Tetsuji Tanimoto

Sensitive enzyme-linked immunosorbent assay for adult T-cell leukemia-derived factor and normal value measurement.

Four different monoclonal antibodies against recombinant adult T‐cell leukemia‐derived factor (ADF), identical to thioredoxin, were established and used for the determination of ADF concentration in serum. Using two of the monoclonal antibodies, we developed a two‐step enzyme‐linked immunosorbent assay (ELISA) for ADF. This ELISA showed a highly specific reactivity on ADF with no cross‐reactivity to several proteins with homologue sequence on the active center. The detection limit of the assay was 2.0 ng/ml (mean ± 2 SD). The intra‐ and interassay coefficients of variation (CV) were 0.81–3.74% (n = 8) and 4.78–6.97% (n = 7), respectively. The normal value of ADF mean concentration from 145 healthy donors was 40.8 ng/ml.

Enhancement of fibroblast growth factor-induced diacylglycerol formation and protein kinase C activation by colon tumor-promoting bile acid in Swiss 3T3 cells: Different modes of action between bile acid and phorbol ester

A small amount (50–200 μM) of deoxycholate (DOC), a colon tumor‐promoting bile acid, did not show a direct effect on protein kinase C activity in a cell‐free system, but enhanced fibroblast growth factor (FGF)‐induced diacylglycerol formation and protein kinase C activation in Swiss 3T3 cells. DOC potentiated both reactions induced by submaximal doses of FGF but showed little effect on the maximal levels of the reactions. DOC alone was inactive in eliciting both reactions in the absence of FGF. DOC did not affect the binding of FGF to the cells. Since it has been described that diacylglycerol serves as a messenger for the activation of protein kinase C in the action of FGF in Swiss 3T3 cells [(1985) FEBS Lett. 191, 205‐210], these results suggest that a small amount of DOC increases the sensitivity to FGF of diacylglycerol formation and thereby potentiates protein kinase C activation in this cell line. This action of DOC was in marked contrast to that of 12‐O‐tetradecanoylphorbol‐13‐acetate, a potent tumor‐promoting phorbol ester, which directly activated protein kinase C in cell‐free and intact cell systems.

Enhancement of collagen-induced phosphoinositide turnover by thromboxane A2 analogue through Ca2+ mobilization in human platelets

In human washed platelets, collagen‐induced phosphoinositide turnover was inhibited by indomethacin, an inhibitor of thromboxane A2 (TXA2) formation, particularly at lower doses of collagen. This inhibition was counteracted by the addition of 9,11‐epithio‐11,12‐methano‐TXA2 (STA2), a stable analogue of TXA2 as well as by the Ca2+ ionophore A23187. STA2 and A23187 did not stimulate phosphoinositide turnover markedly, but significantly increased cytoplasmic free Ca2+ concentrations. The actions of STA2 were blocked by 13‐azaprostanoic acid, a TXA2 receptor antagonist. These results suggest that TXA2 is generated during the action of collagen and increases cytoplasmic free Ca2+ which then stimulates phosphoinositide turnover in cooperation with collagen.

Similar physical and kinetic properties of rat brain synaptic membrane and cytosol phosphoinositide phospholipases C

Phosphoinositide phospholipase C (PLC) was extracted from the synaptic membrane fraction of rat brain by 1% sodium deoxycholate. The molecular weight and sedimentation coefficient of the membrane PLC were about 160,000 and 6.7 as estimated by gel filtration and sucrose density gradient centrifugation, respectively. These values of the membrane PLC were identical with those of the cytosol PLC of the same tissue. Moreover, the membrane PLC showed the substrate specificity for phosphatidylinositol, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate and the sensitivity to Ca2+, sodium deoxycholate and N-ethylmaleimide similar to those of the cytosol PLC. These results indicate that the rat brain synaptic membrane PLC is indistinguishable from the cytosol PLC in physical and kinetic properties.

Small molecular weight GTP-binding proteins and signal transduction.

We have separated multiple GTP-binding proteins (G proteins) having Mr values of about 20,000 (small Mr G proteins) from bovine brain membranes, purified to near homogeneity and characterized two novel G proteins designated as smg p25A and smg p21, the c-Ki-ras protein (c-Ki-ras p21) and the two rho proteins (rho p20 and rho p21). smg p25A is present abundantly in brain and adrenal medulla. This G protein is also found in rat pheochromocytoma PC-12 cells, and its mRNA level increased after differentiation of the cells into neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP. These results suggest that smg p25A plays an important role in the regulation of neuronal functions. In contrast, smg p21 is found in most tissues. This G protein has the same putative effector domain as ras p21s, suggesting that smg p21 exerts the actions similar and/or antagonistic to those of ras p21s. In fact, smg p21 has been found to be identical with the protein encoded by the Krev-1 gene recently isolated as a gene suppressing the transforming action of Ki-ras p21 in NIH/3T3 cells. On the other hand, rho p20 and rho p21 are ADP-ribosylated by an ADP-ribosyltransferase contained or contaminated in botulinum toxin type C1, presumably C3. Botulinum ADP-ribosyltransferase C3 has recently been shown to induce morphological changes similar to those induced by ras p21 in fibroblasts. Thus, small Mr G proteins are part of a huge network of intracellular regulatory systems and play important roles in the regulation of various cell functions including cell transformation, proliferation and differentiation.

Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes

The direct binding protein(s) of ras p21 was (were) investigated in inside‐out vesicles of human erythrocyte ghosts using the pure v‐Kirsten (Ki)‐ras p21 synthesized in E. coli. The bound ras p21 was detected immunochemicallly using an anti‐v‐Ki‐ras p21 monoclonal antibody. ras p21 bound to vesicles. Prior digestion of the vesicles with trypsin reduced this binding significantly. When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS‐polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6. ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein. These results indicate that v‐Ki‐ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell‐free system.