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1.
Blood ; 95(4): 1283-92, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10666201

ABSTRACT

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)


Subject(s)
Actins/blood , Apoptosis , Blood Platelets/cytology , Cytoskeleton/physiology , Leukocytes/cytology , Wiskott-Aldrich Syndrome/blood , Adenosine Diphosphate/pharmacology , Blood Platelets/drug effects , Blood Platelets/pathology , Cell Survival/drug effects , Cytochalasin B/pharmacology , Dinoprost/pharmacology , Humans , In Vitro Techniques , Kinetics , Leukocytes/drug effects , Leukocytes/pathology , Lymphocytes/drug effects , Lymphocytes/pathology , Lymphocytes/physiology , Mutation, Missense , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Necrosis , Point Mutation , Proteins/genetics , Reference Values , Tetradecanoylphorbol Acetate/pharmacology , Thrombocytopenia/blood , Thrombocytopenia/genetics , Thrombocytopenia/pathology , Wiskott-Aldrich Syndrome/genetics , Wiskott-Aldrich Syndrome/pathology , Wiskott-Aldrich Syndrome Protein , fas Receptor/blood , src Homology Domains
2.
Br J Haematol ; 92(4): 968-72, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8616095

ABSTRACT

Arginine vasopressin (AVP) is a neurohypophyseal peptide hormone with protean effects. Previous reports had shown that AVP stimulates platelets, but only at concentrations 3-6 logs higher than the normal plasma concentrations in humans. In this study we tested the hypothesis that AVP, at physiologic concentrations, stimulated the expression of an activation-dependent platelet antigen. Platelets obtained from normal volunteers were incubated with increasing concentrations of AVP and the expression of the activation-dependent platelet antigen P-selectin (CD62) was determined by monoclonal antibodies and flow cytometry. There was a concentration-dependent increase in CD62 expression with increasing AVP concentration; at 1 pm AVP, 24.5% (1.3-88.5%) [median (range)] of platelets expressed CD62. The selective vasopressin V1 receptor antagonist d(CH2)(5)-Tyr(me)AVP (TM-AVP) completely abolished AVP-stimulated CD62 expression. We conclude that AVP can activate platelets at concentrations found in normal humans, at least in vitro, and that this response is mediated by the platelet V1 receptor. AVP may be a physiologic platelet agonist.


Subject(s)
Arginine Vasopressin/pharmacology , Platelet Activation/physiology , Adult , Antidiuretic Hormone Receptor Antagonists , Female , Flow Cytometry , Humans , In Vitro Techniques , Male , Middle Aged , P-Selectin/metabolism
3.
J Lab Clin Med ; 126(4): 401-9, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7561450

ABSTRACT

Desmopressin (1-desamino-8-D-arginine vasopressin (DDAVP)) is a synthetic analog of arginine vasopressin (AVP) and is useful in the treatment of some bleeding disorders. The mechanism of improved hemostasis in patients with platelet dysfunction is uncertain. Platelet-rich plasma samples from 35 normal subjects were incubated with serial dilutions of DDAVP, AVP, and adenosine diphosphate. The expression of the platelet activation-dependent antigen CD62 (P-selectin) was measured by fluorescent-labeled monoclonal antibody and flow cytometry. DDAVP at concentrations of 1.0 to 1000 nmol/L stimulated significant expression of CD62 on normal platelets in vitro. At a pharmacologic concentration of DDAVP (1 nmol/L), 14.1% (0.6% to 45.4%) (median and range) of platelets expressed CD62. There was a strong correlation between DDAVP-induced and AVP-induced CD62 expression (rs = 0.62, p = 0.0008) but not between DDAVP-induced and ADP-induced expression, suggesting a V1 receptor-mediated mechanism. Preincubation of platelets with a vasopressin V1 receptor antagonist completely inhibited CD62 expression in response to DDAVP. We conclude that DDAVP directly activates platelets by interaction with the platelet V1 receptor in vitro. This finding may partially explain in vivo effects of DDAVP on hemostasis.


Subject(s)
Antigens, CD/biosynthesis , Blood Platelets/metabolism , Deamino Arginine Vasopressin/pharmacology , P-Selectin/biosynthesis , Adenosine Diphosphate/pharmacology , Antibodies, Monoclonal , Antidiuretic Hormone Receptor Antagonists , Antigens, CD/blood , Arginine Vasopressin/analogs & derivatives , Arginine Vasopressin/pharmacology , Blood Platelets/drug effects , Dioxolanes/pharmacology , Dose-Response Relationship, Drug , Flow Cytometry , Hemostasis , Humans , In Vitro Techniques , P-Selectin/blood
4.
Am J Hematol ; 49(1): 56-66, 1995 May.
Article in English | MEDLINE | ID: mdl-7741139

ABSTRACT

Previous reports have alluded to variability in the aggregation response of normal human platelets to the neuropeptide arginine vasopressin (AVP). Since it has not been well documented, the current studies were undertaken to characterize this response. AVP (1-100 nM) produced a concentration-dependent aggregation response. Although the aggregation response to 100 nM AVP did not correlate with age or sex, there was a bimodal response distribution based on the presence or absence of a second wave of aggregation. In kinetic studies, the apparent km of AVP was 18.3 +/- 5.4 nM. There was a significant inverse relationship between the maximal aggregation response to 100 nM AVP and the km (r = -0.82). One hundred nanomolar AVP increased the intracellular calcium concentration of platelets by 406 +/- 120 nM in calcium free buffer and by 658 +/- 233 nM in the presence of 1.0 mM CaCl2. The aggregation response to 100 nM AVP correlated most strongly with the transmembrane influx of calcium (r = 0.84). In individuals whom 100 nM AVP was able to generate a second wave of aggregation, the selective protein kinase C inhibitor bis-indolylmaleimide significantly decreased the platelet aggregation response. Thus, there is significant heterogeneity in the aggregation response of normal human platelets to AVP. Based on our kinetic studies and the effects of PKC inhibition on the aggregation response to AVP, we would hypothesize that the variability of the aggregation response of normal human platelets to AVP is related to a polymorphism of the platelet AVP V1 receptor.


Subject(s)
Arginine Vasopressin/pharmacology , Blood Platelets/physiology , Calcium/blood , Platelet Aggregation/drug effects , Age Factors , Anticoagulants/pharmacology , Blood Platelets/drug effects , In Vitro Techniques , Kinetics , Platelet Aggregation Inhibitors/pharmacology , Protein Kinase C/antagonists & inhibitors , Regression Analysis , Sex Characteristics , Time Factors
5.
Blood ; 80(2): 512-6, 1992 Jul 15.
Article in English | MEDLINE | ID: mdl-1320957

ABSTRACT

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin-sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5'-nucleotidase (P5N), 6-phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin-sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.


Subject(s)
Anemia, Sickle Cell/enzymology , Erythrocytes/enzymology , Nucleoside-Phosphate Kinase/blood , Anemia, Sickle Cell/blood , Cell Separation , Hemin/pharmacology , Hexokinase/blood , Humans , In Vitro Techniques , Kinetics , Nucleoside-Phosphate Kinase/deficiency , Reference Values , Thalassemia/blood , Thalassemia/enzymology
6.
Blood ; 77(12): 2774-84, 1991 Jun 15.
Article in English | MEDLINE | ID: mdl-1646049

ABSTRACT

Adenylate kinase (AK) modulates the interconversion of adenine nucleotides (AMP + adenosine triphosphate----2 ADP). We evaluated the fifth kindred with hereditary erythrocyte (RBC) AK deficiency. The proband had chronic hemolytic anemia. Her RBC had undetectable AK activity when measured spectrophotometrically, whereas those of her parents had half-normal AK activity. AK electrophoresis showed only AK-1 in the parents. The activities of pyruvate kinase and phosphoribosylpyrophosphate synthetase were decreased given the young age of the proband's RBC. Despite the absence of spectrophotometric AK activity, the proband's RBC were able to incorporate 14C-adenine into 14C-adenine nucleotides at 50% of the rate expected for her young RBC population, suggesting the possibility of an alternative pathway for the formation of ADP from AMP. Normal hemolysate had AMP:guanosine triphosphate (GTP) phosphotransferase activity, which produced ADP at 8% to 9% of the rate of AK (6.8 +/- 0.8 IU/mL RBC). AMP:GTP phosphotransferase activity was not detectable in the proband's or parent's hemolysates. These additional biochemical defects in the AK-deficient RBC further support the concept that AK deficiency per se may not cause hemolytic anemia. We propose that defects occur in multiple phosphotransferases in the AK-deficient RBC and that these other biochemical defects may produce deleterious lesions that promote the shortened RBC survival in AK deficiency.


Subject(s)
Adenylate Kinase/deficiency , Anemia, Hemolytic/enzymology , Erythrocytes/enzymology , Nucleoside-Phosphate Kinase , Phosphotransferases/blood , Adenosine Diphosphate/blood , Adenosine Monophosphate/blood , Adenosine Triphosphate/blood , Adenylate Kinase/blood , Anemia, Hemolytic/genetics , Child, Preschool , Electrophoresis, Cellulose Acetate , Female , Glutathione/blood , Hemin/pharmacology , Humans , Kinetics , Magnesium/pharmacology , Potassium Chloride/pharmacology , Sulfhydryl Reagents/pharmacology
7.
Miner Electrolyte Metab ; 17(1): 52-7, 1991.
Article in English | MEDLINE | ID: mdl-1770916

ABSTRACT

The effect of dimethyl sulfoxide (DMSO) on the rate of glucose oxidation by cultured rat glomerular mesangial cells, human erythrocytes and peritoneal exudate cells was studied. Mesangial cells, erythrocytes and peritoneal exudate cells incubated with DMSO showed enhancement of 14CO2 production from D-[1-14C] glucose but not from D-[6-14C] glucose. The concentration of DMSO required to stimulate respiratory burst activity was lowest for erythrocytes and highest for peritoneal exudate cells. Studies utilizing tritiated deoxyglucose revealed that the increased glucose oxidation associated with DMSO exposure was not due to increased transmembrane glucose movement at low concentrations of DMSO, and only partially responsible at high concentrations of DMSO. This study documents the ability of DMSO to specifically enhance the activity of the hexose monophosphate shunt pathway in all cells studied. The precise mechanism whereby DMSO stimulates shunt activity remains unknown.


Subject(s)
Dimethyl Sulfoxide/pharmacology , Erythrocytes/metabolism , Glomerular Mesangium/metabolism , Leukocytes/metabolism , Pentose Phosphate Pathway/drug effects , Animals , Ascitic Fluid/cytology , Carbon Radioisotopes , Cells, Cultured , Erythrocytes/drug effects , Glomerular Mesangium/drug effects , Glucose/metabolism , Humans , Kinetics , Leukocytes/drug effects , Male , Oxidation-Reduction , Rats , Rats, Inbred Strains
8.
Blood ; 76(5): 1008-14, 1990 Sep 01.
Article in English | MEDLINE | ID: mdl-2393709

ABSTRACT

We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.


Subject(s)
Anemia, Sickle Cell/blood , Cytochrome-B(5) Reductase/blood , Erythrocytes/metabolism , Methemoglobin/metabolism , NADH, NADPH Oxidoreductases/blood , NAD/blood , Anemia, Sickle Cell/enzymology , Hemoglobin, Sickle/metabolism , Hexokinase/blood , Humans , Kinetics , Oxidation-Reduction , Reference Values , Thalassemia/blood , Thalassemia/enzymology
9.
Am J Clin Pathol ; 92(5): 589-94, 1989 Nov.
Article in English | MEDLINE | ID: mdl-2816812

ABSTRACT

Bone marrow examinations were performed on 20 patients with acquired immune deficiency syndrome (AIDS) and 39 with AIDS-related complex (ARC). Fever of unknown origin and thrombocytopenia were common in ARC, but anemia and leukopenia were most frequent in AIDS. Changes in stromal cells and perivascular cuffing of plasma cells were found significantly more often in patients with AIDS than in those with ARC. Malignancies were common in both groups. Human immunodeficiency virus (HIV) nucleic acids were detected with the use of a 3H-labeled cDNA probe with an in situ hybridization method in 11 bone marrow samples (three ARC and eight AIDS). Most commonly positive cells were mononucleated, resembling lymphocytes and histiocytes. Endothelial cells, interdigitating reticulum cells, nucleated red blood cells, and immature myeloid cells also had positive results in some instances. The number of HIV-positive cells was not related to the size of the bone biopsies or the clinical diagnoses. The authors postulate that changes in the peripheral blood and bone marrow of these patients may be related to latent persistent infection with HIV.


Subject(s)
AIDS-Related Complex/pathology , Acquired Immunodeficiency Syndrome/pathology , Bone Marrow/pathology , AIDS-Related Complex/microbiology , Acquired Immunodeficiency Syndrome/microbiology , Adipose Tissue/pathology , Adult , Bone Marrow/microbiology , DNA Probes , Erythropoiesis , Female , HIV/genetics , HIV/isolation & purification , Hematopoiesis , Hematopoietic Stem Cells/pathology , Histiocytes/pathology , Humans , Male , Middle Aged , Nucleic Acid Hybridization , Nucleic Acids/analysis , Phagocytosis , Plasma Cells/pathology , Retrospective Studies
10.
J Lab Clin Med ; 114(1): 43-50, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2544652

ABSTRACT

Adenosine triphosphate (ATP) and adenosine diphosphate levels are decreased in erythrocytes from individuals with beta-thalassemia minor. Because 5-phosphoribosyl-1-pyrophosphate (PRPP) is an essential precurosr of adenine nucleotides, we tested the hypothesis that impaired PRPP synthesis is a mechanism for the decreased adenine nucleotide content. Erythrocyte PRPP synthetase activity was significantly decreased, and the Michaelis-Menten constant (Km) for ribose-5-phosphate (R5P) was significantly increased in individuals with alpha-thalassemia minor and those with beta-thalassemia minor. Intact erythrocytes from individuals with alpha-thalassemia and those with beta-thalassemia minor also had an impaired rate of PRPP formation. Both the decrease in PRPP synthetase activity and the impaired PRPP formation were also found in erythrocytes with microcytosis resulting from iron deficiency, indicating that these phenomena may not be specific to thalassemia minor. In all individuals examined, the rate of PRPP formation correlated with ATP content, suggesting that either (1) PRPP synthetase activity is a determinant of ATP content or (2) ATP content is a determinant of PRPP synthetase activity. The depletion of ATP from normal erythrocytes did not affect PRPP synthetase activity, suggesting that ATP content is not a determinant of PRPP synthetase activity. However, a decrease in PRPP synthetase activity did cause an impairment in the rate of adenine nucleotide synthesis, suggesting that PRPP synthetase activity is a determinant of ATP content. Taken together, our results suggest that the decrease in PRPP synthetase activity and the resulting impairment in the rate of PRPP formation are mechanisms for the decreased adenine nucleotide content in thalassemic erythrocytes.


Subject(s)
Adenine Nucleotides/blood , Erythrocytes/enzymology , Phosphotransferases/blood , Ribose-Phosphate Pyrophosphokinase/blood , Thalassemia/blood , Adenosine Diphosphate/blood , Adenosine Triphosphate/blood , Anemia/blood , Hemoglobins/analysis , Humans , In Vitro Techniques , Iron Deficiencies , Kinetics , Reference Values , Reticulocytes/metabolism , Ribose-Phosphate Pyrophosphokinase/isolation & purification , Thalassemia/enzymology
11.
Br J Haematol ; 72(2): 265-71, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2757969

ABSTRACT

The percentage of nicotinamide adenine dinucleotide (NAD) in the oxidized form [NAD+/(NAD+ and NADH); i.e. the NAD+/NADT ratio] is increased in the red cell (RBC) in sickle cell disease. We tested the hypothesis that the increased NAD+/NADT ratio was a determinant of the increased 2,3-diphosphoglycerate (DPG) content of the SCD RBC. Using normal subjects and individuals with sickle cell disease or autoimmune haemolytic anaemia (AIHA), we observed an inverse relationship between the packed cell volume (PCV) and the RBC DPG concentration (r = -0.69) and a direct relationship between the RBC NAD+/NADT ratio and the DPG concentration (r = 0.74). When the effect of the PCV on DPG was removed using analysis of covariance [DPGady(PCV)], the NAD+/NADT ratio had a significant relationship with the DPGadj(PCV) (r = 0.50, P less than 0.001). In in vitro incubation studies, increasing the NAD+/NADT ratio significantly increased the DPG content of both normal and AIHA RBC. Conversely, decreasing the NAD+/NADT ratio decreased the DPG content of normal, AIHA and SCD RBC. Thus, the increased DPG content in the SCD RBC appears to be due, in part, to the increased NAD+/NADT ratio and is not purely a physiologic response to decreased oxygen carrying capacity.


Subject(s)
Anemia, Sickle Cell/blood , Diphosphoglyceric Acids/blood , Erythrocytes, Abnormal/metabolism , NAD/blood , 2,3-Diphosphoglycerate , Anemia, Hemolytic, Autoimmune/blood , Erythrocytes/metabolism , Erythrocytes, Abnormal/drug effects , Hematocrit , Humans , Oxidation-Reduction
12.
Biochim Biophys Acta ; 994(1): 81-8, 1989 Jan 19.
Article in English | MEDLINE | ID: mdl-2535789

ABSTRACT

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.


Subject(s)
Erythrocytes/enzymology , Magnesium/blood , Nucleotidases/deficiency , Phosphotransferases/blood , Pyrimidine Nucleotides/pharmacology , Ribose-Phosphate Pyrophosphokinase/blood , 5'-Nucleotidase , Adenosine Monophosphate/blood , Adenosine Triphosphate/pharmacology , Anemia, Hemolytic, Autoimmune/enzymology , Cytidine Diphosphate/pharmacology , Cytidine Monophosphate/pharmacology , Cytidine Triphosphate/pharmacology , Humans , Kinetics , Macromolecular Substances , Magnesium/pharmacology , Magnesium Chloride , Ribose-Phosphate Pyrophosphokinase/antagonists & inhibitors , Ribosemonophosphates/blood
13.
Blood ; 72(2): 500-6, 1988 Aug.
Article in English | MEDLINE | ID: mdl-2456795

ABSTRACT

RBCs from patients with hemolytic anemia due to pyruvate kinase (PK) deficiency are characterized by a decreased total adenine and pyridine nucleotide content. Because phosphoribosylpyrophosphate (PRPP) is a precursor of both adenine and pyridine nucleotides, we investigated the ability of intact PK-deficient RBCs to accumulate PRPP. The rate of PRPP formation in normal RBCs (n = 11) was 2.89 +/- 0.80 nmol/min.mL RBCs. In contrast, the rate of PRPP formation in PK-deficient RBCs (n = 4) was markedly impaired at 1.03 +/- 0.39 nmol/min.mL RBCs. Impaired PRPP formation in these cells was not due to the higher proportion of reticulocytes. To study the mechanism of impaired PRPP formation, PK deficiency was simulated by incubating normal RBCs with fluoride. In normal RBCs, fluoride inhibited PRPP formation, caused adenosine triphosphate (ATP) depletion, prevented 2,3-diphosphoglycerate (DPG) depletion, and inhibited pentose phosphate shunt (PPS) activity. These results together with other data suggest that impaired PRPP formation is mediated by changes in ATP and DPG concentration, which lead to decreased PPS and perhaps decreased hexokinase and PRPP synthetase activities. Impaired PRPP formation may be a mechanism for the decreased adenine and pyridine nucleotide content in PK-deficient RBCs.


Subject(s)
Anemia, Hemolytic/blood , Erythrocytes/metabolism , Pentosephosphates/biosynthesis , Phosphoribosyl Pyrophosphate/biosynthesis , Pyruvate Kinase/deficiency , 2,3-Diphosphoglycerate , Adenosine Triphosphate/metabolism , Carbon Dioxide/metabolism , Diphosphoglyceric Acids/metabolism , Fluorides/pharmacology , Humans , Pentose Phosphate Pathway
14.
Blood ; 71(2): 512-5, 1988 Feb.
Article in English | MEDLINE | ID: mdl-3337912

ABSTRACT

RBCs from individuals with sickle cell disease are more susceptible to oxidant damage. Because key antioxidant defense reactions are linked to the pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), we tested the hypothesis that the RBC redox potential as manifested by the NADH/[NAD+ + NADH] and NADPH/[NADP+ + NADPH] ratios is decreased in sickle erythrocytes. Our data demonstrate that sickle RBCs have a significant decrease in the NADH/[NAD+ + NADH] ratio compared with normal RBCs (P less than .00005). Interestingly, sickle RBCs also had a significant increase in total NAD content compared with normal RBCs (P less than .00005). In contrast, although sickle RBCs had a significant increase in the total NADP content compared with normal RBCs (P less than .00005), sickle RBCs had no significant alteration in the NADPH/[NADP+ + NADPH] ratio. High reticulocyte controls demonstrated that these changes were not related to cell age. Thus, sickle RBCs have a decrease in NAD redox potential that may be a reflection of their increased oxidant sensitivity. The changes in these pyridine nucleotides may have further metabolic consequences for the sickle erythrocyte.


Subject(s)
Anemia, Sickle Cell/blood , Erythrocytes/metabolism , NADP/blood , NAD/blood , Anemia, Sickle Cell/metabolism , Humans , Oxidation-Reduction
15.
Med Pediatr Oncol ; 16(6): 375-7, 1988.
Article in English | MEDLINE | ID: mdl-3264379

ABSTRACT

Seventy-eight individuals previously treated with chemotherapy for non-Hodgkin's lymphoma were enrolled in a phase II pilot study employing methotrexate 100 mg/M2 iv (day 1), calcium leucovorin 10 mg/M2 iv and/or po q6h (days 2-4), VM-26 (teniposide) 100 mg/M2 iv (days 2 and 9), procarbazine 100 mg/M2 po (days 2-15), and dexamethasone 15 mg/M2po (days 2-8) (MV26PD). Thirty percent of the 78 patients treated had a response to therapy (8% complete, 22% partial). Twenty-four percent of patients with diffuse histiocytic (large cell) lymphoma had a response (12% complete, 12% partial). The estimated failure-free survival was 41% at 3 months and the median survival (death from any cause) was 4.5 months for the entire cohort. Two individuals, including one individual with diffuse histiocytic lymphoma, remain in a complete response for over 900 days. Significant hematologic toxicity and infectious complications were seen in this heavily pretreated group of patients. MV26PD represents an active combination of agents for the treatment of non-Hodgkin's lymphoma. The optimal dosing for MV26PD remains to be determined.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Lymphoma, Non-Hodgkin/drug therapy , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Cyclophosphamide/administration & dosage , Cytarabine/administration & dosage , Dexamethasone/administration & dosage , Drug Evaluation , Female , Humans , Leucovorin/administration & dosage , Leukopenia/chemically induced , Male , Methotrexate/administration & dosage , Middle Aged , Neutropenia/chemically induced , Procarbazine/administration & dosage , Teniposide/administration & dosage , Thrombocytopenia/chemically induced , Vincristine/administration & dosage
16.
Biochim Biophys Acta ; 964(1): 96-104, 1988 Jan 12.
Article in English | MEDLINE | ID: mdl-3334878

ABSTRACT

The erythrocyte can phosphorylate a variety of hexoses. Since it can consume mannose and glucose equivalently in the hereditary deficiencies of hexokinase and phosphoglucose isomerase and since erythrocyte defense against oxidants is impaired in a variety of hereditary hemolytic anemias, we tested the hypothesis that mannose may be a significant alternative to glucose as a fuel for this defense system. Unexpectedly, mannose inhibited defense against oxidants as manifested by increased Heinz body formation when both normal and high-reticulocyte erythrocytes were incubated with acetylphenylhydrazine (APH). Using APH as the oxidant, mannose-incubated erythrocytes had decreased reduced glutathione stability and impaired hexose oxidation by the pentose shunt compared to glucose-incubated erythrocytes. After incubation with mannose and APH, normal erythrocytes showed a decrease in ATP content. Approximately 25% of the consumed mannose accumulated in the erythrocytes as mannose 6-phosphate. Erythrocytes incubated with mannose and APH displayed a significant loss of redox potential as manifested by decreased NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios. Since phosphomannose isomerase is the rate-limiting step for mannose metabolism, our results suggest that mannose impairs erythrocyte defense against oxidants by causing ATP depletion and by impairing the regeneration of reduced pyridine nucleotides by the Embden-Meyerhof and pentose phosphate pathways.


Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic , Anemia, Hemolytic/blood , Autoimmune Diseases/blood , Erythrocytes/metabolism , Glycolysis , Hexoses/blood , Mannose/pharmacology , Erythrocytes/drug effects , Humans , Kinetics , Oxidation-Reduction , Phosphorylation , Reference Values
17.
Am J Hematol ; 26(3): 211-9, 1987 Nov.
Article in English | MEDLINE | ID: mdl-3674003

ABSTRACT

Red blood cell (RBC) antioxidant defense was investigated in eight individuals with hemoglobin E (Six EE and two E-B(+) thalassemia) and compared to that in six individuals with thalassemia and ten normal subjects. Individuals with hemoglobin E had increased incubated Heinz body formation (68% +/- 18%; p less than 0.001) compared to normal and thalassemic RBC (10% +/- 2% and 11% +/- 5%, respectively). Stimulated pentose phosphate shunt activity was increased in the thalassemic and decreased in the hemoglobin E RBC as compared to normal. The 2,3-diphosphoglycerate (DPG) content of the EE RBC was increased to 5.59 +/- 0.69 mumol/ml RBC as compared to normal (4.51 +/- 0.77; p less than 0.001). In the EE RBC, there was a direct correlation between Heinz body formation and DPG content (r = 0.73). Ascorbic and dehydroascorbic acid (0.1 and 1.0 mM) were able to decrease the degree of Heinz body formation in the hemoglobin E RBC. Ascorbic acid (0.1 mM) prolonged the response of the pentose shunt. Thus impaired antioxidant defense may account for the persistence of the hemoglobin E gene in areas where malaria is endemic. Oxidant medications should be used with caution in individuals of Southeast Asian origin.


Subject(s)
Antioxidants/chemistry , Erythrocytes/metabolism , Hemoglobin E/metabolism , Hemoglobins, Abnormal/metabolism , Malaria/blood , Thalassemia/blood , 2,3-Diphosphoglycerate , Ascorbic Acid/pharmacology , Diphosphoglyceric Acids/blood , Erythrocytes/drug effects , Erythrocytes/ultrastructure , Glutathione/blood , Heinz Bodies/drug effects , Heinz Bodies/ultrastructure , Hemoglobin A/metabolism , Humans , Malaria/prevention & control , Pentose Phosphate Pathway/drug effects
18.
Am J Med Sci ; 294(5): 364-8, 1987 Nov.
Article in English | MEDLINE | ID: mdl-3425586

ABSTRACT

A Cambodian woman with hemoglobin E trait (AE) and leprosy developed a Heinz body hemolytic anemia while taking a dose of dapsone (50 mg/day) not usually associated with clinical hemolysis. Her red blood cells (RBCs) had increased incubated Heinz body formation, decreased reduced glutathione (GSH), and decreased GSH stability. The pentose phosphate shunt activity of the dapsone-exposed AE RBCs was increased compared to normal RBCs. Although the AE RBCs from an individual not taking dapsone had increased incubated Heinz body formation, the GSH content and GSH stability were normal. The pentose phosphate shunt activity of the non-dapsone-exposed AE RBCs was decreased compared to normal RBCs. Thus, AE RBCs appear to have an increased sensitivity to oxidant stress both in vitro and in vivo, since dapsone does not cause hemolytic anemia at this dose in hematologically normal individuals. Given the influx of Southeast Asians into the United States, oxidant medications should be used with caution, especially if an infection is present, in individuals of ethnic backgrounds that have an increased prevalence of hemoglobin E.


Subject(s)
Anemia, Hemolytic/chemically induced , Dapsone/adverse effects , Heinz Bodies/ultrastructure , Hemoglobin E/genetics , Hemoglobins, Abnormal/genetics , 2,3-Diphosphoglycerate , Adult , Anemia, Hemolytic/blood , Cambodia , Diphosphoglyceric Acids/blood , Female , Glutathione/blood , Humans
19.
Am J Med Sci ; 294(4): 253-7, 1987 Oct.
Article in English | MEDLINE | ID: mdl-3661621

ABSTRACT

Polymorphonuclear leukocyte (PMN) aggregation and chemotaxis were studied in 27 patients with sickle cell disease (SCD). Pain-free patients with SCD had a significantly impaired aggregation response to stimulation with n-formylmethionyl-leucyl-phenylalanine (FMLP) with or without cytochalasin B (CB), compared with normal volunteers (p less than 0.001). Patients with SCD in vaso-occlusive crisis had PMN aggregation induced by FMLP with or without CB that was significantly increased compared with the cohort of pain-free SCD patients (p less than 0.001). PMN from pain-free patients had normal chemotaxis, whereas patients in vaso-occlusive crisis had a significant impairment in PMN chemotaxis. PMN chemotaxis was inversely related to the PMN aggregation response to FMLP with CB (r = -0.75). Thus, the PMN from pain-free patients with SCD appears to have normal or decreased "stickiness" and to develop increased stickiness during vaso-occlusive crisis. The mechanisms responsible for these changes need further elucidation. Alterations in PMN function may be responsible, in part, for the increased risk of infection noted in individuals with SCD and may play a role in the development of the acute chest syndrome.


Subject(s)
Anemia, Sickle Cell/blood , Chemotaxis, Leukocyte , Adolescent , Adult , Anemia, Sickle Cell/physiopathology , Cell Aggregation/drug effects , Chemotaxis, Leukocyte/drug effects , Child , Child, Preschool , Humans , Neutrophils/physiology , Pain , Vascular Diseases/blood , Vascular Diseases/physiopathology
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