Role of peroxiredoxin-2 in protecting RBCs from hydrogen peroxide-induced oxidative stress.

The role of peroxiredoxin-2 (PRDX2) in preventing hydrogen peroxide-induced oxidative stress in the red blood cell was investigated by comparing blood from PRDX2 knockout mice with superoxide dismutase-1 (SOD1) knockout and control mice. Loss of PRDX2 increased basal levels of methemoglobin and heme degradation (a marker for oxidative stress), and reduced red blood cell deformability. In vitro incubation under normoxic conditions, both with and without inhibition of catalase, resulted in a lag phase during which negligible heme degradation occurred followed by a more rapid rate of heme degradation in the absence of PRDX2. The appreciable basal increase in heme degradation for PRDX2 knockout mice, together with the lag during in vitro incubation, implies that PRDX2 neutralizes hydrogen peroxide generated in vivo under the transient hypoxic conditions experienced as the cells pass through the microcirculation.

Long-term follow-up of allogeneic bone marrow transplantation for patients with chronic phase chronic myeloid leukemia prepared with a regimen consisting of cyclophosphamide, cytarabine and single-dose total body irradiation conditioning.

We evaluated long-term toxicities and outcomes in 96 patients with chronic phase chronic myeloid leukemia treated with a single bone marrow allograft regimen. Conditioning was cytosine arabinoside, cyclophosphamide (120 mg/kg) and single fraction total body irradiation (500 cGy). Median follow-up was 12.8 years (0.4-19.9 years). Graft failure occurred in one patient, nonfatal veno-occlusive disease in 13 patients (14%). Overall incidences of acute (a) and chronic (c) graft-vs-host disease (GVHD) were 77 and 63%. The 100-day and 1-year transplant-related mortality (TRM) were 1 and 9.2%, respectively, with no change through 5 years. Five- and 10-year event-free survival rates were 56 and 49%, overall survival (OS) rates 72 and 70%, respectively. Forty patients have relapsed: 8 cytogenetic (20%), 10 hematologic (25%) and 22 molecular (55%). Most have been salvaged with donor-leukocyte infusion, second transplants and/or imatinib therapy. Survival was worse for patients transplanted >2 years from diagnosis (10-year OS 56 vs 78%, P=0.01), for patients over 50 years old (10-year OS 44 vs 75%, P=0.05) and for patients without cGVHD (10-year OS 53 vs 86%, P<0.001). This regimen resulted in successful engraftment, low risk of TRM and long-term survival. In an era when imatinib is first line therapy, this regimen offers a potentially low-toxicity, highly successful alternative in the event of poor imatinib response.

Long-term follow-up of secondary malignancies in adults after allogeneic bone marrow transplantation.

The purpose of this study was to evaluate the estimated incidence of secondary malignancies post-allogeneic bone marrow transplantation (BMT) in a cohort of adult patients previously reported now with an additional 8.5 years of follow-up. A cohort of 557 patients older than age 16 years underwent allogeneic BMT between June 1970 and November 1993. Histologic reports confirmed the diagnosis of a secondary malignancy. Multivariate Cox proportional hazards method was utilized to investigate predictors for the development of secondary malignancies. In all, 31 patients in this cohort developed a secondary malignancy a median of 6.79 years after their transplant. The estimated cumulative incidence rate of secondary malignancy was 4.2% at 10 years post transplant. When compared to the general population, the estimated observed/expected ratio of new cancer diagnoses was 5.13. On multivariate analysis, older age at the time of transplant was the only significant predictor for development of secondary cancer (P=0.01). The most common malignancies observed were nonmelanomatous skin cancers and squamous cell cancers of the buccal cavity. The risk of developing a secondary malignancy after allogeneic BMT is significant, particularly in older patients. Long-term survivors of transplant require regular monitoring for early signs of cancer, particularly of the skin and oral cavity.

Subdural hematomas during CML therapy with imatinib mesylate.

Seven of one hundred twenty-one patients with chronic myeloid leukemia (CML) treated with imatinib mesylate developed subdural hematomas. All had advanced disease and were treated initially at a dose of 600 mg per day. Three patients had thrombocytopenia (platelet < 10 x 10(9)/l), one had leukocytosis (white blood cell count > 150 x 10(9)/l) and three had neither around the time of diagnosis of the subdural hematomas. Four patients required surgical evacuation. One patient, in blast crisis, died as a consequence of the subdural hematoma. Three patients survived but died of progressive CML. The remaining three patients having recommenced imatinib, are alive and well, and one has achieved a major cytogenetic response. Subdural hematomas must be considered even in mildly symptomatic patients receiving imatinib regardless of their peripheral blood counts. Patients who survive can be cautiously restarted on imatinib. Further studies are required to study the potential relationship between imatinib mesylate and subdural hematomas.

Rescue of interferon induced bone marrow aplasia in a patient with chronic myeloid leukemia by allogeneic bone marrow transplant.

A patient with chronic phase Philadelphia chromosome positive CML, developed severe protracted bone marrow hypoplasia after interferon therapy. This complication did not respond to two courses of immunosuppressive therapy with anti-thymocyte globulin, cyclosporin A and prednisone. The patient continued to be transfusion dependent with persistence of Philadelphia chromosome. Allogeneic BMT restored normal hematopoeisis.

Amyloid insulin interaction with erythrocytes.

Erythrocyte membrane interactions with insulin fibrils (amyloid) have been investigated using centrifugation, fluorescence spectroscopy, light scattering, and flow cytometric techniques. The results indicate that insulin fibrils are having moderate affinity to erythrocyte membrane. However, analysis of the apparent dissociation constants of human erythrocyte membranes (leaky and resealed vesicles) with amyloid insulin reveal that the insulin binding is drastically reduced on attaining the fibrillar state compared with native insulin. To understand the role of insulin receptors on erythrocytes binding to amyloid, we have studied the interaction of biotinylated forms of denatured and amyloidic insulin with erythrocytes. FITC-streptavidin was used as a counter staining in flow cytometry measurements. We found that insulin fibrils bind 10 times more with erythrocyte membranes than with amylin and denatured insulin.

The reduction of nitroblue tetrazolium by red blood cells: a measure of Red Cell membrane antioxidant capacity and hemoglobin-membrane binding sites.

The reduction of nitroblue tetrazolium (NBT) with intact Red Blood Cells (RBCs) is biphasic with an initial rapid reduction followed by a slower second phase. This biphasic kinetics has been explained with the initial rapid phase attributed to antioxidants in the red cell which reduce membrane bound NBT and the slower phase associated with the reaction of NBT with membrane bound hemoglobin. This model has been confirmed by a utilization of a number of red cell modifications which either increase the red cell antioxidants (vitamin C and vitamin E) or damage the red cell membrane (cumene hydroperoxide and N-ethylmaleimide). The utilization of this assay for human blood samples was investigated by studying a series of 20 human subjects ranging between 34 and 87 years of age. It was possible to fit all of these samples with two adjustable parameters which reflect the red cell membrane antioxidant capacity (x) and the hemoglobin membrane interactions (m). The antioxidant capacity shows a significant (p < .002; R = -.67) decrease with age. This finding is consistent with a decrease in the level of antioxidants in aged subjects. In addition, the number of hemoglobin membrane sites are negatively correlated with the antioxidant capacity (p < .02; R = -.52) suggesting that the oxidative stress associated with reduced antioxidants results in increased hemoglobin-membrane interactions.

Direct measurements of hemoglobin interactions with liposomes using EPR spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy was used to compare the rates of autoxidation at 37 degrees C of acellular and liposome-encapsulated hemoglobin (LEH) crosslinked between alpha chains with bis (3,5-dibromosalicyl) fumarate (alphaalphaHb). This method avoids the difficulties inherent in using conventional ultraviolet-visible (UV-vis) spectroscopy caused by the high turbidity of liposome suspensions. Rate constants of 0.039/h and 0.065/h were obtained for the alphaalphaHb and LEH samples, respectively. Similar oxidation measurements with alphaalphaHb using UV-vis spectroscopy gave a rate constant comparable to that obtained with EPR spectroscopy. Indirect measurement of the oxidation kinetics of LEH utilizing extraction of alphaalphaHb with chloroform from partially oxidized LEH samples was unreliable because the amount of extractable hemoglobin was inversely proportional to the degree of oxidation. EPR measurements showed a shift in the g value and substantial enhancement in the intensity of the bis-histidine low-spin B complex for the encapsulated hemoglobin, indicating a perturbation of this low-spin complex. We suggest that lipid-associated perturbations are responsible for the enhancement of the oxidation observed with the LEH samples compared to the unencapsulated material.

The origin of red cell fluorescence caused by hydrogen peroxide treatment.

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.

Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation.

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.

Heme degradation during autoxidation of oxyhemoglobin.

Two fluorescent heme degradation compounds are detected during autoxidation of oxyhemoglobin. These fluorescent compounds are similar to fluorescent compounds formed when hydrogen peroxide reacts with hemoglobin [E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun. 247, 592-596 (1998)]. Low levels of heme degradation in the presence of superoxide and catalase are attributed to a reaction involving the superoxide produced during autoxidation. The inhibition of most of the degradation by catalase suggests that the hydrogen peroxide generated during autoxidation of oxyhemoglobin produces heme degradation by the same mechanism as the direct addition of hydrogen peroxide to hemoglobin. The formation of the fluorescent degradation products was inhibited by the peroxidase substrate, ABTS, which reduces ferrylhemoglobin to methemoglobin, indicating that ferrylhemoglobin is produced during the autoxidation of hemoglobin. It is the transient formation of this highly reactive Fe(IV) hemoglobin, which is responsible for most of the heme degradation during autoxidation.

Hemodynamic changes during aging associated with cerebral blood flow and impaired cognitive function.

This study investigates the age associated changes in hemorheological properties and cerebral blood flow. Partial correlations indicate that part of the age-dependent decrease in flow velocities can be attributed to a hemorheological decrement resulting in part from enhanced oxidative stress in the aged. A possible link with Alzheimer's pathology is suggested by the augmented hemorheological impairment resulting from in vitro incubation of red cells with amyloids. These results suggest that in aging, oxidative stress as well as amyloids may influence the fluid properties of blood, resulting in a potential decrement in blood flow and oxygen delivery to the brain. Animal intervention studies further demonstrate that altered hemorheological properties of blood can actually influence cognitive function. The relationships shown to exist between hemorheology, blood flow, amyloids, oxidative stress, and cognitive function suggest that these factors may be one of the mechanisms operating in the complex etiology of Alzheimer's disease.

Brain multiparametric responses to carbon monoxide exposure in the aging rat.

The multiparametric monitoring system was applied to study the effects of 2000 ppm carbon monoxide (CO) on brain functions in vivo in the aging rat. The vasodilatory (non hypoxic) effects of CO on CBF in normal adult rats, which were shown in concentrations of 1000-2000 ppm involved the effect of nitric oxide (NO). Energy metabolism was evaluated by optical monitoring of CBF and mitochondrial function by fluorometry of NADH. Ionic homeostasis was evaluated by monitoring the extracellular level of K(+) and H(+) and the DC steady potential. Seven aging rats (24 months) were exposed to 2000 ppm for 60 min and 120 min of recovery, while five control rats were exposed to air under the same conditions. A comparison between the CO group and the control group showed that the changes in CBF, NADH and light reflectance were not statistically significant while extracellular K(+) was elevated and tissue pH became more acidic. Thus, the typical CO induced increase in CBF, was not recorded in the aging rats. We concluded that the brain vasodilatory response to CO was not active in the aging rat, while the ionic homeostasis responses were similar to those found in the adult rat.

Fourier transform Raman approach to structural correlation in hemoglobin derivatives.

In order to obtain information on the structural aspects of hemoglobin (Hb), Fourier transform Raman (FT-R) measurements on various ferrous, ferric derivatives and nickel reconstituted Hb (NiHb) has been made. FT-R spectra for these derivatives were obtained by laser excitation in the near infrared region (NIR) (1064 nm) whereby the wave-number region (600-1700 cm-1) related to both porphyrin ring modes and some globin modes were monitored. Comparison of various modes was made based on previous resonance Raman (RR) results. The wave-number shifts with respect to changes in oxidation state and spin state are very similar to those observed by RR. Additional bands at 1654, 1459, and 1003 cm-1 for deoxyHb and at 1656, 1454, and 1004 cm-1 for oxy Hb can be correlated to globin modes. The shift in the position of these bands for the binding of oxygen can be related to changes in conformation during the transformation. The presence of two distinct sites in NiHb could be monitored by the use of FT-R technique.

Curvature of dinucleotide poised for formation of trinucleotide in transcription with Escherichia coli RNA polymerase.

A frequently used schematic model of transcriptional elongation shows an RNA polymerase molecule moving along a linear DNA. This model is of course highly idealized and not compatible with promoter sequences [Gralla, J. D. (1991) Cell 66, 415-418; Schleif, R. (1992) Annu. Rev. Biochem. 61, 199-223] and regulatory proteins [Koleske, A. J., and Young, R. A. (1995) Trends Biochem. Sci. 20, 113-116; Dunaway, M., and Dröge, P. (1989) Nature 341, 657-659; Müller, H. P., Sogo, J. M., and Schaffner, W. (1989) Cell 58, 767-777] located some distance away from the point of transcription initiation [Karsten, R., von Hippel, P. H., and Langowski, J. (1995) Trends Biochem. Sci. 20, 500-506]. These circumstances lead to the expectation of curvature along the DNA strand and require looping between sometimes distant points. We have now shown curvature in a dinucleotide formed at the very onset of transcription when it is poised for reaction with a mononucleotide to form a trinucleotide. The curvature became evident from the demonstration that a metal ion bound with a mononucleotide in the i+1 (elongation) site is approximately equidistant from bases at the 5' end (i-1 site) and 3' end (i site) of the dinucleotide. Similar results were obtained with three different dinucleotides and four mononucleotides. Curvature of the RNA initiate may reflect curvature of the DNA to which it is bound. These studies show curvature to be a significant feature in the interaction between DNA template and RNA elongate even at the very beginning of transcription.

Cold acclimation-induced increase of systolic blood pressure in rats is associated with volume expansion.

To investigate the mechanisms of cold-induced hypertension, the systolic blood pressure (SBP) and average daily water consumption were measured weekly in 6-month-old male Wistar rats; they were subsequently acclimated to thermoneutrality (26 degrees C for 7 weeks), to cold temperature (6 degrees C for 9 weeks), and then again reacclimated to 26 degrees C for 5 weeks. Circulating plasma volume and whole blood viscosity were measured in subgroups of rats at the end of acclimation to 26 degrees C after 2 days, after 1, 6, and 8 weeks of cold, and after 2 and 5 weeks of rewarming. The control values obtained at the end of thermoneutral period were: SBP = 130.8 +/- 18.6 mm Hg, plasma volume = 41.9 +/- 4.64 mL/kg, whole body viscosity at shear rate of 22.5 per sec = 6.7 +/- 0.48 cps, and daily water consumption = 42.25 +/- 16.81 mL. After 48 h of cold exposure there was almost a 50% increase in plasma volume that persisted to a lesser degree throughout the whole period of cold exposure (P < .05). After 2 weeks of cold exposure the daily water consumption increased and SBP began to increase. After 6 weeks of cold exposure the SBP was 30 mm Hg above that of the control level (P < .001) and was accompanied by a 25% increase in whole blood viscosity (P < .05). At the end of 8 weeks of cold exposure the plasma volume was 56.8 +/- 9.51 mL/ kg and the whole blood viscosity was 8.0 +/- 1.79 cps at the 22.5 per sec shear rate. During the 5 weeks of rewarming the elevation of SBP and increased whole blood viscosity persisted, whereas the increased daily water consumption and expanded plasma volume returned to normal. Therefore, the acclimation to cold is accompanied by the development of a volume-associated hypertension, which is sustained after rewarming without volume expansion.

Molecular dynamics of human methemoglobin: the transmission of conformational information between subunits in an alpha beta dimer.

Spectroscopic studies indicate an interaction of the distal histidine with the heme iron as well as the transmission of distal heme perturbations across the alpha1beta1 interface. Molecular dynamics simulations have been used to explain the molecular basis for these processes. Using a human methemoglobin alpha beta dimer, it has been shown that at 235 K after 61 ps, a rearrangement occurs in the alpha-chain corresponding to the formation of a bond with the distal histidine. This transition does not take place in the beta-chain during a 100-ps simulation and is reversed at 300 K. The absence of the distal histidine transition in the isolated chains and with the interface frozen indicate the involvement of the alphabeta interface. A detailed analysis of the simulation has been performed in terms of RMS fluctuations, domain cross-correlation maps, the disruption of helix hydrogen bonds, as well changes in electrostatic interactions and dihedral angles. This analysis shows that the rearrangements in the alpha-chain necessary to bring the histidine closer to the iron involve alterations primarily in the CD loop and at the interface. Communication to the beta-chain distal pocket is propagated by increased interactions of the alpha-chain B helix with the beta-chain G-GH-H segment and the flexibility in the EF loop. The G helices shown to be involved in propagation of perturbation across the alpha1beta1 interface extend into the alpha1beta2 interfaces, providing a mechansim whereby distal interactions can modulate the T<==>R transition in hemoglobin.

Breath ethane as a marker of reactive oxygen species during manipulation of diet and oxygen tension in rats.

Breath ethane, O2 consumption, and CO2 production were analyzed in 24-mo-old female Fischer 344 rats that had been fed continuously ad libitum (AL) or restricted 30% of AL level (DR) diets since 6 wk of age. Rats were placed in a glass chamber that was first flushed with air, then with a gas mixture containing 12% O2. After equilibration, a sample of the outflow was collected in gas sampling bags for subsequent analyses of ethane and CO2. The O2 and CO2 levels were also directly monitored in the outflow of the chamber. O2 consumption and CO2 production increased for DR rats. Hypoxia decreased O2 consumption and CO2 production for the AL-fed and DR rats. These changes reflect changes in metabolic rate due to diet and PO2. A significant decrease in ethane generation was found in DR rats compared with AL-fed rats. Under normoxic conditions, breath ethane decreased from 2.20 to 1.61 pmol ethane/ml CO2. During hypoxia the levels of ethane generation increased, resulting in a DR-associated decrease in ethane from 2.60 to 1.90 pmol ethane/ml CO2. These results support the hypothesis that DR reduces the level of oxidative stress.

Maze learning impairment is associated with stress hemopoiesis induced by chronic treatment of aged rats with human recombinant erythropoietin.

Mean cell volume (MCV) of erythrocytes has been reported to increase with age in humans, and to be negatively correlated with memory performance in humans and rats. We evaluated hematological changes in 21-mo old male Fischer 344 rats undergoing a 3-mo twice weekly subcutaneous injection of human recombinant erythropoietin (EPO). A baseline hematocrit (HCT) was obtained initially and repeated at monthly intervals to determine the effectiveness of EPO treatment. At 24-mo of age and after 3 mo EPO treatment, the rats were tested for their ability to learn a 14-unit T maze. Following maze testing, blood was drawn for hematologic analyses, including HCT, MCV, maximum swollen cell volume (MCVS), mean cell transit time (MCTT), and the membrane shear modulus of elasticity (G), the latter a derived measure of the relative elasticity of the red cell membrane. After 1 mo EPO treatment, HCT significantly increased compared to saline-injected controls. After 2 mo treatment, HCT began to decline but remained elevated above baseline levels even after 3 mo treatment. After 3 mo EPO treatment, MCV was significantly lower in EPO-treated rats compared to controls. These changes imply altered hemopoiesis to produce cells which undergo shrinkage associated with accelerated cellular aging. The lower MCV would have predicted a shorter MCTT which instead was unchanged. This observation suggested the presence of an additional factor contributing to the MCTT. The G, which measures the membrane contribution to deformability, very significantly increased with EPO treatment. This finding indicates an increased contribution of membrane properties to the MCTT after EPO treatment, which cancels the expected decrease in MCTT for smaller cells. After 3 mo of EPO treatment, aged rats exhibited significantly impaired maze learning compared to controls. A relationship between, changes in erythrocyte membrane properties and impaired function was indicated by a significant correlation (r=0.67, p <0.04) between G and errors in the 14-unit T-maze. These findings suggest that stress-induced erythropoiesis produces accelerated aging in the red blood cell population that may have functional implications (i.e., impaired learning ability).

Superoxide produced in the heme pocket of the beta-chain of hemoglobin reacts with the beta-93 cysteine to produce a thiyl radical.

The role of the beta-93 cysteine residue in the hemoglobin autoxidation process has been delineated by electron paramagnetic resonance. At low temperatures (8 K) after incubation at 235 K, free radical signals were detected. An analysis of the free radical spectrum produced implies that, besides the superoxide radical expected to be formed during autoxidation, an isotropic free radical is produced with a giso of 2.0133. This g value is consistent with that expected for a sulfur radical. Blocking the beta-93 sulfhydryl group with N-ethylmaleimide was found to eliminate the formation of the isotropic radical, but not the superoxide. This finding confirms the assignment of the isotropic radical as a thiyl radical originating from the oxidation of the cysteine SH group. A kinetic analysis of the time course for the formation of both the superoxide and thiyl radicals is consistent with a reversible electron transfer process between superoxide in the heme pocket of the beta-chains and the cysteine residue. This reaction is expected to produce both a thiyl radical and a peroxide. Direct evidence for peroxide production comes from the detection of a transient Fe(III) heme peroxide complex. The significance of the electron transfer process producing a thiyl radical is discussed. It is shown that the formation of the thiyl radical decreases the rate of autoxidation for the beta-chain and reduces heme degradation attributed to the reaction of superoxide with the heme. The insights gained from these low-temperature studies are believed to be relevant to room-temperature autoxidation.