Biomedical subjects

D J Lew

Publications and source records attributed to D J Lew.

41 records · Page 3Linked to original sources

PubMed1991

Two distinct alpha-interferon-dependent signal transduction pathways may contribute to activation of transcription of the guanylate-binding protein gene.

The promoter of the gene encoding a cytoplasmic guanylate-binding protein (GBP) contains two overlapping elements: the interferon stimulation response element (ISRE), which mediates alpha interferon (IFN-alpha)-dependent transcription, and the IFN-gamma activation site (GAS), which is required for IFN-gamma-mediated stimulation. The ISRE binds a factor called ISGF-3 that is activated by IFN-alpha but not by IFN-gamma. The GAS binds a protein that is activated by IFN-gamma, which we have termed GAF (IFN-gamma activation factor; T. Decker, D. J. Lew, J. Mirkovitch, and J. E. Darnell, Jr., EMBO J., in press; D. J. Lew, T. Decker, I. Strehlow, and J. E. Darnell, Jr., Mol. Cell. Biol. 11:182-191, 1991). We now find that the GAS is also an IFN-alpha-responsive element in vivo and that IFN-alpha (in addition to activating ISGF-3) rapidly activates a GAS-binding factor, the IFN-alpha activation factor (AAF). The AAF has characteristics very similar to those of the previously described GAF. Through the use of inhibitors of protein synthesis and inhibitors of protein kinases, the activation conditions of AAF, GAF, and ISGF-3 could be distinguished. Therefore, not only do IFN-alpha and IFN-gamma stimulate transcription of GBP through different receptors linked to different signaling molecules, but occupation of the IFN-alpha receptor apparently leads to the rapid activation of two different DNA-binding proteins through the use of different intracellular pathways.

Base Sequence↗

PubMed1990

Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3.

Interferon-alpha (IFN alpha) and interferon-gamma (IFN gamma) each induce in susceptible target cells a state of resistance to viral replication and reduced cellular proliferation, presumably through different mechanisms: these two polypeptides are unrelated by primary sequence and act through distinct cell-surface receptors to induce expression of largely non-overlapping sets of genes. However, acting in concert, they can produce synergistic interactions leading to mutual reinforcement of the physiological response. In HeLa cells, this synergistic response was initiated by cooperative induction of IFN alpha stimulated genes (ISGs). These normally quiescent genes were rapidly induced to high rates of transcription following exposure of cells to IFN alpha. Although they were only negligibly responsive to IFN gamma, combined treatment of cells with IFN gamma followed by IFN alpha resulted in an approximately 10-fold increase in ISG transcription. ISG transcription is dependent upon ISGF3, a positive transcription factor specific for a cis-acting regulatory element in ISG promoters. IFN gamma treatment induced increased synthesis of latent ISGF3, which was subsequently activated in response to IFN alpha to form approximately 10-fold higher levels than detected in cells treated with IFN alpha alone. ISGF3 is composed of two distinct polypeptide components, synthesis of one of which was induced by IFN gamma, increasing its cellular abundance from limiting concentrations to a level which allowed formation of at least 10 times as much active ISGF3. Cell lines vary in their constitutive levels of the inducible component of ISGF3 and in the ability of IFNs to increase its synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗

PubMed1989

Interactions of alpha- and gamma-interferon in the transcriptional regulation of the gene encoding a guanylate-binding protein.

Transcriptional regulation of the gene encoding a guanylate-binding protein (GBP) by the two interferon (IFN) types was studied. GBP gene transcription was regulated by alpha IFN in a manner identical to that of previously described IFN-stimulated genes (ISGs): rapid induction, without a need for protein synthesis, followed by a protein synthesis-dependent suppression of transcription to basal levels within 6 h. Transcriptional induction by gamma IFN was equally rapid and independent of ongoing protein synthesis but remained at elevated levels for greater than 24 h. Experiments employing combined treatments with IFNs of both types revealed that induction of the GBP gene by gamma IFN overrides the alpha IFN-induced active repression and reverses the alpha IFN-induced repressed state. Moreover, the alpha IFN-mediated repression of ISG54, a gene normally responsive to only alpha IFN, is also reversed by gamma IFN. Induction of GBP by gamma IFN is presumably mediated by a factor different from the recently described activator Interferon Stimulated Gene Factor 3 (ISGF3) because induction of this factor was not observed upon treatment of cells with gamma IFN. Finally, a complex set of reinforcing or synergistic effects were observed when induction of the GBP gene was evoked by a combined treatment with the two IFN types.

Cycloheximide↗

PubMed1989

Alpha interferon and gamma interferon stimulate transcription of a single gene through different signal transduction pathways.

Interferons (IFNs) play a key role in the defense against virus infection and the regulation of cell growth and differentiation, in part through changes in specific gene transcription in target cells. We describe several differences between the signal transduction events that result in transcriptional activation of the human gene coding for a guanylate-binding protein (GBP) by alpha interferon (IFN-alpha) and gamma interferon (IFN-gamma). Activation by IFN-alpha was rapid, transient, and cycloheximide resistant. Activation by IFN-gamma was slower, sustained, and delayed by cycloheximide. IFN-gamma led to the formation of a stable intracellular signal which led to continued GBP transcription even if the ligand was withdrawn, whereas IFN-alpha-induced GBP transcription decayed rapidly if IFN-alpha was withdrawn. Perturbations of signaling pathways involving classical second messengers (cyclic AMP, Ca2+, protein kinase C) did not induce GBP transcription. However, various kinase inhibitors blocked the transcriptional response to IFN-gamma but not IFN-alpha, suggesting that a specific and possibly novel kinase is involved in gene activation by IFN-gamma.

Calcium↗

PubMed1984

Dehydration and delayed proton equilibria of red blood cells suspended in isosmotic phosphate buffers. Implications for studies of sickled cells.

PO4 buffers isosmotic with plasma or phosphate-buffered saline solution with a substantial proportion of PO4 are often used to wash and suspend red blood cells in studies of respiratory or sickling behavior. Measurements of sequential changes in mean cell hemoglobin concentration, pH, and ion content of red blood cells suspended in 295 mOsm Na-phosphate, pH 7.4, at 23 degrees or 37 degrees C, showed (1) rapid, persistent cell dehydration (mean cell hemoglobin concentration greater than 40 gm/dl) caused initially by Cl- efflux and later by replacement of monovalent Cl- by divalent HPO=4; and (2) temporary reversal of membrane pH gradients with normalization time (30 to 120 minutes) dependent on factors controlling the rate of phosphate-chloride exchange. Sequential equilibration of red blood cells in isosmotic citrate (impermeable) followed by PO4 demonstrated the two stages of the observed shifts in PO4 alone, and red blood cells suspended in 0.15 mol/L 32PO4 at 37 degrees C showed PO4 influx consistent with pH equilibrium kinetics. Sickle trait red blood cells deoxygenated at 37 degrees C, pH 7.4, in plasma or 10 mmol/L HEPES-buffered saline solution showed only 6% to 20% sickling. In isosmotic PO4, mean cell hemoglobin concentration was 40 to 41 gm/dl with approximately 80% sickling. In phosphate-buffered saline solution containing 70 mmol/L PO4, red blood cells showed smaller, similar changes (mean cell hemoglobin concentration approximately 38 gm/dl) with a longer equilibration period and deoxygenated sickle trait cells showed 40% sickling. The altered properties of red blood cells suspended in PO4 or phosphate-buffered saline solution were neither intended nor appropriate for many studies using these media, particularly with hemoglobin S-containing red blood cells, and interpretations of reported results must be reassessed in light of these findings.

Anemia, Sickle Cell↗