the condition and blood transfusions to stabilize the patient's hemoglobin level. These are quite expensive and have attendant risk factors. However, a bright ray of hope involving research into antisickling properties of medicinal plants has been rewarding. This alternative therapy using phytomedicines has proven to not only reduce crisis but also reverse sickling (in vitro). The immense benefits of phytomedicines and nutraceuticals used in the management of sickle cell anemia are discussed in this paper.
doi:10.1155/2013/269659
PMCID: PMC3586489  PMID: 23476125
11.  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
12.  Moringa oleifera Lam. (Moringaceae) grown in Nigeria: In vitro antisickling activity on deoxygenated erythrocyte cells 
Context:
Traditional medicine, which is more available and affordable for the poor uses medicinal plants for the treatment and management of various ailments, including the sickle cell disease (SCD). About 24 million Nigerians are carriers of this sickled cell gene, while approximately 2.4 million are SCD patients. Moringa oleifera Lam. (Moringaceae) possesses high nutritional value and has been used in folklore medicine to treat various ailments related to pain and inflammation. Chemical, pharmacological and pharmacognostical applications of Moringa oleifera have been reported.
Objective:
This study investigated the antisickling potential of polar and non-polar extracts of the seed, flower and leaf of Moringa oleifera for the first time.
Materials and Methods:
Using crude methanol extract, aqueous extract, ethyl acetate and butanol, the in vitro antisickling activities of Moringa oleifera fractions, were evaluated using erythrocyte cells deoxygenated with 2% sodium metabisulphite. p-Hydroxybenzoic acid and normal saline were employed as positive and negative controls.
Results:
Phytochemical screening revealed the presence of saponins, free anthraquinones, and alkaloids. Extracts of the seed and flower demonstrated a higher (P<0.05) antisickling activity in comparison to the leaf extract. The leaf extract, as well as those of the seed and flower, equally demonstrated a (P<0.05) reversal of sickled erythrocytes.
Discussions and Conclusions:
These findings suggest that Moringa oleifera may play a role in the management of SCD, by incorporation of its fractions into recipes. More extensive biological evaluations and further studies will be necessary for the chemical characterization of the antisickling principles.
doi:10.4103/0975-7406.94812
PMCID: PMC3341715  PMID: 22557922
Moringa oleifera; non-polar fractions; phytochemicals; polar extracts; sickle cell disease
13.  Erythrocyte Hb-S Concentration AN IMPORTANT FACTOR IN THE LOW OXYGEN AFFINITY OF BLOOD IN SICKLE CELL ANEMIA 
Journal of Clinical Investigation  1973;52(2):422-432.
The blood in sickle cell anemia has a very low oxygen affinity and, although 2,3-diphosphoglycerate (2,3-DPG) is increased, there is doubt as to whether this is the only factor responsible. In this study of 15 patients with sickle cell anemia (Hb SS) no correlation was found between oxygen affinity (P50 at pH 7.13) and 2,3-DPG in fresh venous blood. Whole populations of Hb SS erythrocytes were therefore separated, by an ultracentrifuge technique, into fractions of varying density. The packed red cell column was divided into three fractions; a bottom fraction rich in deformed cells or irreversibly sickled cells (ISC), with a very high mean corpuscular hemoglobin concentration (MCHC); a middle fraction containing cells with the highest content of fetal hemoglobin; and a top fraction containing reticulocytes and discoid cells but free of deformed cells. Oxygen affinity was shifted to the right in all layers (mean P50 (pH 7.13)±1SD: top 46.3±2.9 mm Hg: middle 49.8±4.9 mm Hg; bottom 61.0±5.8 mm Hg) compared with normal blood (top 32.1±0.7 mm Hg: bottom 30.1±0.5 mm Hg). 2.3-DPG was increased in the top fraction, but was low or normal in the bottom fraction (top 21.8±3.4 μmol/g Hb: middle 17.7±2.2 μmol/g Hb; bottom 13.8±3.1 μmol/g Hb; normal whole blood 14.3±1.2 μmol/g Hb). The level of 2,3-DPG in top fractions could not account for the degree of right shift of P50, and in the middle and bottom fractions the even greater right shifts were associated with lower levels of 2,3-DPG. Top fraction cells depleted of 2,3-DPG had a higher, but still abnormally low, oxygen affinity. A strong relationship was found between oxygen affinity and MCHC. The fractions with the greatest right shift in P50 had the highest MCHC (top 32.4±2.0; middle 36.2±3.1; bottom 44.6±3.2 g/100 ml, respectively) and the plot of P50 vs. MCHC showed a positive correlation (r = 0.90, P < 0.001).
The red cell population in sickle cell anemia is not homogeneous but contains cells of widely varying Hb F content, 2,3-DPG, and hemoglobin concentration. Paradoxically, the cells with the lowest O2 affinity have the lowest 2,3-DPG, but they also have the highest concentration of Hb S. The dense, deformed cell called the ISC is but the end stage in a process of membrane loss and consequent increase in hemoglobin concentration. The P50 of Hb SS blood is, to a large extent, determined by the presence of these cells (r = 0.85, P < 0.001). Increased concentration of Hb S in the cell favors deoxygenation and crystallization even at relatively high Po2. Lowered affinity for oxygen appears to be closely associated with Hb S concentration and not with 2,3-DPG content.
PMCID: PMC302272  PMID: 4683881
14.  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
15.  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
16.  Erythrocytes in sickle cell anemia are heterogeneous in their rheological and hemodynamic characteristics. 
To understand the contribution to the pathophysiology of sickle cell anemia of the different erythrocyte density types present in the blood of these patients, we have studied the viscosimetric and hemodynamic characteristics of four major classes of hemoglobin SS erythrocytes. We have isolated reticulocytes, discocytes, dense discocytes, and irreversibly sickled cells (fractions I-IV) on Percoll-Renografin density gradients. Bulk viscosity was studied in a coneplate viscosimeter and the hemodynamic studies were performed on the isolated, artificially perfused mesoappendix vasculature of the rat (Baez preparation). Bulk viscosity measurements at shear rates of 230 S-1 demonstrate that when the cells are oxygenated, fraction I (reticulocyte rich) has a higher viscosity than expected from its low intracellular hemoglobin concentration. The rest of the fractions exhibit moderate increases in bulk viscosity pari-passu with the corresponding increases in density (mean corpuscular hemoglobin concentration). When deoxygenated, all cell fractions nearly doubled their bulk viscosity and the deoxy-oxy differences remained constant. The Baez preparation renders a different picture: oxygenated fractions behave as predicted by the viscosimetric data, but, when deoxygenated, cell fractions exhibit dramatically increased peripheral resistance and the deoxy-oxy difference are directly proportional to cell density, thus, the largest increases were observed for fractions III and IV. The differences between the rheological and the hemodynamic measurements are most probably due to the different sensitivity of the two methods to the extent of intracellular polymerization. These results also demonstrate that the hitherto unrecognized fraction III cells (very dense discocytes that change shape very little on deoxygenation) are as detrimental to the microcirculation as the irreversibly sickled cell-rich fraction IV. They may, however, induce obstruction by a different mechanism. As the extent to which these fractions are populated by erythrocytes varies considerably from patient to patient, the distribution function of cell densities in each sickle cell anemia patient might have consequences for the type of pathophysiological events occurring in their microcirculation.
Images
PMCID: PMC1129157  PMID: 6874947
17.  Partitioning of benzene in blood: influence of hemoglobin type in humans and animals. 
Environmental Health Perspectives  2002;110(3):255-261.
Earlier studies have shown that air/blood partition coefficients (PCs) for many volatile organic chemicals (VOCs) are much higher in rat blood than in human blood. It has been suggested that the discrepancy could be attributed to the fact that hemoglobin (Hb) in rat blood exists in a quasi-crystalline form of hydrophobicity greater than that of normal human Hb (HbA) and thus has a higher carrying capacity for VOCs. In the present study, we used benzene as a prototypic VOC to examine its relative partitioning into human and animal blood. Additionally, we sought to ascertain whether the water-insoluble form of hemoglobin (HbS) found in subjects with homozygous sickle cell (SC) disease has a greater VOC-carrying capacity than does HbA blood. At a low-O(2) tension, HbS switches to water-insoluble polymers, which physically deforms the red blood cells (RBCs) to the sickle shape. We equilibrated HbA, HbS, Hartley guinea pig, CD1 mouse, and rat (F-344, Wistar, and Sprague-Dawley) blood and their respective fractions with benzene vapor (80 or 400 ppm) for 3 hr at 37 degrees C in air-tight vials. We introduced benzene vapor into the vial head space that contained air or respiratory mixtures of venous-type (low-O(2)) or arterial-type (high-O(2)) gases. The blood measurements included the PC, Hb, partial pressures of O(2)(pO(2)) and CO(2)(pCO(2), pH, and percentage of SCs. The benzene concentration had no effect on these parameters, and the high- and low-O(2) gas mixtures produced the expected changes in pO(2), pCO(2), and pH. At equilibrium, the low-O(2) HbS blood had approximately 85% SCs compared with roughly 15% with air or high-O(2) gas. PCs for rat and mouse blood were about 100% higher than those for human and guinea pig blood, but the PC for deoxygenated HbS blood was only slightly higher than that for HbA or oxygenated HbS blood. Benzene showed higher affinities for RBCs in the deoxygenated HbS, rat, and mouse blood and higher affinity for plasma in the guinea pig blood. There was no evidence of disproportionate partitioning of benzene into oxygenated HbS or into HbA blood forms. These data suggest that the water solubility of Hb alone appears to have little effect on the VOC-carrying capacity of blood and that the influence of species is large in comparison. These latter differences in partitioning may depend on the number of hydrophobic sites on the surface of the plasma/heme proteins and thus be unique to the species.
PMCID: PMC1240765  PMID: 11882476
18.  Clopidogrel Protects Endothelium by Hindering TNFα-Induced VCAM-1 Expression through CaMKKβ/AMPK/Nrf2 Pathway 
Journal of Diabetes Research  2015;2016:9128050.
Clopidogrel (INN), an oral antiplatelet drug, has been revealed to have a number of biological properties, for instance, anti-inflammation and antioxidation. Oxidative stress plays an imperative role in inflammation, diabetes mellitus, atherosclerosis, and cancer. In the present study, human aortic endothelial cells (HAECs) were employed to explore the anti-inflammatory activity of INN. INN reduced TNFα-induced reactive oxygen species (ROS) generation and time-dependently prompted the expression and activity of heme oxygenase 1 (HO-1). Cellular glutathione (GSH) levels were augmented by INN. shHO-1 blocked the INN suppression of TNFα-induced HL-60 cell adhesion. The CaMKKβ/AMPK pathway and Nrf2 transcriptional factor were implicated in the induction of HO-1 by INN. Additionally, TNFα dramatically augmented VCAM-1 expression at protein and mRNA levels. INN treatment strikingly repressed TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Compound C, an AMPK inhibitor, and shNrf2 abolished TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Our data suggest that INN diminishes TNFα-stimulated VCAM-1 expression at least in part via HO-1 induction, which is CaMKKβ/AMPK pathway-dependent.
doi:10.1155/2016/9128050
PMCID: PMC4707324  PMID: 26824050
19.  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
20.  Therapeutic Strategies to Alter Oxygen Affinity of Sickle Hemoglobin 
The fundamental pathophysiology of sickle cell disease involves the polymerization of sickle hemoglobin in its T-state which develops under low oxygen saturation. One therapeutic strategy is to develop pharmacologic agents to stabilize the R-state of hemoglobin, which has higher oxygen affinity and would be expected to have slower kinetics of polymerization, potentially delaying the sickling of red cells during circulation. This therapeutic strategy has stimulated the laboratory investigation of aromatic aldehydes, aspirin derivatives, thiols and isothiocyanates that can stabilize the R-state of hemoglobin in vitro. One representative aromatic aldehyde agent, 5-hydoxymethyl-2-furfural (5-HMF, also known as Aes-103) increases oxygen affinity of sickle hemoglobin and reduces hypoxia-induced sickling in vitro and protects sickle cell mice from effects of hypoxia. It has completed pre-clinical testing and has entered clinical trials. The development of Hb allosteric modifiers as direct anti-sickling agents is an attractive investigational goal for the treatment of sickle cell disease.
doi:10.1016/j.hoc.2013.11.001
PMCID: PMC4195245  PMID: 24589263
Sickle cell; 5-HMF; Anti-sickling; R-state; Hemoglobin allosteric effectors
21.  Interaction of Phosphatidylserine-Phosphatidylcholine Liposomes with Sickle Erythrocytes 
Journal of Clinical Investigation  1983;71(6):1570-1580.
The sickle erythrocyte (RBC) is a pathologic RBC that contains multiple membrane abnormalities. Some of these abnormalities have been implicated in the pathophysiology of vasoocclusive crises characteristic of sickle cell disease; others have yet to be defined in terms of their clinical significance. Recent information has shown that sickle RBC adhere abnormally to cultured endothelial cells yet little is known about the ways in which sickle cells interact with model membranes of defined size and lipid composition. We investigated this phenomenon by interacting sickle RBC with artificial lipid vesicles (liposomes) containing acidic phospholipids. Our results demonstrate that sickle disease (hemoglobin SS) RBC bind more of these liposomes than do normal or sickle trait (hemoglobin AS) RBC and that these differences are accentuated by hypoxia-induced sickling. Binding of liposome phospholipid to sickled RBC was not attributable to phospholipid exchange between liposomes and RBC and was consistent with a mechanism involving both membrane fusion and a stable reversible adhesion of liposomes to the RBC membrane.
Investigations into the mechanism(s) underlying increased liposome binding to sickled RBC suggested that the known reversible translocation of aminophospholipids, phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE), from the inner to the outer leaflet of the reversibly sickled RBC (RSC) plasma membrane during sickling may be a component of increased liposome binding to RSC. This idea was supported from results of experiments in which normal RBC were treated with diamide resulting in the expression of outer leaflet PE and PS and a stimulation of liposome binding to these cells. However, sickle RBC separated according to cell density on stractan gradients showed that irreversibly sickled RBC (ISC) were less capable of liposome binding than were discoid RSC. Since ISC are known to contain elevated levels of outer leaflet aminophospholipids, such a result suggests that other changes in the plasma membrane of sickle cells, in addition to phospholipid reorganization, are probably involved in enhanced liposome binding to these cells. In other experiments, we showed that liposomes containing l-phenylalanine were capable of delivering this antisickling agent into intact sickle RBC as demonstrated by the partial inhibition of hypoxia-induced sickling in vitro. Our results suggest that liposomes can be used as sensitive probes for investigating changes in RBC membrane properties, especially those that affect intermembrane interactions, and that liposomal transport systems may have significant implications in the therapy of sickle cell disease.
PMCID: PMC370363  PMID: 6408122
22.  Neurosupportive Role of Vanillin, a Natural Phenolic Compound, on Rotenone Induced Neurotoxicity in SH-SY5Y Neuroblastoma Cells 
Vanillin, a phenolic compound, has been reported to offer neuroprotection against experimental Huntington's disease and global ischemia by virtue of its antioxidant, anti-inflammatory, and antiapoptotic properties. The present study aims to elucidate the underlying neuroprotective mechanism of vanillin in rotenone induced neurotoxicity. Cell viability was assessed by exposing SH-SY5Y cells to various concentrations of rotenone (5–200 nM) for 24 h. The therapeutic effectiveness of vanillin against rotenone was measured by pretreatment of vanillin at various concentrations (5–200 nM) and then incubation with rotenone (100 nM). Using effective dose of vanillin (100 nM), mitochondrial membrane potential, levels of reactive oxygen species (ROS), and expression patterns of apoptotic markers were assessed. Toxicity of rotenone was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, release of cyt-c, and enhanced expressions of proapoptotic and downregulation of antiapoptotic indices via the upregulation of p38 and JNK-MAPK pathway proteins. Our results indicated that the pretreatment of vanillin attenuated rotenone induced mitochondrial dysfunction, oxidative stress, and apoptosis. Thus, vanillin may serve as a potent therapeutic agent in the future by virtue of its multiple pharmacological properties in the treatment of neurodegenerative diseases including PD.
doi:10.1155/2015/626028
PMCID: PMC4664805  PMID: 26664453
23.  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
24.  The in vitro antisickling and antioxidant effects of aqueous extracts Zanthoxyllum heitzii on sickle cell disorder 
Background
Several plant extracts from Rutaceae family are currently used to the management of sickle cell disorder (SCD) in the African. Few reports have shown that extracts from Zanthoxyllum or Fagara genus demonstrated anti-sickling property. This study investigates the in vitro antisickling and antioxidant properties of extracts from Zanthoxyllum heitzii.
Methods
The sickling of red blood cells (RBCs) was induced using sodium metabisulfite (2%) followed by treatment with extracts at different concentrations. The osmotic fragility tests permits to explore the effect of Z. heitzii extracts on haemoglobin S solubility and sickle cells membrane stability. For each extract, qualitative phytochemical tests were used to identify the presence of alkaloids, tannins, saponins, flavonoids, glycosides and phenols, while some quantitative methods such as Folin, Ferric Reducing Antioxidant Power (FRAP) and diphenyl 1, 2 picryl hydrazyl (DPPH) were used to determine the antioxidant potential of these extracts.
Results
Sodium metabisulphite increased the sickling of RBCs from 29.62 to 55.46% during 2 h. Treatment of sickling cells with extracts at different concentrations showed that a decrease of the percentage of sickling cells was found in both induced and non induced sickling cells. The fruits extract of Z. heitzii demonstrated the best anti-sickling property. The same extract at 250 μg/mL showed the best membrane cell stability compared to others. All the extracts revealed an antioxidant and anti-radical activities although lesser compared to the standard.
Conclusion
The fruit extract of Z. Heitzii demonstrated the most significant antisickling effect with a potential for use in the clinical management of SCD.
doi:10.1186/1472-6882-13-162
PMCID: PMC3708797  PMID: 23829696
Sickling cells; Reversibility; Antioxidant; Hemolysis; Zanthoxyllum heitzii
25.  Effects of aromatic compounds on the production of bacterial nanocellulose by Gluconacetobacter xylinus 
Background
Bacterial cellulose (BC) is a polymeric nanostructured fibrillar network produced by certain microorganisms, principally Gluconacetobacter xylinus. BC has a great potential of application in many fields. Lignocellulosic biomass has been investigated as a cost-effective feedstock for BC production through pretreatment and hydrolysis. It is well known that detoxification of lignocellulosic hydrolysates may be required to achieve efficient production of BC. Recent results suggest that phenolic compounds contribute to the inhibition of G. xylinus. However, very little is known about the effect on G. xylinus of specific lignocellulose-derived inhibitors. In this study, the inhibitory effects of four phenolic model compounds (coniferyl aldehyde, ferulic acid, vanillin and 4-hydroxybenzoic acid) on the growth of G. xylinus, the pH of the culture medium, and the production of BC were investigated in detail. The stability of the phenolics in the bacterial cultures was investigated and the main bioconversion products were identified and quantified.
Results
Coniferyl aldehyde was the most potent inhibitor, followed by vanillin, ferulic acid, and 4-hydroxybenzoic acid. There was no BC produced even with coniferyl aldehyde concentrations as low as 2 mM. Vanillin displayed a negative effect on the bacteria and when the vanillin concentration was raised to 2.5 mM the volumetric yield of BC decreased to ~40% of that obtained in control medium without inhibitors. The phenolic acids, ferulic acid and 4-hydroxybenzoic acid, showed almost no toxic effects when less than 2.5 mM. The bacterial cultures oxidized coniferyl aldehyde to ferulic acid with a yield of up to 81%. Vanillin was reduced to vanillyl alcohol with a yield of up to 80%.
Conclusions
This is the first investigation of the effect of specific phenolics on the production of BC by G. xylinus, and is also the first demonstration of the ability of G. xylinus to convert phenolic compounds. This study gives a better understanding of how phenolic compounds and G. xylinus cultures are affected by each other. Investigations in this area are useful for elucidating the mechanism behind inhibition of G. xylinus in lignocellulosic hydrolysates and for understanding how production of BC using lignocellulosic feedstocks can be performed in an efficient way.
doi:10.1186/1475-2859-13-62
PMCID: PMC4126184  PMID: 24884902
Gluconacetobacter xylinus; Phenolic compound; Bacterial cellulose; Inhibitor

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