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Biomedical subjects

A N Schechter

Publications and source records attributed to A N Schechter.

At least 73 records · Page 4Linked to original sources
PubMed1992

Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation.

The rheological properties of normal erythrocytes appear to be largely determined by those of the red cell membrane. In sickle cell disease, the intracellular polymerization of sickle hemoglobin upon deoxygenation leads to a marked increase in intracellular viscosity and elastic stiffness as well as having indirect effects on the cell membrane. To estimate the components of abnormal cell rheology due to the polymerization process and that due to the membrane abnormalities, we have developed a simple mathematical model of whole cell deformability in narrow vessels. This model uses hydrodynamic lubrication theory to describe the pulsatile flow in the gap between a cell and the vessel wall. The interior of the cell is modeled as a Voigt viscoelastic solid with parameters for the viscous and elastic moduli, while the membrane is assigned an elastic shear modulus. In response to an oscillatory fluid shear stress, the cell--modeled as a cylinder of constant volume and surface area--undergoes a conical deformation which may be calculated. We use published values of normal and sickle cell membrane elastic modulus and of sickle hemoglobin viscous and elastic moduli as a function of oxygen saturation, to estimate normalized tip displacement, d/ho, and relative hydrodynamic resistance, Rr, as a function of polymer fraction of hemoglobin for sickle erythrocytes. These results show the transition from membrane to internal polymer dominance of deformability as oxygen saturation is lowered. More detailed experimental data, including those at other oscillatory frequencies and for cells with higher concentrations of hemoglobin S, are needed to apply fully this approach to understanding the deformability of sickle erythrocytes in the microcirculation. The model should be useful for reconciling the vast and disparate sets of data available on the abnormal properties of sickle cell hemoglobin and sickle erythrocyte membranes, the two main factors that lead to pathology in patients with this disease.

Anemia, Sickle Cell↗
PubMed1991

Cloning of the human erythropoietin receptor gene.

We have isolated and characterized a genomic clone of the human erythropoietin (Epo) receptor from a placental genomic library using a cDNA probe for the murine Epo receptor. The coding region spans about 6.5 kb with seven intervening sequences ranging in size from 81 bp to 2.1 kb. A stretch of 123 purines is found in the 5' region from -456 to -578 upstream from the first codon and flanking the Alu repetitive sequences located further upstream. The human Epo receptor contains a palindromic sequence 5' of the translated region that consists of an almost perfect inverted repeat of 12 nucleotides (CAGCTGC(G/C)TCCG) centered about G at -92 from the first codon. An inverted SP1 binding site (CCGCCC) and an inverted GATA-1 binding site (TTATCT) are located at positions -151 and -179, respectively, and CACCC sequences are located at -585 and further upstream. No TATA or CAAT sequences are in this 5' flanking region. However, this region as far as -275 has a 72% GC content compared with an overall GC content of 56%. A 1-kb BamHI fragment of the human Epo receptor containing 700 bp of sequences 5' of the coding region was transcribed in an in vitro transcription assay; initiation of transcription appeared to be around 132 +/- 5 just downstream from the inverted SP1 site at -151. T1 analysis of human Epo receptor messenger RNA also maps the site of transcription initiation to this region. Within 180 nucleotides 5' to the first exon are three regions with 70% or greater homology with the murine Epo receptor. The study of this gene, including its similarities with the murine Epo receptor, should help elucidate aspects of the transcriptional and possible translational control of the Epo receptor in human erythroid cells and thus its role in signal transduction and erythroid differentiation.

Amino Acid Sequence↗
PubMed1991

Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction.

We have developed a technique to diagnose the alpha- and beta-thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma-globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)-thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha-globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

Base Sequence↗
PubMed1991

Increased protein binding to a -530 mutation of the human beta-globin gene associated with decreased beta-globin synthesis.

Although some cases of the syndrome of hereditary persistence of fetal hemoglobin (HPFH) have been correlated with mutations causing a change in the binding of trans-acting factors to DNA sequences flanking the gamma-globin gene, this mechanism has not been described in beta-thalassemias upstream of the canonical promoter of the beta-globin gene. In this report we describe such a change in binding of a protein that may explain a silent carrier phenotype of beta-thalassemia. We have previously demonstrated the binding of a protein (BP1) derived from a nuclear extract of human K562 cells to DNA 5' to the human beta-globin gene in a region having a negative regulatory function. The binding of BP1 in this region can be detected by DNAse I footprinting and by gel mobility shift analysis. We have now compared binding of BP1 to the normal sequence and a mutated sequence (+ATA/-T at -530 bp from the cap site) from the silent carrier of beta-thalassemia. Using mobility shift assays we show that BP1 binds about nine times more strongly to the mutated sequence than the normal sequence. These results suggest the possibility that the decreased expression of the beta-globin gene exhibited by the carrier may be due, at least in part, to tighter binding of a protein which functions as a negative control element or repressor.

Base Sequence↗
PubMed1991

Effects of alpha-thalassemia and sickle polymerization tendency on the urine-concentrating defect of individuals with sickle cell trait.

A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity of the concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal alpha-globin genotype (alpha alpha/alpha alpha), and those who were either heterozygous (-alpha/alpha alpha) or homozygous (-alpha/-alpha) for gene-deletion alpha-thalassemia, because alpha-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the alpha alpha/alpha alpha genotype than in the -alpha/alpha alpha or -alpha/-alpha genotypes (P less than 0.05). After dDAVP, the urine osmolality was greater in patients with the -alpha/-alpha genotype than with the -alpha/alpha alpha genotype (882 +/- 37 vs. 672 +/- 38 mOsm/kg H2O) (P less than 0.05); patients with the -alpha/alpha alpha genotype had greater concentrating ability than individuals with a normal alpha-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P less than 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and O (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the alpha-globin genotype.

Adult↗
PubMed1990

Hematologic responses of patients with sickle cell disease to treatment with hydroxyurea.

Because fetal hemoglobin contains gammaglobin chains instead of beta chains, it is not affected by the genetic defect that causes sickle cell disease. Increased levels of fetal hemoglobin decrease the tendency toward intracellular polymerization of sickle hemoglobin that characterizes this disease. Hydroxyurea is one of several cytostatic agents that have been shown to increase the production of fetal hemoglobin in some patients with sickle cell disease. We studied the effects of hydroxyurea administration in 10 hospitalized patients with sickle cell disease, each of whom was treated for three months. Seven patients responded with a 2- to 10-fold increase in fetal hemoglobin, from a mean (+/- SD) of 1.6 +/- 1.6 percent of total hemoglobin to 6.8 +/- 4.7 percent; three patients had fetal-hemoglobin levels of 10 to 15 percent of total hemoglobin. Three did not respond to treatment. Four of the patients who responded were retreated with hydroxyurea after one to four months without treatment and were found to have larger increases in fetal-hemoglobin levels. In most patients, levels were still rising at the end of the study, even after 90 days of therapy. Fetal-hemoglobin levels tended to peak at dosages of hydroxyurea that were myelosuppressive. In the patients who responded to treatment, there were significant increases in the percentage of reticulocytes and erythrocytes containing fetal hemoglobin and in the amount of fetal hemoglobin within these cells. The percentage of dense red cells decreased in the patients who responded to treatment. The tendency toward intracellular polymerization at physiologic oxygen saturation was reduced by about 33 percent in the cells containing fetal hemoglobin, whereas there was no change in the other cells. We conclude that hydroxyurea is effective in increasing the production of fetal hemoglobin, which in this study was found to be associated with a small decrease in hemolysis and an increase in hemoglobin levels despite myelosuppression. Controlled, prospective trials are necessary to establish whether these effects will lead to clinical benefit.

Adult↗
PubMed1990

Microcirculatory adaptations in sickle cell anemia: reactive hyperemia response.

With the technique of laser-Doppler velocimetry, cutaneous blood flows in the forearm of patients with stable sickle cell disease after graded periods of proximal ischemia were compared with normal subjects matched for age, race, and sex, and with patients with anemia caused by beta(+)-thalassemia. In the sickle cell patients the reactive hyperemia was characterized by an increased time interval between the release of the occlusion and the peak amplitude response (time-to-peak) and by a greater period of blood flow above the base-line value (payback ratio) compared with controls. In addition, prolongation of the occlusion period led to an augmentation in the magnitude of the characteristic basal flow oscillations or an induction of this phenomenon at sites not exhibiting it before ischemia. Base-line or ischemia-provoked flow oscillations of either this magnitude or frequency were only observed in normal or thalassemic controls during brief intervals in the rapidly decaying portion of the hyperemic response and in one subject with homozygous hemoglobin C disease. These results would support a model of a local integrative control of microcirculatory blood flow, which appears to become augmented, synchronized, and sustained in sickle cell subjects.

Adaptation, Physiological↗
PubMed1989

A common protein binds to two silencers 5' to the human beta-globin gene.

The temporal sequence of expression of human globin genes during development suggests precise regulation of these genes. Recent studies have characterized a number of DNA sequences within or flanking the human beta-globin gene which are important in its regulation and several proteins which bind to these sequences have been identified. We have found two proteins which bind 5' to the human beta-globin gene. One of these proteins, which we designate BP1, binds to two sequences, one between -550 and -527 bp relative to the cap site, the other between -302 and -294 bp. A second protein, BP2, binds to sequences between -275 and -263 bp. The binding sites for both BP1 and BP2 are in two regions which function as silencers in a transient expression assay using the human erythroleukemia cell line K562. These results and others presented here suggest that BP1 may act as a repressor protein. Negative regulation seems to be an important component of tissue and developmental specific globin gene regulation.

Animals↗
PubMed1989

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.

Adenovirus Early Proteins↗
PubMed1989

Identification of a transcriptional silencer in the 5'-flanking region of the human epsilon-globin gene.

We have studied the 5'-flanking sequences required for the transcriptional regulation of human epsilon-globin gene expression. A series of deletion mutants of the human epsilon-globin gene 5'-flanking sequences were constructed and linked to the bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in HeLa cells and the human erythroleukemia K-562 cells. By measuring chloramphenicol acetyltransferase activities and mRNA levels we found that the sequence between -177 and -392 base pairs (bp) relative to the mRNA initiation site exerts a negative effect on epsilon-globin promoter activity. This effect is more pronounced in HeLa cells compared with K-562 cells. To further characterize the negative control region we cloned the DNA sequence between -177 and -392 bp either 5' or 3' of the epsilon-globin promoter and in either orientation. Our data indicate that this negative control region inhibits the epsilon-globin promoter activity in a position- and orientation-independent manner, thus suggesting that it is a silencer. In addition, the silencer also inhibits the expression from the Herpesvirus thymidine kinase promoter. Sequence comparison reveals that there are three short regions within the silencer that share extensive homology with those found in other negative control DNA elements. Our results therefore indicate that an upstream silencer element is present in the epsilon-globin gene and that it may play an important role in the control of epsilon-globin gene expression during development.

Base Sequence↗
PubMed1989

Colloquium on scientific authorship: rights and responsibilities.

The Colloquium on Scientific Authorship was held at the National Institutes of Health (NIH) at a time of extraordinary scrutiny by the public of the ethics of scientists, as represented by intense interest of the press and the Congress of the United States. Indeed, several regulations dealing with scientific misconduct have been proposed during the last year in the Federal Register, and new legislation has been proposed in the Congress. As a result of these concerns, conferences have been organized by the Institute of Medicine, the American Association for the Advancement of Science/American Bar Association, the Council of Biology Editors, and other groups. The colloquium at NIH, which was held May 31, 1988, and sponsored by the Intramural Scientists, focused on publication practices, especially multiple authorship, as contributing to perceived difficulties. The participants suggested various changes in conventions related to authorship that might help prevent future problems.

Authorship↗
PubMed1989

Intracellular polymerization. Disease severity and therapeutic predictions.

The extent of intracellular polymerization of hemoglobin S, leading to loss of erythrocyte deformability and eventual morphological sickling, is primarily determined by oxygen saturation and intracellular hemoglobin concentration and composition. Epidemiological analysis of sickle cell disease severity among the sickle syndromes and studies of the biophysics of intracellular polymerization were used to estimate the potential clinical benefit of various therapeutic strategies. These strategies include those designed to increase deoxyhemoglobin S solubility; to increase erythrocyte volume or water content, thereby reducing the intracellular hemoglobin concentration; or, most recently, to decrease the proportion of hemoglobin S by increasing the amount of non-S hemoglobin. Increasing levels of hemoglobin F is of particular interest due to its "sparing" effect in inhibiting polymerization, the well-characterized epidemiological associations of high levels of hemoglobin F with reduced disease severity, and recent studies of drug-induced increases in hemoglobin F. Our analyses of equilibrium polymer formation at physiological oxygen saturation values suggest that small decreases in polymer formation at intermediate levels of hemoglobin F may give rise to a small decrease in anemia (as associated with homozygous alpha-thalassemia coexistent with sickle cell anemia), but that greater reductions in polymer formation may be necessary to effect a significant improvement in disease severity. Current studies of hydroxyurea-induced increases of hemoglobin F give cautious optimism that therapeutically useful levels may be attainable.

Anemia, Sickle Cell↗
PubMed1989

Hemodynamic studies in sickle cell disease.

With the availability of these noninvasive imaging modalities, one can now obtain more objective information about microcirculatory pathophysiology. As a result, previously held pathophysiologic paradigms in sickle cell disease will undoubtedly require modification or abandonment. These approaches should be viewed as supplements, not substitutes, for more traditional evaluations. Nonetheless, since these techniques are amenable to sequential application, these approaches promise to define disease severity more precisely, in quantitative terms, and should provide the means to follow the results of therapy unambiguously.

Anemia, Sickle Cell↗