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Biomedical subjects

A Dean

Publications and source records attributed to A Dean.

At least 91 records · Page 5Linked to original sources
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

The estimation of the lymphocyte percentage by the Coulter Counter Model S Plus III.

The accuracy of the Coulter Counter Model S Plus III in determining lymphocyte percentage was assessed. In 'routine' adult blood counts accuracy was satisfactory but in infectious mononucleosis, and in chronic lymphocytic leukaemia and other lymphoproliferative disorders, there was a high rejection rate and an often considerable underestimation of the lymphocyte percentage. The S Plus III lymphocyte percentage was of no use in neonates because of inaccuracy and a high rejection rate. In infants the relationship with lymphocyte percentages based on manual differential counts was non-linear, and there was underestimation of some high percentages. The white cell size histogram was useful in altering staff to abnormalities, and also suggested that lymphocytes of neonates and infants were larger than those of adults, with plots being very similar to those of some adults with lymphoproliferative disorders. The S Plus III lymphocyte percentage was useful in reducing the need for differential counts in adult patients but did not replace examination of a blood film or a manual lymphocyte percentage in patients with chronic lymphocytic leukaemia or a lymphoproliferative disorder.

Adult↗
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↗
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

An assessment of the Coulter Counter Models S Plus II and III.

The Coulter Counter Models S Plus II and III have been evaluated. No serious safety hazards were identified. Scientific assessment showed some non-linearity in the Hb which caused the MCH and MCHC to vary as samples were diluted. Precision and carry-over were satisfactory. The results obtained compared well with those of the Coulter Counter Model S, except for WBC; reference methods showed better accuracy on the Models S Plus II and III. Platelet counts agreed with those by phase-contrast microscopy and the lymphocyte percentage was similar to that from the blood film except in the lymphoproliferative disorders. The whole blood and pre-dilute modes gave similar results though the platelet count was slightly higher in the whole blood mode. In the National External Quality Assessment Scheme results were in accordance with those from other Model S Plus Users. Time did not allow a detailed evaluation of the cell volume distribution curves but it was noted that the white cell profile was useful for detecting platelet aggregation. Efficiency assessment showed throughputs of 66 and 93 samples per hour on the Models S Plus II and III respectively. The platelet count was clinically useful as was the lymphocyte percentage measurement which rendered some differentials unnecessary. Rejection of the white cell profile was a helpful index of abnormality on the Model S Plus II but occurred non-specifically on the Model III tested.

Adult↗
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↗
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

Social support scales: a methodological note.

The relationship between social support and depression has implications for researchers studying schizophrenia. In this article, the authors attempt to conceptualize social support and assess the reliability and validity of its various measures. They then evaluate the ability of social support, along with stressors, to explain psychiatric symptoms. Representative data from a community survey of adults, ages 17-70, suggest that social support measures show strong relationships to depression and other psychiatric symptoms. The implications of these and other findings for the study of schizophrenia are discussed.

Depressive Disorder↗
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↗
PubMed1977

The stress-buffering role of social support. Problems and prospects for systematic investigation.

Over the pase 20 years, a sizable body of literature has developed which serves to establish that stressful life events are associated with the onset, incidence, and prevalence of a wide range of physical and psychiatric disorders. As measured by the Holmes and Rahe Social Readjustment Rating Scale, or similar instruments, the stressful life events are fundamentally sociological in nature. Yet, paradoxically, the research has been largely limited in the relevant basic sociological theory and data brought to or yielded from investigation. Recently, however, several prominent researchers have emphasized the importance of studying the role of social support systems as possible buffers or mediators of stress. The most basic objective of this paper is to contribute to the advancement of such studies by clearly identifying key empirical, theoretical and methodological problems and suggesting some approaches to their resolution. Specifically, this paper offers: a) a selective review of the essential status of empirical knowledge; b) an examination of the nature and significance of social support systems; c) clarification of methodological and theoretical problems; and d) detailed proposals for approaching problems of measurement and research design.

Animals↗