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Biomedical subjects

D Housman

Publications and source records attributed to D Housman.

At least 91 records · Page 5Linked to original sources
PubMed1985

Expression of an ouabain resistance gene in transfected cells. Ouabain treatment induces a K+-transport system.

We have investigated the expression of a cloned mouse gene which confers ouabain resistance to African green monkey kidney (CV1) cells. CV1 cells carrying the transfected ouabain resistance (ouaR) gene express an ouabain-inducible K+-transport system. This K+-transport system is not a normal (Na,K)-ATPase since plasma membranes prepared from the transfected cells have significantly reduced Na+-stimulated ATPase activity. RNA sequences homologous to the transfected gene are observed in abundance only following exposure of transfectants to ouabain. The small size of the message induced (1.2 kilobases) also argues that the gene does not code for the alpha-subunit of the (Na,K)-ATPase. Ouabain-treated transfected cells maintain an internal [K+] of 113 mM; a level close to the 139 mM of control cells. However, ouabain-treated transfectants exhibit an internal [Na+] of 61 mM, which is 3-6 times the level in untreated cells (11-21 mM). These results suggest: ouabain resistance can be conferred by a gene which codes for an ouabain-inducible K+-transport system; induction of this transport system by ouabain is due to increased levels of mRNA coded for by the ouabain resistance gene; and the ouabain resistance gene does not encode for the alpha-subunit of the (Na,K)-ATPase.

Animals↗
PubMed1985

Regulation of protein synthesis and accumulation during murine erythroleukemia cell differentiation.

We have examined the repertoire of cytoplasmic proteins present at different times during murine erythroleukemia (MEL) cell differentiation. Our laboratory has developed an improved differentiation system in which the use of rapidly inducing MEL subclones and culture conditions which stabilize terminally differentiated cells results in highly synchronous differentiation and the accumulation of large numbers of cells in the end stages of differentiation. Using two-dimensional gel electrophoresis, the proteins of MEL cell cytoplasm have been fractionated at different times of induction in the improved system. The protein composition of MEL cell cytoplasm changes dramatically during the differentiation program, in contrast to previously reported results. We observe patterns of changes that are consistent with alterations in the relative degradative rates as well as the relative synthetic rates of the different proteins. We find that the rate of incorporation of labeled amino acid into protein is reduced in induced cultures of MEL cells. We demonstrate that the contribution of uninduced cells to the protein patterns observed late in differentiation is minor in our system, and argue that the results previously obtained for differentiating MEL cells were influenced by the heterogeneity of the induced populations.

Animals↗
PubMed1985

Assignment of the gene coding for the T3-delta subunit of the T3-T-cell receptor complex to the long arm of human chromosome 11 and to mouse chromosome 9.

The gene encoding the 20-kDa glycoprotein of the T3-T-cell receptor complex (T3-delta chain) has been mapped to human chromosome 11 by hybridization of a T3-delta cDNA clone (pPGBC#9) to DNA from a panel of human-rodent somatic cell hybrids. In Southern blotting experiments with DNAs of somatic cell hybrids that contained segments of chromosome 11, we were able to assign the T3-delta gene to the distal portion of the long arm of human chromosome 11 (11q23-11qter). By use of a newly developed cDNA clone (pPEM-T3 delta) that codes for the murine T3-delta chain, the mouse T3-delta gene was mapped on chromosome 9. The importance of the T3-delta map position and its relationship to the other genes on the long arm of human chromosome 11 and to those on mouse chromosome 9 is discussed.

Animals↗
PubMed1985

Localization of DNA sequences in region Xp21 of the human X chromosome: search for molecular markers close to the Duchenne muscular dystrophy locus.

Panels of somatic cell hybrid lines carrying various structural rearrangements of the human X chromosome short arm were analyzed with 21 X-chromosome-specific cloned DNA fragments. We mapped these molecular markers to five different regions of the short arm of the X chromosome. The results were confirmed by gene-dosage studies of human lymphoblasts with structurally abnormal X chromosomes. The ornithine transcarbamylase gene and four anonymous DNA sequences map within band Xp21, flanking the presumed locus for Duchenne muscular dystrophy.

Animals↗
PubMed1984

Molecular cloning of the mouse ouabain-resistance gene.

DNA prepared from ouabain-resistant mouse cells was able to transform ouabain-sensitive CV-1 cells to ouabain resistance after DNA-mediated gene transfer. The murine DNA fragment responsible for ouabain resistance was detected on the background of CV-1 DNA by virtue of a repetitive DNA sequence element that reacts positively with a mouse repeat DNA clone. CV-1 DNA is nonreactive with this probe. Southern analysis of several independently derived ouabain-resistant transformants indicates that the mouse ouaR gene is located on a 6.5-kilobase EcoRI restriction fragment. The 6.5-kilobase DNA fragment was initially isolated from a lambda phage library made from a ouabain-resistant secondary transformant and subsequently was subcloned in the plasmid vector pAT153. This plasmid was able to transform wild-type CV-1 cells to ouabain resistance at a frequency of about 10 cells per ng of DNA.

Animals↗
PubMed1984

Chromosome-mediated transfer of the malignant phenotype by human acute myelogenous leukemic cells.

Acute myelogenous leukemia (AML) is a malignancy of the myeloid cells of the bone marrow. Recently, a number of groups have demonstrated that it is possible to study the malignant phenotype at the level of DNA through gene transfer experiments. We have used such an approach to determine whether it is possible to transfer the malignant phenotype of anchorage independence from human AML cells to anchorage-dependent rodent cells, using chromosomes as the source of genetic information. We found that chromosomes isolated from leukemic cell lines were capable of transferring the malignant phenotype of anchorage independence, whereas chromosomes derived from the lymphocytes of normal individuals were not active in this assay. Using Southern blot analysis of the DNA from transferants, we were able to show that the transfer of anchorage independence correlated with the presence of human DNA in the transferants. The pattern of human DNA in the transferants derived from different transfection experiments is compared.

Animals↗
PubMed1983

Ionic regulation of MEL cell commitment.

A key event in the initiation of the dimethyl sulfoxide (DMSO)-induced program of murine erythroleukemia (MEL) cell differentiation is a rise in the level of cytoplasmic calcium ions. Our interest in the present study is whether other inducers of the terminal erythroid differentiation program also act via a calcium-dependent pathway. Inhibition of calcium transport has been found to prevent the induction of MEL cell commitment by DMSO, butyric acid (BA), or hypoxanthine (HX). Enhancement of the calcium flux rate with A23187 or elevation of cytoplasmic calcium levels with FCCP stimulates the kinetics of commitment in response to all three inducers. These results suggest that of the inducers we have tested (DMSO, BA, and HX), all three act to initiate commitment via a common mechanism which involves modulation of cytoplasmic calcium levels.

Animals↗
PubMed1982

Evidence that a Na+/Ca2+ antiport system regulates murine erythroleukemia cell differentiation.

The Na+ and Ca2+ transport properties of cultured murine erythroleukemia (MEL) cells have been investigated. We have previously shown that amiloride prevents dimethyl sulfoxide-induced MEL cell differentiation via inhibition of an essential Ca2+ influx (levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5948-5952). Here we show that external Na+ inhibits Ca2+ influx and stimulates Ca2+ efflux from uninduced MEL cells. Increasing the internal Na+ concentration by a brief incubation of cells with ouabain stimulates the rate of 45Ca2+ influx. Amiloride (40 microM) completely blocks the external Na+-stimulated 45Ca2+ efflux and external Na+-inhibitable 45Ca2+ influx. The same concentration of amiloride had no significant effect on net Na+ uptake. These results suggest that a significant fraction of Ca2+ flux across the MEL cell plasma membrane occurs via a Na+/Ca2+ antiport system and that amiloride prevents differentiation by blocking Ca2+ influx through this system. The importance of a Na+/Ca2+ antiport system for MEL cell differentiation is supported by the following observation: increasing the cellular Na+ level by a brief treatment with ouabain plus monensin accelerates MEL cell commitment as effectively as adding the Ca2+ ionophore A23187. We suggest that dimethyl sulfoxide induces MEL cell differentiation by inhibiting the Na+ pump and consequently allowing Ca2+ influx through the Na+/Ca2+ antiport.

Amiloride↗
PubMed1982

Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation.

The level of cytoplasmic calcium ions appears to be important in the control of murine erythroleukemia (MEL) cell differentiation. Our interest in this study focuses on the relationship between the regulation of calcium concentration and differentiation. We used the fluorescent membrane probe DiOC6 to examine the relationship between MEL cell mitochondria and changes in cytoplasmic calcium levels occurring at the initiation of commitment. Fluorescence microscopy reveals the selective association of DiOC6 with MEL cell mitochondria, where an enhanced fluorescence is observed. Treatment of cells with dimethylsulfoxide (DMSO) or other inducers causes a decrease in mitochondria-associated fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC6 fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit calcium uptake, also prevent the decrease in DiOC6 uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP, a proton ionophore that dissipates mitochondrial membrane potential. FCCP also caused MEL cell mitochondria to release calcium into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period observed when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic calcium levels at the time of commitment of MEL cells to terminal differentiation.

Amiloride↗
PubMed1982

Genetic fine-structure mapping in human chromosome 11 by use of repetitive DNA sequences.

A method is described for mapping of the DNA fragments of a human chromosome produced by restriction enzyme treatment of the total DNA from a hybrid cell containing a single human chromosome. The method involves production and selection of somatic cell mutants containing deletions of the human chromosome and application of a hybridization probe consisting of an individual member copy of a repetitive human DNA family. A linear map has been constructed of 19 marker DNA fragments and 5 immunological and biochemical markers on human chromosome 11, selected as a model chromosome for these studies. This approach appears to be widely applicable, is independent of cytogenetic analysis, promises to be capable of revealing the existence of rearrangements as well as deletions, appears to be amenable to further increase in resolving power, and offers potential application in various human genetic problems.

Cell Line↗
PubMed1982

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end-stage cells.

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.

Animals↗
PubMed1981

Stability of globin mRNA in terminally differentiating murine erythroleukemia cells.

The stability of globin mRNA is terminally differentiating MEL cells has been reevaluated. Previously, it had been reported that globin mRNA has a half-life of approximately 17 hr in terminally differentiating MEL cells. We show that the previous measurements of this parameter were confounded by physical instability of differentiating MEL cells. By using culture conditions that physically stabilize end-stage cells we show that the stability of globin mRNA in terminally differentiating MEL cells is equal to the value observed for ribosomal RNA, a half-life greater than 60 hr.

Animals↗