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Biomedical subjects

A N Schechter

Publications and source records attributed to A N Schechter.

At least 127 records · Page 7Linked to original sources
PubMed1983

DNA methylation and globin gene expression in patients treated with 5-azacytidine.

5-Azacytidine, a cytidine analog, stimulated fetal hemoglobin synthesis in five patients who had either severe beta-thalassemia or sickle cell anemia. After treatment, a reduction in the frequency of methylated cytosine residues was observed at all Hpa II sites examined. Despite causing "global" hypomethylation, 5-azacytidine augmented the synthesis of gamma-globin only. Although gamma-gene hypomethylation and increased gamma-gene expression seem to be linked, hypomethylation near other genes was not sufficient to activate transcription. These data suggest that the gamma genes lie in a unique "preactivational" state responsive to hypomethylation, and that other genes are repressed in bone marrow cells by different mechanisms. DNA hypomethylation and an increased concentration of gamma-mRNA were observed in bone marrow cells 2 days after initiation of treatment, indicating that 5-azacytidine may act directly on differentiated erythroid precursors. This compound probably affects early erythroid progenitors as well, since an increased level of gamma-globin synthesis persists for 1-2 weeks after the drug is stopped. A direct effect on erythroid progenitors was also suggested by in vitro assays: Erythroid colonies derived from progenitor cells obtained on day 2 of treatment produced more Hb F than colonies derived from progenitors obtained before 5-azacytidine was given.

Azacitidine↗
PubMed1983

Abundance and structure of globin mRNA in K562 human leukemia cells.

K562 cells can be reversibly induced by hemin to accumulate embryonic and fetal hemoglobins. The increase in individual globin mRNAs accompanying induction was measured by isolation and in vitro translation of poly A+ RNA from both control and induced cells. epsilon-Globin mRNA showed the greatest increase (threefold) with hemin induction, followed by zeta greater than gamma greater than alpha. These results were corroborated by measuring steady-state mRNA concentrations by a recently developed S1 nuclease mapping procedure. Changes in epsilon-, gamma-, and alpha-globin levels parallel the changes measured by in vitro translation. S1 nuclease mapping studies revealed that epsilon-, gamma-, and alpha-globin transcripts were correctly initiated and processed. In addition, correctly initiated and processed delta-globin transcripts were detected at low levels, increasing 1.5-fold following hemin induction. However, no beta-globin transcripts could be detected by this sensitive and specific assay. The beta-globin gene may be under developmental control in K562 cells, and this cell line may therefore provide a unique assay system for molecules involved in globin gene regulation.

Animals↗
PubMed1983

The effect of phenylalanine derivatives on the solubility of deoxyhemoglobin S. A model class of gelation inhibitors.

Among the class of non-covalent inhibitors of deoxyhemoglobin S gelation, the aromatic amino acids have been shown to be the most effective. We have examined several synthetic chemical modifications of phenylalanine in order to determine the stereospecific constraints for inhibition of gelation by this class of compounds. The phenylalanine derivatives with ring modification by electron-donating groups (NH2, CH3, or OH) inhibited gelation to the same order of magnitude as phenylalanine (10-20% increase in deoxyhemoglobin S solubility at 32 mM). The phenylalanine derivative with the electron-withdrawing group NO2 in the p-position behaved similarly, but the inhibitory effect was eliminated by NO2 in the m- and possibly o-positions. Furthermore, side-chain modifications also eliminated the inhibitory effect. These studies, in conjunction with crystallographic analyses of the binding sites of gelation inhibitors, may provide a rational strategy for finding suitable compounds (whether covalent or non-covalent inhibitors) with appropriate physicochemical and biological properties to pursue as potential therapies with sickle cell disease.

Anemia, Sickle Cell↗
PubMed1982

Molecular and cellular pathogenesis of hemoglobin SC disease.

Solution and cell studies were performed to ascertain why individuals with hemoglobin (Hb) SC have disease whereas those with Hb AS do not. The polymerization of deoxygenated mixtures containing sickle cell Hb (Hb S; alpha 2 beta 2(6)Glu leads to Val) and Hb C (alpha 2 beta 2(6)Glu leads to Lys) was investigated by measurements of delay times and solubilities. In mixtures containing more than 40% Hb S, polymerization takes place by the same mechanism as in solutions of Hb S alone, with no evidence for independent crystallization of Hb C. A detailed comparison of Hb S/Hb C and Hb S/Hb A mixtures with identical concentrations and proportions of Hb S show that there is no significant difference in the tendency of Hb C and Hb A to copolymerize with Hb S. In 50:50 Hb S/Hb C mixtures, polymerization is about 15 times more rapid than in 40:60 Hb S/Hb A mixtures at the same total Hb concentration. Measurements on density-fractionated erythrocytes show that SC cells contain a higher total Hb concentration and a more uniform distribution of reticulocytes compared to normal (AA) or sickle trait (AS) cells. The concentration distribution for C trait (AC) cells is much closer to that of SC cells than to AS or AA cells. It appears, therefore, that the presence of Hb C results in the SC cell beginning its life with an abnormally high Hb concentration. From these findings we conclude that both the larger proportion of Hb S and the higher intracellular Hb concentration contribute to the pathogenesis of Hb SC disease.

Anemia, Sickle Cell↗
PubMed1982

The solubility of hemoglobins A and S reconstituted with various metalloporphyrins.

The solubilities of hemoglobins A and S, reconstituted with various metalloporphyrins, in concentrated phosphate solutions are reported. In HbA the metallo-substituted derivatives have solubilities identical to Fe(II)HbAO2, except for PorHbA, which is less soluble. In HbS the central metal influences the solubility in the order Fe(II)HbSO2 greater than Cu(II)HbS greater than Ni(II)HbS greater than Zn(II)HbS approximately of porphyrinHbS greater than Fe(II)HbS. The changes in solubility properties of metallo-substituted HbS is probably related to conformational changes which alter interactions among hemoglobin molecules.

Hemoglobin A↗
PubMed1982

Determination of sickle hemoglobin polymer in SS and AS erythrocytes.

Using 13C nuclear magnetic resonance techniques, we have studied the polymerization of hemoglobin S and intracellular polymerization in SS and AS erythrocytes. For SS erythrocytes, polymer could be detected at high oxygen saturations (greater than 90%) and increased with decreasing oxygen saturation to 70% of the total hemoglobin at complete deoxygenation. These results are consistent with the non-ideal behavior of hemoglobin S at the high intracellular concentration of 34 g/dl. The amount of polymer in AS erythrocytes was also measured as a function of oxygen saturation and is in agreement with the amount of polymer determined for homolysates using ultracentrifuge sedimentation. In general, these results on intracellular gelation are in good agreement with results predicted by cell-free studies extrapolated to the high intracellular hemoglobin concentration (34 g/dl). The significance of the detection of polymer at high oxygen saturation of SS erythrocytes is discussed in terms of morphologic deformation and erythrocyte transit through the circulation. We propose that intracellular polymerization is important in abnormal cell rheology and manifestation of disease.

Anemia, Sickle Cell↗
PubMed1981

Polymerization of hemoglobin in sickle trait erythrocytes and lysates.

We report 13C/1H magnetic double resonance measurements of polymer in sickle trait erythrocytes and direct measurements of solubility on lysates of these cells as a function of oxygen saturation. Polymerized hemoglobin is detected by these two methods only at oxygen saturation values below 60% and increases to 0.4 of the total hemoglobin at complete deoxygenation. These results confirm the validity of the NMR approach in determining values of polymer fraction. We have also found that the thermodynamic theory, that successfully describes gelation in sickle erythrocytes, does not predict the experimental results for sickle trait erythrocytes and must be refined.

Anemia, Sickle Cell↗
PubMed1981

Induction of hemoglobin accumulation in human K562 cells by hemin is reversible.

Twenty micromolar hemin causes no change in the rate of division of K562 cells but results in accumulation of 11 to 14 picograms of embryonic and fetal hemoglobins per cell. This effect is reversible, and hemoglobin induction in response to hemin, and loss of hemoglobin upon removal of hemin, can be cyclically repeated. The cells can be indefinitely subcultured in the presence of the inducer. Thus, the control of hemoglobin levels in K562 cells does not depend on irreversible differentiation.

Cell Differentiation↗
PubMed1981

Inhibitors of cell division reversibly modify hemoglobin concentration in human erythroleukemia K562 cells.

The human leukemia K562 cell line can be induced by 20 micro M hemin to reversibly accumulate embryonic and fetal hemoglobins without any change in the rate of cell division. When we reduced the rate of cell division by glutamine starvation or addition of hydroxyurea, the cells increased by tenfold the basal hemoglobin level of 0.3-0.5 pg Hb/cell. The combined effects of hemin and inhibitors of cell division permitted K562 cells to attain levels of hemoglobin (26-34 pg Hb/cell) close to that found in normal red cells. This superinduction was reversible and cells could be recycled indefinitely. Furthermore, electrofocusing experiments show that the three primary hemoglobin species produced by these cells (Hb Gower 1, Hb Portland, and fetal Hb), were induced, or reinduced, synchronously by inhibitors of cell division but asynchronously by hemin. Differing effects of hemin and inhibitors of cell division were observed in the absence of irreversible differentiation and suggest different molecular mechanisms controlling globin gene expression.

Cell Transformation, Neoplastic↗
PubMed1980

Tritium labeling of proteins to high specific radioactivity by reduction methylation.

The third component of human complement, hemoglobin A2, and IgG have been radiolabeled with tritium to specific activities of 179, 103, and 89 Ci/mmol, respectively, by reductive methylation. The labeling procedure is mild, requiring only brief exposure to formaldehyde (10 to 12 mM) and tritiated sodium borohydride (3 to 5 mM), and specific for the alpha amino groups of NH2-terminal residues and epsilon-amino groups of lysyl residues. The extent of modification for each protein ranged between 5 and 16% at available amino groups based on a stoichiometric reactivity of 2 mol of formaldehyde/mol of primary amine. Although the degree of substitution required to obtain these specific activities was high, no significant loss of antigenic or biological activities was apparent. Each labeled protein was 90 to 95% precipitable with antiserum elicited to the unreacted protein. The hemolytic activity of C3 was reduced by only 16%; the oxygen affinity of HbA2 was minimally decreased, and the phosphorylcholine binding affinity of an IgA myeloma protein (TEPC-15) was not significantly affected. Precise double antibody radioimmunoassays were developed with each tritiated protein capable of detecting less than 10(-8) g of antigen. Labeled protein samples have been stored for time periods up to 1 year without appreciable deterioration.

Complement C3↗
PubMed1980

Third component of human complement: appearance of a sulfhydryl group following chemical or enzymatic inactivation.

Treatment of human C3 with hydroxylamine or hydrazine at physiological pH and ionic strength totally abrogates the intrinsic ability of this protein to sustain classical pathway induced hemolysis of sheep red blood cells. Concomitant with the loss of this function the appearance of a single sulfhydryl group can be followed by titration with the sulfhydryl-specific reagents p-(chloromercuri)benzoate, [1-14C]iodoacetamide, 2,2'-dipyridyl disulfide, and 5,5'-dithiobis(2-nitrobenzoic acid). These reagents have also been used to follow the appearance of a free sulfhydryl group on conversion of C3 to C3b with bovine trypsin. Autoradiography of the electrophoretogram of separated alpha-, alpha'-, and beta-polypeptide chains of inactivated, [1-14C]carboxamidomethylated C3 samples has shown that the reactive sulfhydryl group is present in the alpha chain of C3 and in the alpha' chain of C3b, respectively. Digestion of the radiolabeled protein with porcine elastase has localized this sulfhydryl group to a 28 000-dalton fragment of the alpha chain with immunochemical and functional reactivities of the C3d domain. Autoradiographic analysis of a hydrolysate prepared from radioalkylated C3 and subjected to high-voltage paper electrophoresis has shown the labeled amino acid to be [1-14C]-S-(carboxymethyl)cysteine. The susceptibility of native C3 to rapid and irreversible inactivation by nitrogen nucleophiles with the parallel appearance of a cysteinyl residue may indicate the presence of an internal thiol ester. The relationship of the proposed thiol ester to the ability of nascent C3b to acylate cell surface components and carbohydrate polymers is discussed within the context of a transesterification reaction.

Chloromercuribenzoates↗