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Biomedical subjects

A N Schechter

Publications and source records attributed to A N Schechter.

At least 109 records · Page 6Linked to original sources
PubMed1985

Irreversibly sickled erythrocytes in sickle cell anemia: a quantitative reappraisal.

Irreversibly sickled cells (ISCs), considered by some to be of major pathophysiologic significance, have been reported to comprise between 5-50% of the total red cell population in patients with homozygous sickle cell anemia. Since the deformation of erythrocytes containing sickle hemoglobin is highly dependent on the concentration of hemoglobin in the deoxy conformation, any method established to enumerate the true ISC count requires the hemoglobin to be in the full oxy or liganded conformation. Because the oxygen dissociation curve for sickle erythrocytes is significantly shifted to the right, extremely high partial pressures of oxygen are required to approach full saturation. On the other hand, fully liganding the hemoglobin in the oxy conformation with carbon monoxide (CO) can be readily accomplished. We found that there is a significant reduction in the average number of sickled forms in the peripheral blood of sickle cell anemia patients after incubation in CO (to a value of 6.5 +/- 3.5%) when compared to conventional methods for ISC preparations. These results suggest that fully liganded erythrocytes should be used in quantitating ISCs in studies of the pathophysiology of this disease, especially since ISCs are likely to affect rheology differently from reversibly sickling cells.

Anemia, Sickle Cell↗
PubMed1985

Noninvasive techniques to evaluate the vaso-occlusive manifestations of sickle cell disease.

Although the pathophysiologic manifestations of sickle cell disease have been assumed to result from microvascular occlusion consequent to in situ sickling of erythrocytes, actual blood vessel obstruction have been rarely demonstrated in vivo. Recent observations utilizing sophisticated biophysical techniques to study the intracellular hemoglobin S polymerization process has led to major revisions in this previously held pathophysiologic paradigm, but in vivo correlations are still lacking. With the development of new noninvasive imaging and para-imaging methods, it is now technically possible and feasible to characterize both regional organ perfusion and tissue biochemistry in quantitative terms. In addition, these modalities promise to clarify pathogenesis of the disease through definition of the events responsible for the progression from tissue ischemia and infarction through the resolution phase. Since these noninvasive techniques are amenable to sequential applications, they should facilitate objective evaluations of clinical trials of therapeutic agents designed to prevent or delay the vaso-occlusive manifestations of sickle cell disease.

Anemia, Sickle Cell↗
PubMed1985

Conjunctival sign in sickle cell anaemia: an in-vivo correlate of the extent of red cell heterogeneity.

A consecutive series of 22 stable adult inpatients with sickle cell anaemia were examined for the presence and severity of spontaneous 'comma' signs of the conjunctiva. Fifteen patients had severe conjunctival signs (more than 10 commas in the worse eye). The presence of severe conjunctival signs was associated with a broader distribution of intraerythrocytic haemoglobin concentrations (p = 0.0005). The patient group with severe conjunctival signs was not found to be significantly different from the group without such signs for age, sex, haemoglobin value, reticulocyte count, alpha-globin gene number, percentage fetal haemoglobin, or the proportion of very dense cells (CHC greater than 37 g/dl). Thus the singular heterogeneity of the erythrocytes in sickle cell disease may be indicative of the factor(s) responsible for the diagnostic comma sign.

Adult↗
PubMed1985

Alpha thalassemia changes erythrocyte heterogeneity in sickle cell disease.

Homozygous alpha-thalassemia has the beneficial effect in sickle cell anemia of reducing the hemolytic severity while changing several other hematological parameters. We examined in detail the cellular basis of some of these hematologic alterations. We find that the broad distribution in erythrocyte density and the large proportion of dense cells associated with sickle cell anemia are both reduced with coexisting alpha-thalassemia. Measurements of glycosylated hemoglobin levels as a function of cell density indicate that the accelerated increase in cell density, beyond normal cell aging, in sickle cell anemia is also reduced with alpha-thalassemia. The patients with homozygous alpha-thalassemia and sickle cell disease have slightly lower levels of hemoglobin F than the nonthalassemic sickle cell patients. Examination of hemoglobin F production revealed that the proportion of hemoglobin F containing reticulocytes remained unchanged, as did the proportion of hemoglobin F in cells containing hemoglobin F (F cells). Preferential survival of F cells occurs in sickle cell anemia, with or without alpha-thalassemia, and the slight difference in hemoglobin F levels appear to reflect differences in numbers of circulating F cells. Thus, in sickle cell disease with coexisting alpha-thalassemia, the change in the erythrocyte density profile, possibly due to inhibition of polymerization-related increases in cell density, explains the hematological improvement.

Anemia, Sickle Cell↗
PubMed1985

Hemoglobin S polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes.

We examined the extent to which the intracellular polymerization of sickle hemoglobin (HbS) can account for the severity of anemia and of vaso-occlusive manifestations in the various sickling syndromes. Polymer formation in sickle cell disease depends principally on the intraerythrocytic hemoglobin composition and concentration. In our studies, the polymer fraction in sickle red cells was determined from reported mean values for hemoglobin composition and mean corpuscular hemoglobin concentration (MCHC) in 12 groups of patients with sickle hemoglobinopathies (homozygotes for HbS, with and without coexistent alpha-thalassemia or various forms of the hereditary persistence of fetal hemoglobin [HPFH], beta+-, beta 0-, and delta beta-thalassemia, and heterozygotes for HbS with HbA). The calculated HbS polymer fractions at full deoxygenation and at physiologic oxygen saturation values were closely correlated with mean blood hemoglobin concentrations. In addition, polymer fraction correlated with the ranking of the sickling syndromes by vaso-occlusive severity. We find that polymer fraction accounts for about 80% of the variability in hemolytic and clinical severity. The method of analysis presented here provides a quantitative and systematic means of assessing the role of polymer formation in the pathophysiologic manifestations of the sickling syndromes. Our results support the hypothesis that the intracellular polymerization of HbS is the primary determinant of the severity of both anemia and clinical symptomatology in the sickle hemoglobinopathies.

Anemia, Sickle Cell↗
PubMed1985

Cell heterogeneity in sickle cell disease: quantitation of the erythrocyte density profile.

An increasing body of experimental evidence demonstrates that intracellular hemoglobin concentration and composition is a primary determinant of pathophysiology in sickle cell disease. To quantitate more precisely the heterogeneous distribution of intracellular hemoglobin concentrations in a given individual with this disease, we have calibrated the phthalate ester separation technique by using discontinuous Stractan density gradients to isolate subpopulations of red cells of relatively uniform corpuscular hemoglobin concentration values. We find that blood from individuals with sickle cell anemia exhibits a markedly broader distribution of corpuscular hemoglobin concentration values, containing both very light and very dense cells, than the red cell density profile from normal individuals. This increased breadth of cell densities in patients with sickle cell anemia remains even after exclusion of the very light and very dense subpopulations. In patients with stable sickle cell anemia, there appears to be minimal variation in the distribution of cell densities that are unimodal but skewed toward higher density values. The phthalate ester method can conveniently be used to follow changes in cell densities during vaso-occlusive events, to monitor therapy targeted at modifying intracellular hemoglobin S concentrations, and in sequential applications in large field trials designed to determine the relationship between red cell heterogeneity and specific manifestations of the sickle cell syndromes.

Adult↗
PubMed1984

Periodic microcirculatory flow in patients with sickle-cell disease.

We have applied the technique of laser-Doppler velocimetry to compare patterns of cutaneous blood flow in the forearms of patients with stable sickle-cell disease, with the patterns in normal subjects matched for age, race, and sex, and in patients with anemia due to beta+-thalassemia. The mean resting blood flow in the patients with sickle-cell disease was comparable to that of the control groups but was associated with large, local oscillations in flow with periods of 7 to 10 seconds and peak-to-trough magnitudes about half the mean flow. Oscillations occurred simultaneously at sites separated by 1 cm but were independent in phase and frequency. Since these laser-Doppler measurements represent the average flow pattern in about 1 mm3 of skin (i.e., in approximately 50 to 70 capillary loops), these results suggest that microcirculatory flow in patients with sickle-cell disease proceeds by synchronization of rhythmic flow in large domains of microvessels. These findings indicate that periodic flow may be a compensatory mechanism to offset the deleterious altered rheology of erythrocytes containing polymerized hemoglobin S, and suggest that laser-Doppler velocimetry may be a useful method to investigate microvascular physiology in patients with sickle-cell disease.

Adult↗
PubMed1984

A beta-globin gene, inactive in the K562 leukemic cell, functions normally in a heterologous expression system.

The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for beta-globin; failure to produce beta-globin could result from an acquired mutation in each of the beta-globin genes or from an alteration in the regulatory factor environment of the beta-globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 beta-globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 beta-globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the beta-globin gene showed that K562 cells contain two different beta-globin alleles, both of which are inactive. A K562 beta-globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 beta-globin gene was transcribed in COS cells as efficiently as a normal beta-globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5' and 3' termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of beta-globin gene expression results not from an alteration in the beta-globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in beta-globin gene expression.

Alleles↗
PubMed1984

Preparation and properties of nickel hemoglobin.

Hemoglobin A reconstituted with nickel protoporphyrin IX (NiHbA) has been prepared and characterized. Kinetics of its reaction with p-mercuribenzoate and with haptoglobin, absorption and circular dichroism spectra, and x-ray crystallographic properties have been investigated as probes of its structural conformation. The results suggest that NiHbA exists in a structure that is similar to the deoxy, or T-state of HbA. It is proposed that NiHbA and its derivatives may serve as a useful model for future studies of hemoglobin allosteric changes.

Crystallization↗
PubMed1984

Control of oxygen affinity of hemoglobin in K562 cells induced by hemin.

The oxygen affinity of hemoglobin in K562 cells induced by hemin and the relationship between levels of 2,3-diphosphoglycerate (2,3-DPG) and hemoglobin have been investigated. Absorption spectra of induced cells indicate that the hemoglobin is oxygenated; oxygen dissociation curves are symmetric, with a P50 of 20 +/- 0.9 mm Hg, Hill coefficient of 2.5, and a normal temperature dependence. The intracellular pH measured by phosphorus 31 nuclear magnetic resonance (NMR) was 7.3. The amount of 2,3-DPG was determined by an enzymatic method and by 31P NMR. The level of 2,3-DPG in uninduced K562 cells, containing 0.5 pg of hemoglobin per cell, was low (5 +/- 0.5 mumole/10(8) cells), but increased to 64 +/- 5 mumole/10(8) cells upon induction of hemoglobin accumulation (to a final level of 20 pg hemoglobin/cell). For several experiments, there was a closely coordinated relationship between 2,3-DPG and hemoglobin levels, at about 1:1 stoichiometry of the two molecules. The time course of induction of hemoglobin, and of 2,3-DPG levels, are very similar; both processes are reversible. These data suggest that induction of hemoglobin synthesis in K562 cells by hemin results in hemoglobin-containing cells with normal oxygenation properties and that 2,3-DPG and hemoglobin levels are coordinately controlled in these cells. Elucidation of the mechanism of this effect should be of importance in understanding the erythroid-like differentiation of these cells.

Adenosine Triphosphate↗
PubMed1984

Non-uniformity of intracellular polymer formation in sickle erythrocytes: possible correlation with severity of hemolytic anemia.

Intact sickle erythrocytes were fractionated to obtain a more uniform population in mean corpuscular hemoglobin concentration (MCHC). Polymer fraction at complete deoxygenation varied with increasing MCHC and, at varying oxygen saturation, was in good agreement with theoretical predictions based on solubility and the non-ideal behavior of concentrated hemoglobin solution and of water. These data and the theoretical analysis indicate that, in uniform sickle erythrocytes at 34 g/dl, polymer is only detected below 84% oxygen saturation. Non-uniformity of unfractionated sickle erythrocytes causes this critical oxygen saturation to shift to higher values (over 90%). The implication is that polymer may exist in arterial blood, a possibility which must be considered in therapeutic strategies. Recently, we showed that polymer formation correlated with the severity of hemolysis for 12 sickle syndromes varying from sickle trait to hemoglobin SS (African) disease.

Anemia, Sickle Cell↗
PubMed1983

Receptor-mediated endocytosis of transferrin in K562 cells.

Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.

Ammonium Chloride↗
PubMed1983

Inducible transcription of five globin genes in K562 human leukemia cells.

We studied the abundance and structure of globin mRNAs present in K562 cells both before and after induction of hemoglobin synthesis by hemin. In vitro translation of poly(A)+ RNA from K562 cells generated protein products corresponding to alpha, A gamma-, G gamma-, epsilon-, and zeta-globin mRNAs. Individual globin mRNAs increased 1.5- to 3-fold after induction. Similar results were obtained by measuring steady-state mRNA concentrations of induced and uninduced cells by using S1 nuclease mapping. Globin gene transcripts were correctly initiated and processed. In addition, S1 nuclease analysis revealed the presence of delta-globin mRNA in both control and induced cells. A small percentage of delta-globin transcripts appeared to be initiated upstream from the normal initiation site. beta-Globin mRNA was not detected in any studies. The results (i) suggest that hemin induction of K562 cells is mediated at a transcriptional level and (ii) reveal the dissociation of delta- and beta-globin gene expression in K562 cells compared with normal erythroid cells.

Cell Line↗
PubMed1983

Intracellular polymerization of sickle hemoglobin. Effects of cell heterogeneity.

To determine the extent to which the broad distribution in intracellular hemoglobin concentrations found in sickle erythrocytes affects the extent of intracellular polymerization of hemoglobin S, we have fractionated these cells by density using discontinuous Stractan gradients. The amount of polymer formed in the subpopulations was experimentally measured as a function of oxygen saturation using 13C nuclear magnetic resonance spectroscopy. The results for each subpopulation are in very good agreement with the theoretical predictions based on the current thermodynamic description for hemoglobin S gelation. We further demonstrate that the erythrocyte density profile for a single individual with sickle cell anemia can be used with the theory to predict the amount of polymer in unfractionated cells. We find that heterogeneity in intracellular hemoglobin concentration causes the critical oxygen saturation for formation of polymer to shift from 84 to greater than 90%; polymer is formed predominantly in the dense cells at the very high oxygen saturation values. The existence of polymer at arterial oxygen saturation values has significance for understanding the pathophysiology of sickle cell anemia. The utility of these techniques for assessing various therapeutic strategies is discussed.

Anemia, Sickle Cell↗
PubMed1983

5-Azacytidine increases gamma-globin synthesis and reduces the proportion of dense cells in patients with sickle cell anemia.

We previously demonstrated that 5-azacytidine can selectively increase gamma-globin synthesis in a patient with beta +-thalassemia, prompting us to treat two patients with sickle cell anemia and two additional patients with beta + thalassemia. 5-Azacytidine (2 mg/kg/day) was continuously infused for 7 days with no apparent clinical toxicity. The gamma/beta-globin biosynthetic ratio increased fourfold to sixfold in the bone marrow cells of each patient after treatment and remained elevated for 7-14 additional days. Hypomethylation of DNA near the gamma-globin genes in bone marrow cells was demonstrated 2 days after beginning the 5-azacytidine infusion. The peripheral blood fetal hemoglobin (HbF) level increased from 6.0% to 13.7% in one patient with sickle cell anemia and from 1.6% to 8.9% in the second. Stractan gradient analyses of peripheral blood from patients with sickle cell anemia revealed a marked decrease in the percentage of dense cells (cells that contain increased amounts of HbS polymer when deoxygenated) following treatment. These observations provide an impetus to investigate the effects of repeated courses of 5-azacytidine in a small group of severely ill patients to determine whether this drug may have a role in the treatment of patients with sickle cell anemia and beta-thalassemia.

Adult↗