Biomedical subjects

S Ralph

Publications and source records attributed to S Ralph.

21 records · Page 2Linked to original sources

PubMed1991

Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

Amino Acid Sequence↗

PubMed1987

Structural studies of T200 glycoprotein and the IL-2 receptor.

Immunological analysis of the cell surface of hematopoietic cells has led to the identification of many different cell membrane molecules, some of which have well-defined functions as receptors. In general, however, the role of most lymphocyte cell surface molecules remains ill-defined even in cases in which antibody inhibition studies have given some insight into the biological processes in which they participate. Here we describe molecular and biochemical studies of T200 glycoprotein (leukocyte-common antigen) and the IL-2 receptor which illustrate the kinds of approaches that can be currently used to characterize individual molecules. T200 glycoprotein is a large Mr glycoprotein found exclusively on leukocytes. However, the exact Mr varies in a cell-type-specific fashion and this property is conserved between different species. Comparison of the rat, mouse and human cDNA sequences show that the large cytoplasmic portion of the molecule is well-conserved, approximately 90%, whereas the exterior portion is only about 50% homologous. Cell-type-specific differences in the primary sequence of the molecule have been identified in the N-terminal portion of the molecules. In contrast to T200, the function of the IL-2 receptor is well-known. The interaction of IL-2 with its receptor provides a growth signal that determines the magnitude and duration of T-cell responses. Limited proteolysis studies provide the first direct biochemical evidence that the external region of the IL-2 receptor consists of two independent domains. 125I-labeled IL-2 has been chemically crosslinked to the receptor and proteolytic cleavage of the crosslinked product indicates that IL-2 is selectively bound to the N-terminal domain of the receptor.

Animals↗

PubMed1987

Regulation of transferrin receptor expression in myeloid leukemia cells.

Surface transferrin (Tf) receptors are displayed by cultured human hematopoietic cells and provide Fe required for cell growth. Cell cycle status, cell density in culture, exposure to Fe, and differentiation alter Tf receptor display by myeloid leukemia cells. To investigate mechanisms controlling Tf receptor expression, rates of receptor synthesis and steady-state mRNA levels were measured in HL60 promyelocytic cells grown in serum and serum-free media or after differentiation in response to dimethylsulfoxide (DMSO). Although surface binding sites were unchanged during the first three days in culture with serum or in serum-free media containing Tf, by the third day, rates of receptor biosynthesis and steady-state mRNA levels declined, consistent with cell density-dependent, receptor regulation. Cells grown with soluble Fe instead of Tf showed reduced Tf binding sites, rates of receptor synthesis, and Tf receptor mRNA. When cells grown with Fe were subcultured, Tf receptor mRNA levels increased within 15 minutes and peaked by 24 hours. This was followed by a decline in receptor and gene expression so that by three days cells grown in the presence of Fe expressed approximately four times fewer receptors and had half the rates of Tf receptor synthesis and mRNA levels of cells grown in serum or Tf. Cells treated with DMSO showed a rapid decline in surface receptors, receptor synthesis, and steady-state mRNA levels. Modulation of Tf receptor expression was not due to redistribution between the cell surface and an internal receptor pool. In each instance, concurrent assessment of N-ras transcripts showed that changes in Tf receptor mRNA levels were not due to generalized alterations in protein synthesis. Exposure of cells grown in Fe or treated with DMSO to cycloheximide did not alter Tf receptor mRNA levels, thereby suggesting that receptor expression was not regulated by posttranscriptional processes dependent on protein synthesis. Actinomycin D inhibition of Tf receptor mRNA was compatible with a transcript half-life of approximately 2.2 hours. Nuclear transcription studies showed reduced rates of Tf receptor transcription after culture with Fe or exposure to DMSO. The present studies show complex patterns of Tf receptor gene regulation in cultured myeloid leukemia cells and demonstrate that transcriptional regulation is a major mechanism controlling Tf receptor gene expression in response to Fe and differentiation.

Animals↗