Evaluation of expression of transferred genes in differentiating myeloid cells: expression of human glucocerebrosidase in murine macrophages.

The retroviral vector LGSN, in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was tested for expression in the murine myelomonocytic leukemia cell line M1 before and after induction of differentiation with interleukin-6 (IL-6). Southern analysis of the seven transduced clones selected for neomycin resistance in Geneticin (G-418 sulfate) demonstrated one to eight copies of intact provirus with rearrangements in only two clones. Absolute levels of human GC RNA and protein increased with increased copy numbers of provirus in the clones. Upon induction with IL-6 of the seven transduced clones to the macrophage phenotype, there was no significant change, overall, in RNA levels but some increase in human GC protein levels could be detected. Although this was the average trend, considerable clonal variation in RNA and protein levels was observed upon induction. Transduction of the M1 cells did not interfere with the ability of the cells to differentiate from blasts to macrophages as seen by the appearance of membrane receptors for the constant region of immunoglobulins (Fc gamma RI) and lysozyme production in the differentiated M1 cells. Thus, the M1 cell line can be used for testing retroviral vector expression in myeloid lineages at early and late stages of differentiation. This rapid in vitro testing of potential retroviral vectors will be beneficial for gene therapy of disorders that affect differentiated macrophages such as Gaucher's disease.

Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 s after stimulation with three types of human thrombin: alpha-thrombin, gamma-thrombin, which is proteolytically active but does not bind to the high-affinity sites, and di-isopropyl fluorophosphate-derivatized (DIP)-alpha-thrombin, an active site-inhibited analogue that does bind to the high-affinity site. Large doses of gamma-thrombin evoked moderate aggregation and serotonin release, but minimal phosphorylation of the 20 and 47 kDa proteins. The initial (1.5-3.0 s) increase in cystolic free calcium concentration ([Ca2+]i) indicated by Indo-1 was also diminished, but by 5 s was nearly as high (1.0 microM) as with alpha-thrombin. A large dose of DIP-alpha-thrombin, on the other hand, induced minimal aggregation, serotonin secretion and [Ca2+]i response within 6 s. There was, however, a transient dephosphorylation of the 20 kDa protein. When combined, gamma- and DIP-alpha-thrombin were approximately additive in their ability to induce aggregation and serotonin secretion, but strongly synergistic in phosphorylating the 20 and 47 kDa proteins. The [Ca2+]i increase was not, however, enhanced over that induced by gamma-thrombin alone. These results demonstrate that phosphorylation of either the 20 or 47 kDa proteins is not correlated with [Ca2+]i dynamics and is neither required nor directly involved in platelet aggregation and secretion induced by thrombin. The high-affinity binding activity of thrombin is not necessary for rapid platelet Ca2+ influx, aggregation and serotonin release within the first critical seconds of activation.

Effect of epinephrine on rapid ADP-induced aggregation, protein phosphorylation, and cytoplasmic calcium dynamics of platelets: a quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation with a low concentration of adenosine 5'-diphosphate (ADP) (0.5 mumol/L), epinephrine (15 mumol/L), or a combination of the two. Compared with ADP alone, the combination nearly doubled the rate of aggregation as measured by the loss of single particles. Our aim was to determine whether the aggregation, protein phosphorylation, and cytoplasmic calcium responses to this potentiating combination of ADP and epinephrine were analogous to those caused by a maximal concentration of ADP (10 mumol/L) and also to determine whether cyclooxygenase was involved in this potentiation. Phosphorylation of myosin light chain (MLC) and the 47 kd protein were analyzed during the first 5 seconds of platelet response and were related to the progress of aggregation. Unlike aggregation, ADP-induced phosphorylations of both MLC and the 47 kd protein were inhibited rather than potentiated by epinephrine. Pretreatment of the platelets with indomethacin did not affect aggregation or MLC phosphorylation, but increased 47 kd phosphorylation at 0.6 seconds and eliminated it at 3 seconds. Cytoplasmic free calcium ([Ca2+]i) of indo-1-loaded platelets were monitored with our continuous-flow fluorescence approach. Although epinephrine itself did not change [Ca2+]i, it potentiated the [Ca2+]i rise induced by a low dose of ADP. A comparison of these results with platelet activation caused by 10 mumol/L ADP indicates that MLC and 47 kd phosphorylations are not correlated with [Ca2+]i dynamics and are not required or directly involved in platelet aggregation.

ADP causes subsecond changes in protein phosphorylation of platelets.

We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched-flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.