Search

We compared nitric oxide production and nitrosyl hemoglobin steady state concentrations during the early phases of endotoxemic and hemorrhagic shock of equivalent severity. Sprague-Dawley rats were randomly assigned to (1) sham-operated control, (2) hemorrhage, and (3) intravenous endotoxin. Electron paramagnetic resonance spectroscopy was used to measure NO in the vasculature (binding to hemoglobin) and in the liver (binding to cytochrome P450). Despite similar changes in cardiorespiratory variables and identical microvascular pO(2), nitrosyl hemoglobin concentrations were significantly higher in endotoxemic rats than in rats in hemorrhagic shock, suggesting increased rates of NO production. A substantial venous minus arterial concentration gradient was observed for nitrosyl hemoglobin. This increased in line with the plasma total nitrite + nitrate concentration. Nitrosyl hemoglobin formation is likely to occur predominantly in the venous pool, suggesting that removal of NO from hemoglobin in the presence of oxygen may be faster than previously thought. In the liver, an increase in intracellular heme-NO complexes was detected in endotoxemic rats compared with rats in hemorrhagic shock; this was associated with increased reduction of the mitochondrial respiratory chain and is suggestive of NO inhibition of mitochondrial respiration.

Low oxygen induction of the bacterial (Vitreoscilla) hemoglobin gene (vgb) by the Arc system was investigated, as the presumptive vgb Crp site was found to have 73% identity to the Escherichia coli consensus ArcA site. The role of ArcA by itself and with Fnr was examined in E. coli using the wild type vgb promoter and promoter mutants affecting the Fnr and Crp (presumptive ArcA) sites and E. coli strains with all combinations of fnr+/fnr-, arcA+/arcA- genotypes. High-level transcription required both ArcA and Fnr systems to be functional; low oxygen induction required at least one of ArcA and Fnr to be intact. Levels of Vitreoscilla hemoglobin protein (VHb) followed the same trends as seen with mRNA, although the relative decreases in the mutants relative to wild type were less than with transcription. Growth of cells was stimulated by VHb, generally to a greater extent as VHb levels increased.

Hemoglobin I (HbI) from Lucina pectinata is a monomeric protein composed of 143 amino acids with high sulfide affinity. Its unique heme pocket contains three residues not commonly found in vertebrate globins: Phe 29 (B10), Gln 64 (E7), and Phe 68 (E11), which are thought to be important for high affinity for hydrogen sulfide. Recombinant HbI (rHbI) and several site-directed mutants were cloned and expressed in Escherichia coli yielding high amounts of protein. The highest rHbI protein yield was obtained when the HbI cDNA was cloned into the pET28 (a+) expression vector, transformed into BLi5 cells, the induction performed with 1 mM IPTG at 30 degrees C and TB medium was supplemented with 30 microg/mL hemin chloride and 1% glucose. The highest yield obtained of HbI was 32 mg/L of culture using Fernbach flasks. UV/Visible spectral analysis showed that rHbI binds heme and ESI-MS shows that its molecular weight corresponds to the expected size. Kinetic studies with H2S confirmed that rHbI and HbI have identical binding properties, where the kON for the clam's Hb is 2.73x10(4)M-1s-1 and for rHbI is 2.43x10(4)M-1s-1.

Iron deficiency remains a public health challenge. The neurocognitive deficits that are associated with iron deficiency are most worrisome. Moreover, iron deficiency-associated elevated blood lead levels magnify the problem. Pediatricians must strive to prevent iron deficiency.

Several agents are known to induce differentiation in the human erythroleukemic cell line K562. In this work we have studied the ability of hyperthermia to induce differentiation in the K562 cell line. K562 cells were treated with hyperthermia in the range of 41-45 degrees C. Cell proliferation and the plating efficiency of heat treated cells along with their hemoglobin synthesis was measured and compared with controls. Hyperthermia severely inhibited the growth of K562 cells in the suspension culture in a time- and temperature-dependent manner. Sixty minutes of heating and 44 and 40 min of heating at 45 degrees C totally inhibited the growth of the cells. The number of clonogenic cells also decreased as a result of heat treatment. Extended periods of heating for more than 2 h at 41 degrees C resulted in thermal adaptation. Hyperthermia-induced hemoglobin synthesis by these cells, only at 42 and 43 degrees C. Maximum induction was observed after heat treatment for 80 min at 43 degrees C and 180 min at 42 degrees C. At lower temperature, although the fraction of surviving cells was high, but no signs of hemoglobin synthesis could be observed. At temperatures higher than 43 degrees C, the fraction of surviving cells decreased rapidly and also no signs of hemoglobin synthesis could be detected. At the two selective temperatures, hemoglobin synthesis started 4 days after heat treatment. The results showed that hyperthermia caused cytotoxicity and growth arrest and induced differentiation as judged by hemoglobin synthesis and reduced clonogenicity in this cell line. This is the first time that a physical agent has been shown to induce differentiation in erythroleukemic cell lines.

Developmental-stage-specific regulation and physiological levels of expression of the globin genes can be recaptured in transgenic mice carrying a YAC/BAC- or cosmid-based construct. By contrast, proper developmental regulation and high-level expression cannot be achieved coordinately in transgenic mice carrying a more manipulated construct, such as a plasmid-based globin gene construct. These differences provide us an opportunity to define the requirements for a developmentally regulated, high-level expression of the globin genes in vivo. To achieve this, as a first step, we studied maximum transcriptional potentials of the beta-globin genes at various stages of development. microLCR-enhanced expression of the epsilon-, gamma-, and beta-globin genes driven by their minimal promoters was estimated and compared with that in betaYAC transgenic mice. Quantitative measurements of steady state mRNA levels of the epsilon-, gamma-, and beta-globin genes showed that the microLCR was able to enhance expression of each beta-like globin gene to levels similar to those in the betaYAC mice. Moreover, transcriptional potentials of each globin gene were unchanged during the entire course of development. These observations indicate that the highest level of expression of the globin genes can be achieved in both embryonic and definitive erythropoiesis regardless of developmental specificity of the genes. This finding implies that transcription suppression is the major mechanism of the developmental specificity of the expression of the beta-like globin genes.

In a recent investigation, expression of a double Vitreoscilla haemoglobin (two fused VHb molecules) in Escherichia coli grown in shake flasks resulted in higher final cell density and considerably higher levels of ribosomes and tRNA. In this study, we have investigated the E. coli transcriptome in cells expressing native VHb, double VHb and control cells lacking VHb by hybridising mRNA from the different constructs to high-density oligonucleotide arrays. Within the 95% confidence interval, 4 and 5% of all detected genes in native VHb cells were up- and down-regulated, respectively; in double VHb cells the corresponding numbers were 6 and 10%, respectively. Dividing the data into different functional groups revealed that genes involved in energy metabolism, central intermediary metabolism and cell processes were the most affected at the mRNA level. Particularly, the up-regulation of genes involved in translation and posttranslational modification observed in double VHb cells demonstrates a strong relationship between the regulation of ribosomal genes and the actual number of ribosomes.

Hydroxyurea is a relatively new treatment for sickle cell disease. A portion of hydroxyurea's beneficial effects may be mediated by nitric oxide, which has also drawn considerable interest as a sickle cell disease treatment. Patients taking hydroxyurea show a significant increase in iron nitrosyl hemoglobin and plasma nitrite and nitrate within 2 h of ingestion, providing evidence for the in vivo conversion of hydroxyurea to nitric oxide. Hydroxyurea reacts with hemoglobin to produce iron nitrosyl hemoglobin, nitrite, and nitrate, but these reactions do not occur fast enough to account for the observed increases in these species in patients taking hydroxyurea. This report reviews recent in vitro studies directed at better understanding the in vivo nitric oxide release from hydroxyurea in patients. Specifically, this report covers: (1) peroxidase-mediated formation of nitric oxide from hydroxyurea; (2) nitric oxide production after hydrolysis of hydroxyurea to hydroxylamine; and (3) the nitric oxide-producing structure-activity relationships of hydroxyurea. Results from these studies should provide a better understanding of the nitric oxide donor properties of hydroxyurea and guide the development of new hydroxyurea-derived nitric oxide donors as potential sickle cell disease therapies.

Of the four known globin genes that exist in the fresh-water crustacean Daphnia magna, several are individually induced by hypoxia, lending pale normoxic animals a visible red color when challenged by oxygen deprivation. The promoter regions of the Daphnia globin genes each contain numerous hypoxia response elements (HREs) as potential binding sites for hypoxia-inducible factors (HIFs). Daphnia HIF, bound to human HRE sequences, was detected in extracts from hypoxic (red), but not normoxic (pale), animals. Taking advantage of the phylogenetically conserved HIF/HRE recognition, we employed heterologous transfections of HIF-expressing human and Drosophila cells to model HIF signaling in Daphnia. These experiments revealed that three functional HREs within the promoter of the D. magna globin-2 gene cooperate for maximal hypoxic induction of a downstream luciferase reporter gene. Two of these three cis-elements, at promoter positions -258 and -107, are able to specifically bind human, Drosophila, or Daphnia HIF complexes in vitro. The same two sites are also necessary for maximal induction of reporter transcription under low oxygen tension in the presence of either endogenous human or overexpressed Drosophila HIF proteins. The third motif of the globin-2 gene promoter, a CACGTG palindrome at position -146, functions as a docking site for an unknown constitutive transcription factor. In human cells, this -146 complex interferes with HIF occupancy at the adjacent -107 HRE and thus controls the extent of HIF-mediated hypoxic activation of the downstream target.

Objective: To evaluate the effect of hemolysis on the measurement of cardiac troponin T (cTnT) using the Elecsys 2010 immunoanalyzer., Methods: cTnT concentrations were measured using the Elecsys 2010. Interference studies were conducted by mixing serum of known cTnT concentration with either hemolysates or serum to which purified hemoglobin (Hgb) had been added. Hemolysates were prepared by mechanical disruption. PROBIT analysis was conducted to determine the probability of a cTnT result of >0.1 microg/l being decreased to <0.1 microg/l due to hemoglobin interference., Results: Purified hemoglobin as well as serum-hemolysates mediated a concentration-dependent inhibition of cTnT determination with the Elecsys 2010. With every 1 g/l increase in hemoglobin, the probability of cTnT >0.1 is decreased by 2.5%., Conclusions: A concentration-dependent negative bias in the measurement of cTnT is associated with increasing hemoglobin concentrations in serum.

Co-expression of Vitreoscilla hemoglobin (VHb) can enhance production of foreign proteins in several microorganisms, including Escherichia coli. Production of foreign proteins [green fluorescent protein (GFP) and organophosphorous hydrolase (OPH)] has been examined in two typical industrial E. coli strains, W3110 (a K12 derivative) and BL21 (a B derivative). In particular, we investigated the effects of VHb co-expression and media glucose concentration on target protein production. We employed the nar O(2)-dependent promoter for self-tuning of VHb expression based on the natural changes in dissolved O(2) levels over the duration of culture. Foreign protein production in strain BL21 was decreased by a high glucose concentration but co-expression of VHb had no effect on this. In contrast, co-expression of VHb in strain W3110 overrode the glucose-induced repression and resulted in steady expression of foreign proteins.

The differentiation induction of K562 chronic myelogenous leukemia (CML) cells by crambescidin 800, a pentacyclic guanidine alkaloid isolated from a marine sponge, was examined. Crambescidin 800 increased hemoglobin production in K562 cells at concentrations of 0.15-1 microM and arrested the cell cycle of K562 cells at the S-phase. The expression of p21 was detected after 24-h treatment with crambescidin 800, and an increase of the expression was observed after 48-h treatment, but there was no remarkable change in the expression level of p27. This evidence indicates that crambescidin 800 induced the differentiation of K562 cells into erythroblasts accompanied by cell cycle arrest at the S-phase. Furthermore, crambescidin 800 induced a morphological change with neurite outgrowth in Neuro 2A cells at a 0.03-0.1 microM concentration.

It was hypothesized that pentoxifylline might improve the response to recombinant human erythropoietin (rh-Epo) in anemic renal failure patients. Sixteen patients with ESRD and rh-Epo-resistant anemia, defined by a hemoglobin of <10.7 g/dl for 6 mo before treatment and a rh-Epo dose of > or =12,000 IU/wk, were recruited. They were treated with oral pentoxifylline 400 mg o.d. for 4 mo. Ex vivo T cell generation of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) from the patients was assessed before treatment and 6 to 8 wk after therapy. A total of 12 of 16 patients completed the study. Before therapy, the 12 patients' mean hemoglobin concentration was 9.5 +/- 0.9 g/dl. After 4 mo of pentoxifylline treatment, the mean hemoglobin concentration increased to 11.7 +/- 1.0 g/dl (P = 0.0001). Baseline ex vivo T cell expression of TNF-alpha decreased from 58% +/- 11% to 31% +/- 23% (P = 0.0007) after therapy. Likewise, IFN-gamma expression decreased from 31% +/- 10% to 13% +/- 10% (P = 0.0002). Pentoxifylline therapy may significantly improve the hemoglobin response in patients with previously rh-Epo-resistant anemia in renal failure. This may occur due to inhibition of proinflammatory cytokine production, which could interfere with the effectiveness of rh-Epo.

In serum-free WEHI-3 supernatants an activity was detected inducing haemoglobin synthesis in human and murine erythroleukaemia cell lines. The absolute numbers of benzidine-positive cells induced with either DMSO or WEHI-3-conditioned medium were comparable. Terminal differentiation was not observed. An expression library from WEHI-3 RNA aided by PCR cloning revealed an open reading frame corresponding to a 209 amino acid protein. This was 100% identical to a sequence from human stimulated peripheral blood mononuclear cells. In contrast to human RNA, mouse RNA exhibited multiple bands of pre-mRNA in Northern blots. The gene was provisionally termed erythroid differentiation regulator (edr). In mammalian cells EDR is mostly expressed as a 56 kDa dimer showing higher activity than the recombinant monomer. The activity profile is bell-shaped. Expression was observed in many normal mouse tissues, yet in haematopoiesis it was largely confined to CD34+ cells. It was enhanced by a series of stimuli such as phorbol ester, and transformed cells generally showed a higher level of EDR expression than normal ones. The protein is localized at the inner side of the cytoplasmic membrane and is released in part via vesicles. In view of the broad range of EDR-expressing tissues the function obviously exceeds haemoglobin synthesis induction. Involvement in cell survival and growth control has been observed and will be dealt with in detail elsewhere.

A saturated nitric oxide (NO) solution (1.88 mM) infused i.v. in the anesthetized pig at a dose of 68 nmol/kg/min for 24 min resulted in a time-dependent increase of nitrosylhemoglobin [HbFe(II)NO] as determined by electron spin resonance (ESR), reaching a C(max) of 7.99 +/- 0.42 microM at the end of the infusion, compared to 1.13 +/- 0.42 microM before (p < 0.01). This indicates that NO i.v. is efficiently bioconserved as HbFe(II)NO (approximately 34% of the NO dose) and to a greater extent than by the oxidative pathway (approximately 24% of the NO dose), as determined by measuring plasma nitrites/nitrates (chemiluminescence) and Met-Hb (ESR analysis). When the NO infusion was stopped, HbFe(II)NO declined with a t(1/2) of 15 min, indicating that it is a stable storage form of NO, able to deliver NO distally to the site of administration. No significant differences were observed in systemic and pulmonary vascular resistances during and after NO infusion, but PO(2) showed a significant decrease 15 and 30 min after the infusion. Thus, in normoxic/physiological conditions, HbFe(II)NO does not induce significant NO-dependent vasorelaxation.

The hemoglobins found in unicellular organisms show a greater chemical reactivity, protect cells against oxidative stress and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than VHb counterparts.

Estimating the risk of in-hospital mortality in the newborn intensive care unit can provide important information for health-care providers, and illness severity scores have been devised to provide mortality risk estimates. Calculation of illness severity scores is time-consuming, and the information used to predict mortality is collected only for the first 12 to 24 h of life. A noninvasive continuous measure that uses information collected throughout the hospitalization and that requires no data entry could be less costly and more informative. We have previously shown that the abnormal heart rate characteristics (HRC) of reduced variability and transient decelerations accompany neonatal illness such as late-onset sepsis. We hypothesized that more frequent and severe abnormal HRC are associated with an increased risk of death. We tested this hypothesis in two ways. Using data on infants older than 7 d of age, we first determined the association of the HRC index with death in the next week. Second, we devised a cumulative HRC score and determined its association with in-hospital death. There were 37 deaths in the 685 patients. The major findings were 1) the HRC index showed highly significant association with death in the succeeding 7 d (receiver-operating characteristic area > 0.7, p < 0.001), and 2) the cumulative HRC was highly significantly associated with neonatal in-hospital mortality (receiver-operating characteristic area > 0.80, p < 0.001). In both analyses, HRC added information to birth weight, gestational age, and postnatal age (p < 0.01). The HRC index provides independent information about the risk of neonatal death in the upcoming 7 d, and the cumulative HRC is an estimate of the risk of in-hospital neonatal mortality.

The differentiation of K562 chronic myelogenous leukemia (CML) cells by smenospongine, which is a sesquiterpene aminoquinone isolated from a marine sponge, was examined. Smenospongine increased hemoglobin production in K562 cells at concentrations of 3-15 microM. In addition, flow cytometric analysis of smenospongine-treated K562 cells with FITC-labeled glycophorin A antibody showed an increase of glycophorin A expression, a marker for erythroid differentiation. Cell-cycle analysis showed G1 arrest in K562 cells after treatment with smenospongine for 24 h. The effect on expression of CIP/KIP family cyclin-dependent kinase inhibitors was investigated by Western blotting analysis and the result showed increased expression of p21, which is known to play an important role in differentiation. Furthermore, smenospongine was also found to inhibit the phosphorylation of Crkl, a substrate of Bcr-Abl tyrosine kinase, which is known as a causative protein of CML. In conclusion, our investigation indicated that smenospongine induced the differentiation of K562 cells into erythroblasts along with cell-cycle arrest at G1 phase and the mechanism might be attributed to the increased expression of p21.

Anemia is a common complication of cancer that has been associated with poor response to treatment and decreased survival in a number of malignancies. By chart review the effects of prognostic factors (e.g. age, body mass index, tumor stage) and hemoglobin levels, measured prior to and during adjuvant (724/996; 72.7%) and primary radiotherapy (185/996; 18.6%) in 996 endometrial cancer patients treated between 1986 and 1998 were investigated using Kaplan-Meier survival on disease-free and overall survival. Preoperative hemoglobin levels were of no prognostic value. Patients with normal hemoglobin had an improved survival during primary radiotherapy (p<0.05). Anemia and course of hemoglobin during adjuvant radiotherapy were significantly associated with poor survival in patients undergoing adjuvant radiotherapy (p=0.001). Cox regression analysis confirmed these findings for adjuvant treatment. Local recurrences were also less frequently observed in patients with normal hemoglobin levels (p=0.044). Hemoglobin levels prior to and during radiotherapy seem to be important with respect to treatment outcome for endometrial cancer. This finding supports the use of measures to maintain adequate hemoglobin levels (transfusions, erythropoietin) to improve local control and patient survival.

Tellimagrandin I is a hydrolysable tannin compound widely present in plants. In this study, the effect of tellimagrandin I on chemically induced erythroid and megakaryocytic differentiation was investigated using K562 cells as differentiation model. It was found that tellimagrandin I not only inhibited the hemoglobin synthesis in butyric acid (BA)- and hemin-induced K562 cells with IC50 of 3 and 40microM, respectively, but also inhibited other erythroid differentiation marker including acetylcholinesterase (AChE) and glycophorin A (GPA) in BA-induced K562 cells. Tellimagrandin I also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of CD61 protein, a megakaryocytic marker. RT-PCR analysis showed that tellimagrandin I decreased the expression of erythroid genes (gamma-globin and porphobilinogen deaminase (PBGD)) and related transcription factors (GATA-1 and NF-E2) in BA-induced K562 cells, whereas tellimagrandin I induced the overexpresison of GATA-2 transcription factor that played negative regulation on erythroid differentiation. These results indicated that tellimagrandin I had inhibitory effects on erythroid and megakaryocytic differentiation, which suggested that tannins like tellimagrandin I might influence the anti-tumor efficiency of some drugs and the hematopoiesis processes.

STI571 is a specific tyrosine kinase inhibitor of Abl kinase. It was previously reported that STI571 induced hemoglobin synthesis in the chronic myelogenous leukemia (CML) cell line K562. However, its mechanisms remain unknown. In this study, we demonstrated that STI571 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and dephosphorylation of extracellular signal-regulated kinase (ERK) in K562 cells. In contrast, the phosphorylation of c-Jun N-terminal kinases (JNK) in K562 cells was not altered by STI571. We also found that STI571 induced all the myeloid (CD11b, CD13), megakaryocytic (CD41a, CD42), and erythroid (glycophorin-A) markers on K562 cells. A p38 MAPK-specific inhibitor, SB203580, inhibited the STI571-induced multi-lineage differentiation of K562 cells, indicating that p38 MAPK is crucial for this differentiation. In contrast, SB203580 did not overcome the inhibitory effect for proliferation of K562 cells, indicating that p38 MAPK activation by STI571 does not affect cell numbers. Among the hematopoietic transcription factors, the expression level of c-myb mRNA was clearly downregulated after incubation with STI571 in K562 cells. STI571-induced downregulation of c-myb mRNA was prevented by the pretreatment of K562 cells by SB203580. Our data provides insights into how p38 MAPK and ERK pathways are involved in STI571-induced differentiation of K562 cells., (Copyright 2003 Wiley-Liss, Inc.)

Cardiac T2* (magnetic resonance imaging relaxation parameter) is abnormally low in approximately 40% of adults with thalassemia major (TM), suggesting myocardial iron deposition, but it is unknown at what age this occurs. To address this question, we measured cardiac T2* and function in 19 young patients (aged 7-26 years) with TM as well as 17 patients receiving long-term transfusions for sickle cell anemia (SCA) matched for age, sex, and liver iron content. Cardiac T2* was normal in all of the SCA patients but was low (high iron) in 8 of 19 TM patients. Abnormal T2* was observed only in the TM patients receiving transfusions for 13 years or longer and was correlated with ferritin but not liver iron levels. Cardiac dysfunction was present in 3 of the 8 patients with low T2*. Cardiac T2* changes have a long latency relative to liver iron accumulation. Total transfusional burden is a significant independent risk factor for low cardiac T2* and may partially account for differences observed between patients with SCA and TM.

Haemoglobin acts as an important protein for oxygen carrier in all living beings. Purification of donkey haemoglobin was done using PEG 4000 as a separating medium. Crystallization was achieved using hanging drop vapor diffusion method using 2.8 M phosphate buffer, pH 6.8. Data collection was done using mar345 image plate detector system. The crystals grown under 2.8 M phosphate buffer are monoclinic with space group C2 and cell dimensions a=107.664 A, b=63.084 A, c=54.042 A, and beta=111.747 degrees.

Objective: To assess the validity of using the measurement of haemoglobin as a primary screen test at point-of-care in general practice., Methods: 1100 consecutive full blood counts carried out at Hammersmith Hospital haematology laboratory on blood samples sent by the general practitioners in the area were reviewed. Where haemoglobin was in the normal range all the parameters of a full blood count were checked and a blood film was screened in order to identify any abnormal features occurring in the absence of anaemia., Results: Haemoglobin was normal in 81% of the samples, and in 81% of this set none of the blood count components showed any abnormal features (i.e. outside two standard deviations of normal reference range). The remaining 19% of cases included 32 with iron deficiency, 26 with high MCV, 84 with leucocyte abnormalities (neutrophilia, neutropenia, lymphocytosis, eosinophilia, monocytosis) and 19 with platelet counts either too high or too low. The predictive value of a normal full blood count from a normal haemoglobin was 0.81. However, when the limits of normal reference values were set at three standard deviations only 7% of the cases showed abnormal features and the predictive value of normality from a normal haemoglobin increased to 0.92., Conclusion: There are now simple and suitable devices available for measuring haemoglobin at point-of-care, away from a laboratory. This provides a useful screening test in general practice as a full blood count would, as a rule, be required only in the small proportion of cases where anaemia is detected or the patient's history and/or clinical signs specifically indicate the need for this further investigation. This approach should contribute to more efficient, convenient and economical practice without compromising clinical management.

Targeting BCR-ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) on TF-1/bcr-abl (which was generated by transduction of a bcr-abl fusion gene into the TF-1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF-1/bcr-abl cells but not in the parental TF-1 cells. However, GM-CSF, a survival factor of the parental TF-1 cells, protected TF-1/bcr-abl cells from imatinib-induced apoptosis in a dose-dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM-CSF, accompanied by upregulation of Bcl-xL and downregulation of p27/Kip1. In addition, although untreated TF-1/bcr-abl cells had lost responsiveness to both GM-CSF and EPO and showed autonomous growth, GM-CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib-treated TF-1/bcr-abl cells differentiated into hemoglobin-positive cells in the presence of EPO, as in the case for the parental TF-1 cells. Taken together, imatinib-treated CML cells may differentiate into mature cells in the presence of differentiation-inducing cytokines such as EPO.

Insulinoma is a rare, almost always benign endocrine tumor of the pancreas, clinically characterized by hyperinsulinemic, hypoglycemic episodes. Surgical excision is the therapy of choice, which may lead to postpancreatectomy diabetes mellitus in the case of extensive pancreatic resection. We present the cases and the metabolic follow up of two patients, 81 and 73 years old, with insulinoma localized close to the main duct in the pancreatic neck. Both patients underwent an 80% left pancreatectomy, avoiding a pancreatico-enteric anastomosis. In order to prevent postpancreatectomy diabetes, the islets from the tumor-free part of the resected pancreas were isolated and injected via a right colic vein into the portal system. After a follow up of 6 and 3 years respectively, both patients remained insulin-independent without any dietary restrictions. Fasting and glucagon-stimulated C-peptide-levels and glycosylated hemoglobin remained within normal range. There were no signs of recurrent insulinoma. Liver biopsy performed in one patient at 1 year after autotransplantation, showed intact, insulin-producing islets within the portal spaces. In conclusion, autologous islet transplantation can preserve the insulin secretory reserve after extended left pancreatectomy for the treatment of benign tumors in the pancreatic neck.

The influence of bacterial hemoglobin, VHb, on dechlorination and degradation of 2-chlorobenzoate (2-CBA) by recombinant Burkholderia sp. under variable oxygen availability with an initial dissolved oxygen concentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Ability to express VHb was provided to recombinant Burkholderia by transformation with the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded in cultures with 85% and 70% of total volume as headspace air in closed reactors by both wild type and recombinant Burkholderia. The recombinant cultures were able to dechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 degrees C compared to more than 120 hours for wild type cultures. The rate and extent of CBA degradation by recombinant cultures with 40% of total volume as headspace air was higher than those achieved by wild type cells at the end of the 168 hours of incubation period, 98 and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40% of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinant strains, whereas it was non-stoichiometric (molar ratio = 0.24) for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorination and degradation pathway for recombinant cells under hypoxic conditions. The degradation and dechlorination ability of both types of cells was also investigated in continuous reactor studies by varying the dilution rate under hypoxic conditions. Regarding potential of the recombinant strain for 2-CBA degradation in either open ecosystems or closed bioreactor bioremediation systems, the stability of the plasmid containing vgb in the recombinant cells was also studied; the plasmid was 100% stable at 0.025 h(-1) dilution rate (approximately 1.7 d hydraulic retention time), even after one month.

Anemia is a common complication of cancer that has been associated with poor response to treatment and decreased survival in many malignancies. A retrospective chart review was undertaken to determine the effects of hemoglobin (Hb) levels, measured prior to initiation of chemotherapy and before each chemotherapy cycle, and clinical prognostic factors (e.g., age, tumor stage, residual tumor size, hematologic parameters, and type of health insurance) in 250 ovarian cancer patients treated between 1985 and 1998. All patients were scheduled to receive at least 6 courses of systemic chemotherapy. None of the patients received recombinant human erythropoietin. The difference between observed overall survival and its predicted value was computed by multiple regression analysis for each patient with respect to prognostic factors. Hemoglobin levels prior to and during chemotherapy were identified as a prognostic factor for overall survival. Hemoglobin levels > or =12 g/dl were significantly associated with prolonged overall survival (P<0.001). In addition, Hb level correlated with scheduled completion of chemotherapy, overall therapeutic success, tumor stage, age at diagnosis, and residual tumor size (all, P<0.005). Hemoglobin level represents an important prognostic factor for patients with ovarian cancer. This finding supports the use of measures to maintain adequate Hb levels, such as treatment with recombinant human erythropoietin, to improve patient survival.

The ribosome and tRNA levels of Escherichia coli cells, transformed with a native or mutated Vitreoscilla hemoglobin genes (vhb), were investigated using asymmetrical flow field-flow fractionation (AFFFF). Mutagenesis of rhb by error-prone PCR was carried out to alter the growth behavior of microaerobically cultivated native VHb-expressing E. coli. A VHb mutant, pVMT1, was identified, which was able to reach a remarkably high final A600 of 15, the value of which being 160% higher than that of a VHb control carrying pVHb8 (A600 5.8). AFFFF revealed that cells expressing mutant vhbs showed up to a doubling in the number of active 70S ribosomes cell(-1), an almost 3-fold increase in the number of tRNAs cell(-1), and up to a 26% increase in the mass fraction of active 70S ribosomes.

Fixed dosing is potentially more convenient than weight-based dosing for both patients and physicians. Therefore, this open-label, randomized (1:1), multicenter study was conducted to compare the effectiveness, safety, and quality-of-life benefits of fixed vs. weight-based dosing of epoetin alpha in anemic cancer patients undergoing chemotherapy. Five hundred forty-six anemic patients undergoing platinum-based chemotherapy for solid malignancies were enrolled. Patients received epoetin alpha, either a fixed dose of 10,000 IU or a weight-based dose of 150 IU/kg, administered subcutaneously 3 times weekly for up to 12 weeks. Endpoints were transfusion requirements over days 29-84, change in hemoglobin (Hb) level from baseline, and change in quality-of-life (QOL) scores from baseline as measured using the Cancer Linear Analog Scale (CLAS). Five hundred and thirty-two patients received at least 1 dose of epoetin alpha, and 510 of these (255 in each treatment group) were considered evaluable for efficacy. At day 84, rates for freedom from transfusion were similar between the fixed-dose and the weight-based dose group (84% vs. 87%, respectively, p=0.32), as calculated by the lifetable method. These rates were also similar between patients in the 45-63 kg weight group receiving the fixed 10,000 IU dose or 7,000-9,000 IU on a per-weight basis (83% vs. 87%, respectively), and those in the 70-100 kg weight group receiving the fixed 10,000 IU dose or 11,000-15,000 IU on a per-weight basis (85% vs. 83%, respectively). Mean Hb increases from baseline to last observation were 2.10 g/dl [95% confidence intervals (CI95) 1.85-2.35] in the 10,000 IU group (from 9.64-11.74 g/dl) and 2.06 g/dl (CI95 1.82-2.30) in the 150 IU/kg group (from 9.70-11.76 g/dl). QOL results were similar for both groups and cumulative data have been reported. For 275 patients (in both groups combined) with CLAS QOL scores both at baseline and 29-98 days thereafter, the QOL index (average of scores for the 3 QOL parameters: energy level, ability to do daily activities and overall QOL) increased by 10.4 mm (CI95 7.5-13.2), from 46.2 mm at baseline to 56.6 mm at the final observation. QOL improvements were directly associated with Hb increases (p<0.001, multiple linear regression analysis) within all chemotherapy response classes. Epoetin alpha was well tolerated in both groups. Fixed (10,000 IU) and weight-based (150 IU/kg) dosing regimens of epoetin alpha demonstrated similar efficacy in maintaining freedom from transfusion, increasing Hb levels, and improving QOL in anemic cancer patients undergoing platinum-based chemotherapy. QOL improvements were directly associated with Hb increases. These findings support the use of a fixed-dose regimen of epoetin alpha, which may offer greater convenience for physicians and patients than weight-based dosing with this agent.

Hydroxyurea (HU) enhances fetal hemoglobin (Hb) production. An increase in total Hb level has been repeatedly reported during HU treatment in patients with sickle cell disease and in several patients with beta-thalassemia intermedia. Effects in patients with beta-thalassemia major are controversial. We now report a marked elevation of total Hb levels with HU that permitted regular transfusions to be stopped in 7 children with transfusion-dependent beta-thalassemia. The median follow-up was 19 +/- 3 months (range, 13-21 months). We conclude that HU can eliminate transfusional needs in children with beta-thalassemia major, which could be particularly useful in countries such as Algeria, where supplies of blood or chelating agents are limited.

The quantity and quality of the haemoglobin (Hb) of Daphnia magna is related to oxygen partial pressure in the water. Both the dynamics of hypoxia-induced Hb gene transcription, as well as Hb properties in animals incubated long-term at hyperoxia, normoxia and hypoxia, were investigated. Examination of Hb gene (dhb1-dhb3) transcription showed the expression of dhb2 and especially dhb3 to increase markedly approximately one hour after the onset of hypoxia, whereas dhb1 was expressed more or less constitutively. At an incubation close to anoxia, an onset of dhb3 transcription was found already after two minutes. In long-term incubated animals, concentration and oxygen affinity of Hb were lower at higher oxygen partial pressures. With decreasing oxygen availability, the subunit composition of Hb macromolecules changed. The share of the dhb2-encoded subunit, DHbF, increased already during moderate hypoxia. The increase of dhb3 mRNA (encoding DHbC) may be related to a transient increase of DHbC in the first days of hypoxia and/or to an additional coding of dhb3 for DHbD. The rise of DHbD, and particularly DHbA, only at severe hypoxia coincided with the increase of Hb oxygen affinity. The dhb1-encoded subunits DHbB and DHbE showed either a relatively moderate increase or even a decrease in concentration at hypoxia. In small animals with restricted homeostasis capabilities such as Daphnia, adaptation of the protein equipment seems to be a more effective strategy than allosteric modulator control.

Purpose: Recent findings indicating that many genes related to cancer development are silenced by an aberrant DNA methylation suggest that inhibitors of this process may be effective cancer therapeutics. In this study we investigated the efficacy of low-dose 5-aza-2'-deoxycitydine (DAC), a methylation inhibitor, with interleukin (IL) 12, one of the most potent cytokines with antitumor activity., Experimental Design: Mice inoculated with L1210 leukemia cells or with B16F10 melanoma cells were treated with 7 daily injections of low-dose DAC (0.2 mg/kg) and/or 7 daily doses of IL-12 (100 ng/dose). Scid/scid mice as well as monoclonal antibodies against CD4, CD8, and NK1.1 were used to investigate the mechanisms of the antitumor effects of the combination treatment. The activity of murine lymphocytes was measured with enzyme-linked immunospot and (51)Cr release assays., Results: Treatment with DAC or IL-12 given alone produced moderate antitumor effects. In both tumor models combined treatment resulted in potentiated antitumor effects and produced 70% long-term survivors among mice inoculated with L1210 cells. The antitumor efficacy of combined treatment was abrogated in scid/scid mice, and after depletion of CD4(+) and CD8(+) T cells. Mice inoculated with B16F10 melanoma cells had significantly delayed tumor growth after combined treatment with DAC and IL-12. Strong antitumor effect correlated with a significant activation of lymph node-derived CD8(+) and CD4(+) cells. Transient neutropenia was observed in mice under treatment of DAC alone, but remarkably this effect was not potentiated by IL-12., Conclusions: This study provides the first evidence that antitumor effects of DAC can be strongly potentiated by IL-12 and could be beneficial in an effective low-dose-based antitumor therapy.

Hemoglobins in unicellular organisms, like the one here in the bacterium Vitreoscilla, have greater chemical reactivity than their homologues in multicellular organisms. They can catalyze redox reactions and may protect cells against oxidative stress. The ability of Vitreoscilla hemoglobin to complement deficiencies of terminal cytochrome oxidases in Escherichia coli also suggests that this hemoglobin can receive electrons during respiration. In this study, a recombinant strain of Enterobacter aerogenes engineered to produce the Vitreoscilla Hb was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress produced by exogenously applied hydrogen peroxide was characterized in terms of cell growth, survival and the activities of two key antioxidant enzymes (catalase and superoxide dismutase). The influence of the physiological state of the cells and different media upon these culture dynamics was determined. Results showed that the hemoglobin-expressing strain is quite distinct in terms of growth/survival properties and activity of antioxidant enzymes from that of non-hemoglobin counterparts.

We studied the effect of certain differentiation inducers-thymidine, sodium butyrate, and dimethyl sulfoxide--on the cells of parental line K562 and the derived sublines resistant to quinoline xenobiotics 2-(4-dimethylaminostyryl)quinoline 1-oxide or 4-nitroquinoline 1-oxide. The cells of the both derived sublines demonstrate cross-resistance to these xenobiotics, while the subline resistant to 2-(4-dimethylaminostyryl)quinoline 1-oxide is also resistant to ethidium bromide and colchicine. Treatment of cells of the sublines with thymidine but neither sodium butyrate nor dimethyl sulfoxide for 2 or 4 days considerably increases intracellular content of hemoglobin as compared to the parental cells K562. The revealed effect is due to increased hemoglobin content in the cells rather than to the increased proportion of hemoglobin-containing cells, which results from decreased proliferation rate observed in all cases.

Our previous assembly studies to form hemoglobin hetero-dimers and -tetramers using a coupled transcription/translation cell-free system suggested that alpha-globin chains bind to nascent non-alpha chains during and/or soon after translation to promote hemoglobin formation [Adachi et al., J. Biol Chem. 2002 (277) 13415]. In this report effects of CN-hemin on subunit assembly were studied using this cell-free system. Addition of CN-hemin and excess unlabeled heme-containing partner chains during synthesis leads to formation of radiolabeled heme-containing alpha(h)beta(h) hetero-dimers. In contrast, in the absence of added CN-hemin, unlabeled heme-containing alpha or beta chains can assemble with newly synthesized radiolabeled beta- and alpha-globin chains to form heme-containing alpha(h)beta(h) and semi-alpha (alpha(h)beta(0)) or semi-beta (alpha(0)beta(h)) hetero-dimers, respectively. These results suggest the existence of semi-hemoglobins as intermediates prior to formation of heme-containing alpha(h)beta(h) and indicate transfer of heme from alpha and/or beta chains into semi-hemoglobin hetero-dimers to form heme-containing hetero-dimers.

Erythropoiesis requires the stepwise action on immature progenitors of several growth factors, including stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (Epo). Epo is required to sustain proliferation and survival of committed progenitors and might further modulate the level of expression of several erythroid genes, including globin genes. Here we report a new SCF-dependent immortalized mouse progenitor cell line (GATA-1 ts SCF) that can also grow in either Epo or IL-3 as the sole growth factor. When grown in SCF, these cells show an "open" chromatin structure of the beta-globin LCR, but do not significantly express globin. However, Epo or IL-3 induce globin expression and are required for its maintainance. This effect of IL-3 is unexpected as IL-3 was previously reported either to be unable to induce hemoglobinization, or even to antagonize it. This suggests that GATA-1 ts SCF cells may have progressed to a stage in which globin genes are already poised for expression and only require signal(s) that can be elicited by either Epo or IL-3. Through the use of inhibitors, we suggest that p38 may be one of the molecules modulating induction and maintenance of globin expression., (Copyright 2003 Wiley-Liss, Inc.)

Bcl-X(L) is essential for the survival and normal maturation of erythroid cells, especially at the late stage of erythroid differentiation. It remains unclear whether Bcl-X(L) serves only as a survival factor for erythroid cells or if it can induce a signal for differentiation. We have previously shown that dimethyl sulfoxide (DMSO) induction of erythroid differentiation in murine erythroleukemia (MEL) cells correlates with delay of apoptosis and specific induction of Bcl-X(L). In this study, we investigate the contribution of Bcl-2 and Bcl-X(L) to survival and erythroid differentiation by generating stable MEL transfectants expressing these antiapoptotic regulators. Overexpression of Bcl-2 completely prevented apoptosis of MEL cells before and after DMSO induction, whereas overexpression of Bcl-X(L) only delayed it. Overexpression of Bcl-2 or Bcl-X(L) neither induced spontaneous erythroid differentiation nor accelerated DMSO-induced differentiation. Inhibition of Bcl-X(L) by antisense transcripts accelerated apoptosis in DMSO-treated MEL cells and blocked the synthesis of hemoglobin without altering the growth arrest associated with terminal erythroid differentiation. An antisense oligonucleotide to Bcl-X(L) did not induce apoptosis in MEL cells overexpressing Bcl-2 but greatly decreased their hemoglobin synthesis when treated with DMSO, suggesting that Bcl-X(L) is necessary for erythroid differentiation independently of its antiapoptotic function. Importantly, Bcl-X(L) antisense transcripts prevented heme synthesis but not globin mRNA induction in DMSO-treated MEL cells. Furthermore, inhibition of hemoglobin synthesis by Bcl-X(L) antisense was reversed by addition of exogenous hemin. Finally, Bcl-X(L) localized to mitochondria during MEL erythroid differentiation, suggesting that it may mediate a critical mitochondrial transport function related to heme biosynthesis.

The aim of this work was to describe the factors influencing the levels of three antioxidant markers -total antioxidant status, erythrocyte copper/zinc superoxide dismutase (SOD), whole-blood selenium glutathione peroxidase--and to establish their reference intervals in supposedly healthy subjects. The studied population included 463 subjects, i.e., 223 adults and 140 children aged 20 to 65 and 4 to 19 years, respectively. The effect of factors such as age, gender, body mass index, alcohol and tobacco consumption, menopause, drug intake, trace elements, transferrin, ferritin, albumin, bilirubin, haptoglobin, total proteins, uric acid, haemoglobin, and mean corpuscular volume of erythrocytes have been studied for the three antioxidant markers. Total antioxidant status (TAS) was higher in men than in women whatever the age (p < 0.001). Albumin and uric acid in men, women and girls, and total proteins in boys were significant determinants of TAS levels. Mean corpuscular volume of erythrocytes were negatively and significantly associated with SOD activity in men and in women (p < 0.01) but not in children. Among the studied determinants, none were found to influence the selenium glutathione peroxidase activity in the four groups. Reference intervals including the 90% confidence intervals were established by age and sex for the three antioxidant markers.

Previous studies of the interactions of NO with human hemoglobin have implied the predominance of reaction channels that alternatively eliminate NO by converting it to nitrate, or tightly complex it on the alpha subunit ferrous hemes. Both channels could effectively quench NO bioactivity. More recent work has raised the idea that NO groups can efficiently transfer from the hemes to cysteine thiols within the beta subunit (cysbeta-93) to form bioactive nitrosothiols. The regulation of NO function, through its chemical position in the hemoglobin, is supported by response to oxygen and to redox agents that modulate the molecular and electronic structure of the protein. In this article, we focus on reactions in which Fe(III) hemes could provide the oxidative requirements of this NO-group transfer chemistry. We report a detailed investigation of the reductive nitrosylation of human met-Hb, in which we demonstrate the production of S-nitroso (SNO)-Hb through a heme-Fe(III)NO intermediate. The production of SNO-Hb is strongly favored (over nitrite) when NO is gradually introduced in limited total quantities; in this situation, moreover, heme nitrosylation occurs primarily within the beta subunits of the hemoglobin tetramer. SNO-Hb can similarly be produced when Fe(II)NO hemes are subjected to mild oxidation. The reaction of deoxygenated hemoglobin with limited quantities of nitrite leads to the production of beta subunit Fe(II)NO hemes, with SNO-Hb produced on subsequent oxygenation. The common theme of these reactions is the effective coupling of heme-iron and NO redox chemistries. Collectively, they establish a connectivity between hemes and thiols in Hb, through which NO is readily dislodged from storage on the heme to form bioactive SNO-Hb.

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.

A pure population of young reticulocytes was isolated from the blood of healthy blood donors by an immunomagnetic technique. The young reticulocytes isolated had a larger mean corpuscular volume (MCV) but lower mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) than reticulocytes in peripheral blood. The cells were incubated in vitro for 5 days with hemolysis less than 5%. Maturation of reticulocytes was studied with 4 different systems: new methylene blue-stained smears and flow cytometry after auramine O, oxazine 750, or thiazole orange staining. The maturation process from reticulocytes to erythrocytes had a half-life of 30 hours estimated by visual counting. Reticulocytes analyzed with flow cytometers had half-lives of 20, 28, and 29 hours with Coulter Epics XL, Sysmex R-1000, and Bayer H*3 devices, respectively. Total maturation time was 4 to 5 days. During maturation, the reticulocyte MCV gradually decreased with a concomitant increase in reticulocyte MCHC. The reticulocytes synthesized hemoglobin, and the MCH of the cells increased approximately 7% during the incubation period. Taking into account loss of hemoglobin-containing vesicles during reticulocyte maturation, we estimated that more than 20% of hemoglobin in erythrocytes was synthesized in reticulocytes after release from bone marrow.

Purpose of the Study: To determine the effects of choline on red blood cell membrane properties and NO metabolism., Material and Methods: Aliquots of venous blood from eleven healthy subjects were incubated in vitro with choline concentrations 10(-10) to 10(-3) M. The following parameters were determined: erythrocyte deformability, aggregation and membrane lipid fluidity, plasma K+, Na+, Ca2+, total blood haemoglobin and methemoglobin concentrations. Additionally, plasma and intra-erythrocyte nitrites concentrations were measured., Results: Choline increases erythrocyte deformability at lower shear stresses, decreases erythrocyte aggregation, increases membrane lipid fluidity, and decreases of Na+ plasma concentrations. We also find an increase of nitrites concentration both in the plasma and in the intra-erythrocyte compartment., Conclusion: Choline induces changes on erythrocyte membrane properties, Na+ plasma concentration, and NO metabolites concentrations.

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells., (Copyright 2002 Wiley-Liss, Inc.)