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Biomedical subjects

E J Benz

Publications and source records attributed to E J Benz.

At least 91 records · Page 5Linked to original sources
PubMed1982

Abnormally spliced messenger RNA in erythroid cells from patients with beta+ thalassemia and monkey cells expressing a cloned beta+-thalassemic gene.

The reduced beta-globin synthesis characterizing the beta+ thalassemia phenotype has been shown to be caused by anomalous processing within the small intervening sequence (IVS1) of the beta-globin mRNA precursor. The beta-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3' junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3'-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with beta+ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned beta+-thalassemic gene preferentially generated the anomalously spliced RNA when expressed in monkey kidney cells. The anomalously spliced RNA constituted approximately 80%--90%, and normal beta RNA approximately 10%--20%, of the total beta mRNA. In contrast, the small amount of beta mRNA present in reticulocytes from such patients consisted predominantly of normal beta mRNA. These results suggest that the reduced amount of normally functioning beta mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.

Animals↗
PubMed1982

Constitutive expression of platelet glycoproteins by the human leukemia cell line K562.

The human leukemia cell line K562 was derived from a patient with chronic granulocytic leukemia. This cell line has subsequently been shown to possess phenotypic markers typical of erythroid and myeloid cells. Using a rabbit antiserum directed against purified platelet glycoproteins (PGPs), we have obtained evidence for the constitutive expression of PGPs on the surface of K562 cells. PGPs expressed have been tentatively identified as IIa and III based on their apparent migration in a 7% sodium dodecylsulfate polyacrylamide gel. K562 may become an important tool for the study of early events involved in megakaryocytic differentiation.

Animals↗
PubMed1982

Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562.

The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3-4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3-5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

Amino Acid Sequence↗
PubMed1981

beta-Thalassemia present in cis to a new beta-chain structural variant, Hb Vicksburg [beta 75 (E19)Leu leads to 0].

Hemoglobin Vicksburg was discovered in a 6-year-old Black boy who had been anemic since infancy. Examination of his hemolysate revealed 87.5% Hb F, 2.4% Hb A2, and 7.6% Hb Vicksburg, which had the electrophoretic and chromatographic properties of Hb A. Structural analysis of Hb Vicksburg demonstrated a deletion of leucine at beta 75(E19), a new variant. Hb Vicksburg was neither unstable nor subject to posttranslational degradation. The alpha/non-alpha biosynthetic ratio was 2.6. Because the proband appeared to be a mixed heterozygote for Hb Vicksburg and beta 0-thalassemia, Hb Vicksburg should have comprised the major portion of the hemolysate. Thus, Hb Vicksburg was synthesized at a rate considerably lower than would be expected on the basis of gene dosage. There was no reason to suspect abnormal translation of beta Vicksburg mRNA; in individuals with Hb St. Antoine (beta 74 and beta 75 deleted), the abnormal hemoglobin comprised 25% of the hemolysate in the simple heterozygote yet was unstable. Deletion of beta 75, therefore, would not in itself appear to lead to diminished synthesis. There was a profound deficit of beta Vicksburg mRNA when measured by liquid hybridization analysis with beta cDNA. The most plausible explanation for the low output of Hb Vicksburg is that a mutation for beta +-thalassemia is present in cis to the structural mutation.

Child↗
PubMed1981

Molecular analysis of the beta-thalassemia phenotype associated with inheritance of hemoglobin E (alpha 2 beta2(26)Glu leads to Lys).

Inheritance of the gene for betaE-globin is associated with hypochromia and microcytosis, reminiscent of typical heterozygous beta-thalassemia. Patients with hemoglobin (Hb)E-beta-thalassemia exhibit clinical phenotypes of severe beta-thalassemia, a circumstance not encountered in other compound heterozygous states for structural beta-chain mutations and beta-thalassemia. We have analyzed the kinetics of globin synthesis and the levels of globin messenger (m) RNA accumulation in patients with Hb E-beta-thalassemia and Hb E trait. The initial rate of beta-globin synthesis (betaE/alpha=0.20-0.34) was less than expected on the basis of gene dosage, or comparable studies of other compound heterozygous states for beta-thalassemia and structurally abnormal beta-chains. betaE-globin synthesis was not only reduced during short-term incubations (1-5 min), but also remained relatively unchanged during long-term pulse or chase incubations up to 5h. Analysis of globin mRNA by cell-free translation and molecular hybridization confirmed that the unexpectedly low levels of betaE-globin synthesis were associated with comparable reduction in the levels of beta-globin mRNA. In Hb E-beta-thalassemia the betaA + betaE (alpha globin nRNA ratio observed were substantially lower than those obtained from reticulocytes of patients with heterozygous beta-thalassemia, or Hb S-betaO-thalassemia, while in Hb E trait, the betaA + betaE/alpha mRNA ratio was in the ranged observed for beta-thalassemia trait. The betaE-globin gene specifies reduced accumulation of betaE-globin mRNA, a property characteristic of other forms of beta-thalassemia. The beta-thalassemia phenotype associated with inheritance of Hb E is thus determined at the level of beta-globin mRNA metabolism.

Globins↗
PubMed1981

Posttranscriptional defects in beta-globin messenger RNA metabolism in beta-thalassemia: abnormal accumulation of beta-messenger RNA precursor sequences.

The production of beta-globin messenger RNA (mRNA) in beta-thalassemic erythroblasts was studied during pulse-chase incubations with [3H]uridine. Globin [3H]mRNA was quantitated by molecular hybridization to recombinant DNA probes complementary to globin mRNA and mRNA precursor sequences. Each of six patients with beta +-thalassemia produced normal amounts of globin alpha and beta [3H]mRNA during a 20-min pulse incubation, but the beta/alpha [3H]mRNA ratio declined to steady-state levels during a chase incubation, suggesting posttranscriptional defects in beta-globin mRNA metabolism. beta-globin mRNA precursor production was estimated by measurement of [3H]RNA sequences hybridizing to a pure DNA probe containing only the large intervening sequence (intron) of the beta-mRNA precursor. Four of the patients exhibited abnormal accumulation of 3H-beta-intron sequences (2-10 times normal), indicating abnormal posttranscriptional processing. In the remaining two patients, one of whom is known to carry a mutation in the small intron of the beta-globin gene, accumulation of large 3H beta-intron RNA and beta-globin [3H]mRNA was normal in nuclei, but the ratio of beta/alpha [3H]mRNA in cytoplasm was reduced, suggesting a different posttranscriptional defect in beta-mRNA processing. These findings imply the existence of heterogeneous posttranscriptional abnormalities in beta-globin mRNA metabolism in different patients with beta-thalassemia. The initial rates of gamma- and delta-mRNA synthesis were low in all patients, suggesting that the low level of expression of these genes in adults is mediated at the transcriptional level.

Adolescent↗
PubMed1980

Embryonic-fetal erythroid characteristics of a human leukemic cell line.

We have studied a number of cell surface, enzyme, and protein markers in the human leukemic K562 cell line. We have confirmed previous observations that these cells accumulate human embryonic hemoglobins after exposure to hemin. In addition, our results demonstrated that these cells possess in the "ininduced" state i surface antigen, lactate dehydrogenase isoenzymes characteristic of embryonic or fetal erythroid cells, fetal and embryonic globin chains, and globin mRNAs. The levels if i antigen, embryonic globin chains, and embryonic globin mRNA increased substantially after exposure of the cells to hemin in suspension culture. In contrast, K562 cells lacked several surface, enzymatic, and functional properties typical of granulocytes, lymphocytes, monocytes, or adult erythroblasts, including HLA surface antigens, surface immunoglobulins, sheep erythrocyte rosetting, phagocytosis, terminal deoxynucleotidyl transferase, carbonic anhydrase, ABO and Rh blood groups, and adult hemoglobins. The K562 cell line therefore exhibits phenotypic properties of embryonic erythroid progenitor cells and a quantitative increase in the expression of some of these properties can be achieved by exposure of the cells to hemin.

Antigens, Surface↗
PubMed1979

Detection of early cardiac dysfunction in patients with severe beta-thalassemia and chronic iron overload.

To detect early left ventricular dysfunction, we used radionuclide cineangiography to determine left ventricular ejection fraction during exercise in 24 patients with transfusion-dependent, congenital anemias, 21 of whom had severe beta thalassemia. Ejection fraction at rest was normal in 21 patients (greater than 45 per cent) and in all patients was 53 +/- 2 per cent (mean +/- S.E.M.)--not significantly different from the value in normal subjects. However, ejection fraction during exercise was normal in only 11 patients (53 +/- 3 per cent in all patients, P less than 0.001 as compared with the normal value). All eight patients who had received fewer than 100 transfusions but only three of 16 (19 per cent, P less than 0.001) who had received 100 or more transfusions had normal responses during exercise. Whereas echocardiographic fractional shortening at rest was normal in 16 of 19 patients studied, eight patients with normal fractional shortening had abnormal ejection-fraction responses to exercise. Thus, radionuclide cineangiography during exercise is a highly sensitive technique for detecting preclinical myocardial dysfunction in patients with systemic iron overload.

Adolescent↗
PubMed1979

Hemoglobin switching in sheep. Synthesis, cloning, and characterization of DNA sequences coding for the beta B, beta C, and gamma-globin mRNAs.

Synthetic double-stranded DNAs (sDNAs) were prepared from sheep globin mRNA templates isolated from reticulocytes producing either hemoglobin B (HbB) (alpha 2 beta B2), HbC (alpha 2 beta C2), or HbF (alpha 2 gamma 2). These DNAs were inserted into the Eco RI site of plasmid pMB9 by the homopolymer tailing method and used to transform Escherichia coli X1776 to tetracycline resistance. Recombinant clones were identified by colony hybridization and further characterized by molecular hybridization and restriction endonuclease analysis. All plasmids analyzed thus far contained either beta- or gamma-globin DNA sequences. Moreover, sDNAs used for cloning yielded restriction endonuclease fragments consistent with the presence of predominantly beta- or gamma-sDNA, indicating that formation of double-stranded alpha-sDNA proceeds much less efficiently under our conditions than the formation of non-alpha-sDNAs. Three recombinant plasmids, pS beta B2, pS beta C69, and pS gamma 56, were selected for detailed study. These were shown to contain, respectively, beta B-, beta C-, and gamma-DNA sequences by molecular hybridization and by protection of the appropriate cDNAs from S1 nuclease digestion. Each contained all of the restriction endonuclease sites defined for the synthetic sDNAs and protected at least 90% of the sequence length of homologous cDNA. Restriction endonuclease maps of the beta B- and beta C-globin genes were identical at all 12 sites that were mapped, whereas four differences were identified in the gamma gene compared to the two others; three of these corresponded to differences in amino acid sequence of the globins. A method was developed to isolate the anti-mRNA strand of the insert for use as a specific molecular hybridization probe analogous to complementary DNA.

Animals↗
PubMed1979

Induction of erythropoietic colonies in a human chronic myelogenous leukemia cell line.

The ability of cells derived from the K562 cell line to generate erythropoietic colonies was studied. The K562 cell line was derived from a patient with chronic myelogenous leukemia 8 yr ago by Lozzio and Lozzio. Rare benzidine-positive colonies formed when these cells were cloned in plasma clots (3 +/- 1/10(4) cells), and their number was not substantially increased by the addition of erythropoietin (9.5 +/- 1/10(4) cells). Sodium butyrate was capable of markedly enhancing the number of benzidine-positive colonies (19.5 +/- 1/10(4) cells) formed, while the combination of sodium butyrate plus erythropoietin exerted a synergistic effect on erythropoietic colony formation (57 +/- 4/10(4) cells). The K562 cell line is a long-term culture system that contains human erythropoietic stem cells. This cell line should be useful in future studies on the cellular and molecular events associated with human erythroid cell differentiation.

Benzidines↗
PubMed1978

Variability in the amount of beta-globin mRNA in beta0 thalassemia.

Globin mRNA isolated from a number of beta0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of alpha- and beta-mRNA by hybridization to purified alpha-and beta-cDNA revealed that in approximately half the cases, there was less than 1% as much beta-mRNA as alpha-mRNA. In the rest of the cases, low levels of beta-like mRNA were detected in amounts 4-12% as abundant as alpha-mRNA. There was variability in the yield of beta-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: beta-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, beta-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the beta0 thalassemia mRNA-beta-cDNA hybrid was measured and found to be slightly lower than that of the authentic beta-mRNA-beta-cDNA hybrid. In none of the cases tested was there synthesis of beta-globin chains directed by beta0 thalassemia mRNA in a cell-free protein-synthesizing system, even when beta-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent alpha chain-specific oligonucleotides without detectable beta chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of beta-mRNA in the same RNA samples. Our results support the concept that beta0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent beta globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional beta or beta-like globin mRNA. The variable amounts of beta-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of beta-like mRNA observed in beta0 thalassemia.

Blood Cells↗