Immunochemical recognition of phosphate ester derivatives of phenol rings is dependent upon the electronic charge of the ester group

The recognition of phosphate and sulphate esters of tyrosine residues has been studied employing antisera with specificity for tyrosine phosphate, and the enzymes aryl sulphatase, and acid and alkaline phosphatases. The ability of tyrosine phosphate, and of phosphate esters of phenol, to inhibit the antiserum was pH dependent. The capacity to effect inhibition appeared to correlate with alterations in the ionisation of the inhibitor. Moreover, the antisera with reactivity for tyrosine phosphate had no reactivity with tyrosine sulphate or sulphate esters of phenol at any pH value studied. The enzymes alkaline phosphatase, acid phosphatase, and aryl sulphatase were also studied. The phosphatases were found not to hydrolyse sulphate ester containing substrate analogues at any pH value in the range 5.0–9.0. In contrast, aryl sulphatase appeared to hydrolyse phosphate esters at pH 5.0 and 7.0, but not at pH 9.0.Abbreviations ABP Azobenzyl phosphonate - KLH-ABP Keyhole limpet haemocyanin derivatised with azobenzyl phosphonate groups - OVA-ABP Ovalbumin derivatised with azobenzylphosphonate groups     

Protein Kinase C-Activating Tumor Promoters Enhance the Differentiation of Astrocytes in Aggregating Fetal Brain Cell Cultures

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a marked, rapid, and sustained increase in the activity of the astrocyte-specific enzyme glutamine synthetase (GS). This effect was accompanied by a small increase in RNA synthesis and a progressive reduction in DNA synthesis. Only mitotically active cultures were responsive to PMA treatments. Since in aggregate cultures astrocytes are the preponderant cell type, both in number and mitotic activity, it can be concluded that PMA induces and/or enhances the terminal differentiation of astrocytes. The developmental expression of GS was also greatly stimulated by mezerein, a potent nonphorbol tumor promoter, but not by 4 alpha-phorbol 12,13-didecanoate, a nonpromoting phorbol ester. Since both tumor promoters, PMA and mezerein, are potent and specific activators of C-kinase, it is suggested that C-kinase plays a regulatory role in the growth and differentiation of normal astrocytes.     

Phorbol Esters Increase GTP-Dependent Adenylate Cyclase Activity in Rat Brain Striatal Membranes

Abstract: 4β-Phorbol 12-myristate 13-acetate (PMA), added to a lysed mitochondrial fraction of rat striatum, stimulates adenylate cyclase activity with an apparent time lag of ～30 s. Half-maximal and maximal enzyme stimulations are obtained with 8 and 200 nM PMA, respectively. The PMA stimulation is GTP dependent, reaching a maximum of ～60% at 50 μ.M GTP, and is associated with disappearance of the enzyme inhibition induced by micromolar concentrations of GTP. Enhancement of enzyme activity by cholera toxin and 3,4-dihydroxyphenylethylamine is amplified by PMA only at micromolar concentrations of GTP. PMA does not affect the enzyme stimulation by forskolin but reverses the inhibition of forskolin-stimulated enzyme by GTP. When guanyl-5′-yl-imidodiphosphate is substituted for GTP, PMA does not modify adenylate cyclase activity. Enzyme inhibition by acetylcholine, Leu-enkephalin, and R(-)N⁶-(2-phenylisopropyl)adenosine is magnified by PMA. Stimulation of adenylate cyclase by PMA is markedly reduced following EGTA treatment, is not observed when adenyl-5′-yl-imidodiphosphate is substituted for ATP as substrate for adenylate cyclase, and is enhanced by l-α-phosphatidyl-l-serine. Like PMA, 4β-phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol stimulate striatal adenylate cyclase, whereas 4β-phorbol and 4β-phorbol 13-acetate are ineffective. The results indicate that phorbol esters increase striatal adenylate cyclase activity by reducing the GTP-induced inhibition of the enzyme, presumably as a result of protein kinase C activation.     

Effects of phorbol esters and a calcium ionophore on angiotensin II binding in rat brain synaptosomes

In previous studies we determined that protein kinase C (PKC) and calcium are important intracellular regulators of neuronal angiotensin II (Ang II) binding sites. In the present study we investigated the effects of the protein kinase C (PKC) agonist phorbol esters (PE) and also a calcium ionophore (A23187) on the specific binding of [¹²⁵I]Ang II to brain synaptosomes prepared from rats of different ages. The rationale was to determine whether the larae changes in the level of brain Ang II specific binding observed in different age rats are due to changes in the regulation of these sites by PKC or by calcium. The present data indicate no qualitative differences in the effects of PE or A23187 on [¹²⁵I]Ang II specific binding to hypothalamic or brain stem synaptosomes, from either 2–5 or 70-day-old rats, i.e. the active PE TPA increased while A23187 decreased Ang II binding in all situations. Thus, the dramatic differences in brain Ang II specific binding seen with age appear not to be due to changes in regulation by PKC or calcium.     

Phenotypic effects of overexpression of PKC beta 1 in rat liver epithelial cells

We have used a previously described retroviral expression vector pMV7-PKC beta 1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold-higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage-independent growth, or tumorigenicity. K16PKC-4 and K22PKC-2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC-4 and K22PKC-2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC-4 and K22PKC-2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC-4 and K22PKC-2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKC beta 1 is not itself sufficient to cause cell transformation.     

Epidermal growth factor-stimulated DNA synthesis requires an influx of extracellular calcium

The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)-induced rat liver epithelial (T51B) cell DNA synthesis. Fura-2-loaded T51B cells responded with an increase in [Ca2+]i to EGF (5-50 ng/ml) that was blocked by low (25 microM) extracellular Ca2+ or by pretreatment with 50 microM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1-50 ng/ml) dose-dependently incorporated [3H]-thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF-stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis.     

HL-60和Raji细胞在TPA作用下原癌基因结构和表达改变

本文观察了TPA诱导前后HL-60和Raji细胞中c-myc,c-H-ras,c-sis和c-erb A,B的表达情况,并对c-myc,c-H-ras的基因结构作了初步的测定。实验结果表明,两株细胞中四种原癌基因的表达水平各不相同。TPA诱导1,伴随HL-60细胞的分化,c-myc和c-H-ras基因表达最下降;c-sis表达量不变。Raji细胞在TPA处理后c-H-ras表达量有所下降,c-myc表达量不变。本文提示,多种癌基因的表达和/或癌基因的表达增加可能是致癌的原因之一。     

Free and conjugated desmethylsterol composition of Zea mays hybrids exposed to mild osmotic stress

Five hybrids of Zea mays (A619 × H60, B73 × MO17, B73 × PA91, B73 × VA17. A632 × H96) were grown hydroponically at osmotic potentials of -0.1 (control), -0.4 and -0.6 MPa. Dry weights of hybrids B73 × VA17 and A632 × H96 decreased significantly at -0.4 and -0.6 MPa. Few changes were observed in the free sterol, steryl glycoside and steryl ester fractions. However, steryl esters of the roots and stems of osmotically treated plants declined in most of the hybrids. The stigmasterol to sitosterol ratio increased in all three steryl fractions in the roots of B73×VA17 and A632×H96 as a result of the osmotic treatments. B73×VA17 and A632×H96 also exhibited the greatest initial electrolyte leakage when leaf dises were subjected to osmotic stress. A632×H96 and possibly B73×VA17 were less tolerant to osmotic treatments than the other hybrids. Modification of sterols may reflect initial events that lead to the stabilization or destabilization of cellular membrances which in turn may affect the tolerance of plants to stressful environments.     

Phorbol ester stimulates the synthesis of sphingomyelin in NIH 3T3 cells. A diminished response in cells transformed with human A-raf carrying retrovirus

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [¹⁴C]choline-labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerP-Cho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of TPA (2–100 nM) had progressively less effect on CerPCho synthesis which correlated with increased hydrolysis of precursor PtdCho. In cells transformed with human or mouse A-raf carrying retroviruses TPA-stimulated PtdCho hydrolysis, but not CerPCho synthesis, suggesting independent regulation of these processes by the TPA-stimulated signal transduction system.