Induction of hemoglobin synthesis in the human leukemia cell line, SPI-802.
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Child↗
Biomedical subjects
A N Schechter
Publications and source records attributed to A N Schechter.
Trans-activation of human globin genes by HTLV-I tax1.
We studied the effects of a known retroviral trans-activating factor, HTLV-I tax1, on transcription of human globin genes. Transfection of HeLa cells by the cloned tax1 gene stimulated activity of both the beta- and epsilon-globin promoters approximately 20-fold, as measured by chloramphenicol acetyl transferase (CAT) assays. Studies of promoter 5'-deletion mutants revealed that the trans-activation response required only 185 base pairs (bp) of beta-globin 5'-flanking sequence or 177 bp of epsilon-globin 5' flanking sequence. These promoter regions contain either two (for beta) or three (for epsilon) copies of the pentanucleotide sequence CTGAC, which is characteristic of previously described tax1-responsive promoters. We also stably transfected tax1 into the erythroid cell line K562. Transfectants expressing tax1 showed increased transcription of epsilon-, gamma-, zeta-, and alpha-globins. This indicates that tax1 can stimulate transcription of globin genes in their native chromosomal location. This was confirmed by measurements of increases in intracellular hemoglobin as determined by an increased percentage of cells staining with benzidine and by spectrophotometric measurements of hemoglobin. The observed trans-activation of globin genes by tax1 may provide insight into normal regulation of globin genes by clarifying cis regulatory sequences. Furthermore, it suggests that the trans-acting effects of tax1 on heterologous genes are more widespread than was previously appreciated.
Base Sequence↗
Nuclease hypersensitivity in the beta-globin gene region of K562 cells.
We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.
Adult↗
Levels of fetal hemoglobin necessary for treatment of sickle cell disease.
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Anemia, Sickle Cell↗
Brain glucose metabolism in neurologically normal patients with sickle cell disease. Regional alterations.
Neurologic dysfunction is a significant source of morbidity and mortality in the sickle cell diseases, occurring with a prevalence of 6% to 34%. Because changes in brain glucose metabolism may precede gross functional or morphologic alterations, we recently applied the technique of positron emission tomography with fluorodeoxyglucose F 18 in an exploratory study to compare six patients with sickle cell disease without prior neurologic abnormalities (and with normal cranial computed tomographic scans) with six healthy age-matched controls, with respect to overall and regional cerebral metabolic rates for glucose. We found no significant difference in the global metabolic rates for the two groups. However, we observed an unusual clustering of abnormal regional cerebral metabolic rates for glucose in the frontal lobes of these subjects. These results support previous observations that frontal lobe involvement may be quite prevalent in sickle cell disease, even among individuals with normal computed tomographic scans.
Adult↗
Use of selective vasodilation in treatment of sickle cell disease.
Individuals with sickle cell disease and its genetic variants demonstrate a modest to severe hemolytic anemia. They are also prone to both acute and chronic vaso-occlusive phenomena, which may lead to irreversible tissue damage, organ failure, and premature death. To better understand these vaso-occlusive phenomena, it would be useful to determine the extent to which arteriolar microvascular obstruction might contribute to the pathogenesis of sickle cell disease. We studied the hematologic and ocular effects of orally administered nifedipine in several steady-state patients and compared these with a case-control group of patients not receiving nifedipine. In the nifedipine-treated patients, a striking reversal in ischemic retinal and conjunctival changes was observed, as well as a significant improvement in color vision performance. In addition, the treated subjects showed a slight but significant decline in the levels of both indirect serum bilirubin and plasma hemoglobin, suggesting a concurrent amelioration in the rate of hemolysis. These observations are consistent with the hypothesis that inappropriate vaso-constriction, or frank vasospasm, perhaps in response to the altered rheology of red cells containing polymerized hemoglobin S, may play an important role in the pathophysiology of sickle cell disease. Furthermore, selective vasodilation may prove to be a useful adjunctive approach to the treatment of the manifestations of the sickle syndromes.
Adult↗
Is there a role for selective vasodilation in the management of sickle cell disease?
To test the hypothesis that microvascular obstruction to blood flow at the level of the arteriole may be significant in individuals with sickle cell anemia, the ophthalmologic effects of orally administered nifedipine were monitored in 11 steady-state patients. Three patients with evidence of acute peripheral retinal arteriolar occlusion displayed a prompt reperfusion of the involved segment. Two other patients showed fading of retroequatorial red retinal lesions. Color vision performance was improved in six of the nine patients tested. The majority of patients also demonstrated a significant decrease in the amount of blanching of the conjunctiva which reflects improved blood flow to this frequently involved area. Such improvements were not observable in a control group of untreated stable sickle cell subjects. These findings support the hypothesis that inappropriate vasoconstriction or frank vasospasm may be a significant factor in the pathogenesis of the microvascular lesions of sickle cell disease and, further, that selective microvascular entrapment inhibition may offer an additional strategy to the management of this disorder. We believe a larger, placebo-controlled study with nifedipine and similar agents is warranted.
Adult↗
Electron microscopic studies of the intracellular polymerization of sickle hemoglobin.
Transmission electron microscopy has been used to study intracellular sickle hemoglobin polymer in unfractionated cells from the arterial and venous blood of patients and after external deoxygenation. We detect polymerized hemoglobin in up to 10% of the cells in the venous circulation, especially in cells that are "cigar-shaped" and appear to be irreversibly sickled. We could not see well-defined polymer in mixed arterial samples; nevertheless, we found electron opaque spots, which could be ferritin granules, hemosiderin, or small aggregates of hemoglobin S. However, upon sequential chemical deoxygenation using 1.0% sodium metabisulphite, polymer formation was seen at oxygen saturation values of 75%-85%. Cells that were physically deoxygenated using gas mixtures containing nitrogen-carbon dioxide-oxygen mixtures were found to contain distinct polymers of deoxyhemoglobin S at oxyhemoglobin saturation values of 50%-75%. As deoxygenation increases, we detect short, randomly arranged polymer in a loose network, with occasional long polymers. Upon further deoxygenation, the length and number of polymer forms increased. Between 0% and 50% saturation, most erythrocytes were full of long, parallel, closely packed polymers that tend to align and run parallel to the cell membrane. In both chemical and physically deoxygenated blood samples, cells were seen at 50%-75% oxyhemoglobin saturation that retained their normal biconcave disc shape, although they contained significant amounts of polymer. The structural changes in sickle erythrocytes seen in vitro due to physical or chemical deoxygenation of cells, may reflect in vivo intracellular changes in the sickle cell patient.
Anemia, Sickle Cell↗
The development of a filtration system for evaluating flow characteristics of erythrocytes.
A complete description of the pathophysiology of sickle cell disease requires a physiologically meaningful measurement of red cell deformability. We have designed and built a system which allows one to determine filtration characteristics of erythrocytes. A dilute red cell suspension is forced through a 3.0-micron polycarbonate Nuclepore membrane with a constant positive pressure of 20 mm Hg. Under these conditions blockage of the pores in the polycarbonate membrane is insignificant and flow is linear. We use the relative number of cells filtered through the membrane as a means of approximating the means deformability of cells in the suspension. Using this system we have compared erythrocytes from various mammals and shown that our technique is sensitive in detecting not only differences in cell deformabilities between mammalian species but also changes in cell deformability of human red cells due to exchange transfusion and application of drugs. There was a positive correlation between cell filtrability and percentage cell recovery (coefficient of correlation, 0.65) and a negative correlation between cell size and filtrability (coefficient of correlation, -0.61). The filtrabilities of normal volunteers and sickle cell disease patients were found to be 71.8 +/- 6.6 and 53.6 +/- 5.0%, respectively. This system is sensitive and reliable, and should be useful in evaluating both the contribution of filtrability to the viability of red cells in vivo and potential therapeutic agents for sickle cell disease.
Anemia, Sickle Cell↗
Retroequatorial red retinal lesions in sickle cell anemia.
We report 2 patients with sickle cell anemia who showed retroequatorial localized red retinal lesions. In 1 patient these lesions were associated with closure of the retinal precapillary arteriole and capillary bed. In both patients these lesions receded during the course of therapy with a selective arteriolar vasodilator, nifedipine. These observations are discussed in relation to the possible pathogenesis of the retinopathy seen in patients with sickle cell anemia.
Adult↗
The development of non-invasive methods to evaluate sickle cell patients in the steady state and in crisis.
Methodologies now exist that promise to disclose fundamental and critical information about the microvascular status of patients with sickle cell disease. Several of these modalities are in an embryonic stage of development, whereas others are close to being ready for application to clinical trials. It is important that these sophisticated diagnostic techniques not supplant our clinical approach to the patient. However, it is hoped that these strategies will allow us to understand better the extreme spectrum of disease manifestations, as well as to provide objective means of monitoring the response to therapy in patients with sickle cell disease.
Anemia, Sickle Cell↗
Predicting therapeutic efficacy for sickle cell anemia.
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Anemia, Sickle Cell↗
Intracellular polymerization of sickle hemoglobin: disease severity and therapeutic goals.
We have demonstrated that the extent of intracellular polymerization of deoxyhemoglobin S can be predicted from knowledge of intracellular hemoglobin concentration, composition and oxygen saturation. Furthermore, we have demonstrated that polymer, which appears to be the main determinant of abnormal red cell rheology, can be detected in sickle erythrocytes at high oxygen saturation values and is not significantly affected by membrane and other cellular constituents. Some of the factors which modify the pathophysiology of sickle cell anemia can be classified as genetic or cellular. To analyze in more detail the genetic factors, we examined 12 sickle syndromes. When the effects of these genotype differences are analyzed for their changes in hemoglobin composition and concentration, we found that polymer formation can account for 80% of the variation in hemolytic and clinical severity. Cell heterogeneity can also modify polymer formation. The premature increases in erythrocyte density (intracellular hemoglobin concentration) in sickle cell anemia increases polymerization tendency. Homozygous alpha-thalassemia in sickle cell patients reduces this increase in cell heterogeneity and improves the hemolytic aspect of the sickle cell disease. For homozygous sickle cell patients we find that the broader density distributions (higher degree of cell heterogeneity) are associated with those cell populations with greater tendency of polymer formation. However, the major utility of our knowledge of intracellular polymerization appears to be its value in defining quantitatively the goals of the major therapeutic approaches with respect to how much inhibition of polymerization would be necessary to achieve various levels of amelioration of disease processes. The primary determinant of the amount of polymer formation within the SS erythrocyte is the extent of oxygen saturation. We measured intracellular polymer formation in SS erythrocytes using carbon-13/proton double nuclear magnetic resonance. As the oxygen saturation is decreased below about 90% oxygen saturation, we begin to see the appearance of polymer which steadily increases with decreasing oxygen saturation. The total intracellular hemoglobin concentration also affects the amount of polymer formed. By examining polymer formation in fractionated subpopulations of SS erythrocytes at various density values (or intracellular hemoglobin concentrations) we demonstrated that the polymer fraction increased with increasing intracellular hemoglobin concentration for any given oxygen saturation.(ABSTRACT TRUNCATED AT 400 WORDS)
Anemia, Sickle Cell↗
The 5'-flanking sequences of human globin genes contribute to tissue specific expression.
The specificity of expression of the isolated 5'-flanking sequences of the human beta- and epsilon-globin genes was examined in the K562 human erythroleukemia cell line, the murine erythroleukemia (MEL) cell, and in nonerythroid cell lines CV-1, HeLa-S3, and WI-38. Globin flanking sequences were active only in the erythroid K562 and MEL cell lines. Furthermore, the upstream sequence of the adult, beta-globin gene was active in MEL cells which express adult globin and not in K562 cells which express embryonic and fetal globins suggesting that tissue specificity resides in these upstream sequences. No expression was observed in K562 cells which have an embryonic-fetal hemoglobin phenotype.
Acetyltransferases↗
Hemoglobin SC disease and alpha-thalassemia. Prolonged survival and mild clinical course.
An 86-year-old black woman admitted for an elective cataract extraction was found to have moderate hypochromic microcytic anemia. Hematologic evaluation disclosed the presence of hemoglobin SC disease and heterozygous alpha-thalassemia-2 (alpha alpha/alpha-). A red cell density profile of the patient's peripheral blood revealed an absence of the typical uniform shift toward higher-density values seen in hemoglobin SC disease, indicating a "normalization" in the distribution of intracellular hemoglobin types. It is suggested that the co-inheritance of hemoglobin SC disease and heterozygous alpha-thalassemia-2, probably by decreasing the tendency toward intracellular hemoglobin S polymerization, contributed to her prolonged survival and relatively mild clinical course.
Aged↗
Deoxyhemoglobin S polymer formation in sickle cells.
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Anemia, Sickle Cell↗
Chemical potential measurements of deoxyhemoglobin S polymerization. Determination of the phase diagram of an assembling protein.
We have used the "osmotic stress" method to determine the phase diagram of deoxyhemoglobin S polymerization. This method involves equilibration, through a semipermeable membrane, of the protein with solutions of inert polymers of known osmotic pressure. With deoxyhemoglobin A and S solutions, in which we have demonstrated achievement of equilibrium, plots of osmotic pressure versus concentration initially agree closely with the results of other methods of measurement of colligative properties. However, once the known solubility value is exceeded for the deoxyhemoglobin S solutions at various temperatures, there is a rapid rise in hemoglobin concentration over a narrow osmotic pressure range and then a more gradual increase in concentration. We believe that these two regions correspond, respectively, to the onset of the polymerization process, and of subsequent continuing growth and compression or alignment of polymer. We derive the thermodynamic values for these processes and show that the behavior of the deoxyhemoglobin S system is analogous to the phase transition for a simple chemical system. These results are relevant to understanding the intracellular polymerization of deoxyhemoglobin S in sickle cell disease, and these concepts are applicable to other protein assembly systems.
Gels↗