1. Novel Evidence of Expression and Activity of Ecto-Phospholipase C γ1 in Human T Lymphocytes
by Sebastiano Miscia, Angela Di Baldassarre, Amelia Cataldi, Rosa Alba Rana, Valerio Di Valerio, and Giuseppe Sabatino
Blood
Volume 91(10):
May 15, 1998
©1998 by American Society of Hematology

2. Sebastiano Miscia et al. Blood 1998;91:3833-3840
©1998 by American Society of Hematology

3. Sebastiano Miscia et al. Blood 1998;91:3833-3840
©1998 by American Society of Hematology

4. Sebastiano Miscia et al. Blood 1998;91:3833-3840
©1998 by American Society of Hematology

5. Sebastiano Miscia et al. Blood 1998;91:3833-3840
©1998 by American Society of Hematology

6. Sebastiano Miscia et al. Blood 1998;91:3833-3840
©1998 by American Society of Hematology

7. FACScan histogram of ecto-PLCγ1+lymphocytes.
FACScan histogram of ecto-PLCγ1+lymphocytes. The percentage of ecto-PLCγ1+ lymphocytes was obtained by calculating the mean ± SD of 10 experiments.
Sebastiano Miscia et al. Blood 1998;91:
©1998 by American Society of Hematology

8. Expression of PLCγ1 at the external leaflet of the plasma membrane in T-cell subsets.
Expression of PLCγ1 at the external leaflet of the plasma membrane in T-cell subsets. PBMCs were double-stained with anti-PLCγ1, evidenced by a FITC-conjugated secondary antibody, and with either PE-labeled anti-CD4, anti-CD8, anti-CD45RA, or anti-CD45RO antibody. (A) Results showed that the enzyme is expressed on the membrane of CD8+ cells (29.2% ± 5.1%), while the percentage of circulating PLCγ1+CD4+lymphocytes is very low (1.1% ± 0.2%, P < .05). (B) When expression of the enzyme on naive and memory cells was considered, PBMCs displayed a significant percentage of PLCγ1+CD45RA+ (27.2% ± 1.7%), whereas the phenotype PLCγ1+CD45RO+was slightly detectable (4.2% ± 0.6%, P < .005). Significance of the results was determined by Student's ttest. n = 10 samples.
Sebastiano Miscia et al. Blood 1998;91:
©1998 by American Society of Hematology

9. Sebastiano Miscia et al. Blood 1998;91:3833-3840
©1998 by American Society of Hematology

10. Sebastiano Miscia et al. Blood 1998;91:3833-3840
©1998 by American Society of Hematology

11. Kinetics of [3H]-lyso-PI uptake.
Kinetics of [3H]-lyso-PI uptake. Transfer of [3H]-lyso-PI from donor vesicles to the cell membrane of lymphocytes was measured by scintillation counting of the incubation medium from EGTA-containing samples. The 100% value was obtained by measuring an aliquot of solution of μmol/L [3H]-lyso-PI (concentration of substrate added to the cells). The kinetics of spontaneous uptake of the lipid by the cells shows that its concentrations in the medium plateaued at about 25% within 5 minutes. Beyond this time, the rate of lyso-PI transfer was evidently reduced. Data are the mean ± SD of at least 3 separate experiments.
Sebastiano Miscia et al. Blood 1998;91:
©1998 by American Society of Hematology

12. Formation of water-soluble radiolabeled products in the absence (a) or presence (b) of EGTA. Assays were performed for the indicated times in the absence or presence of 0.5 mmol/L EGTA. In the absence of EGTA, production increased linearly at least fivefold...
Formation of water-soluble radiolabeled products in the absence (a) or presence (b) of EGTA. Assays were performed for the indicated times in the absence or presence of 0.5 mmol/L EGTA. In the absence of EGTA, production increased linearly at least fivefold over a 5-minute period, plateauing between 5 and 30 minutes. When EGTA was added to the reactions, recovery of radioactivity dramatically decreased, indicating its Ca2+ dependency. Data are the mean ± SD of at least 3 different experiments.
Sebastiano Miscia et al. Blood 1998;91:
©1998 by American Society of Hematology

13. Percentage of radiolabeled inositol phosphates in the water-soluble material (100%).
Percentage of radiolabeled inositol phosphates in the water-soluble material (100%). Lymphocytes in the different experimental conditions were incubated in a reaction mixture containing μmol/L lyso-PI up to 30 minutes. Once the reaction was stopped, the water-soluble products extracted from the incubation medium were chromatographed over a 1-mL column of Dowex AG 1-X8 resin, and the fractions were analyzed by scintillation counting. The peak is evident for Ins(1)P in 90-min interferon-treated samples, while Ins(1:2 cycl)P constantly increases along the time of treatment and accumulates to significant concentrations in 24-hour treated samples and in unstimulated cells. Data are the mean of 3 experiments differing ≤5% SD.
Sebastiano Miscia et al. Blood 1998;91:
©1998 by American Society of Hematology