Cellular Acid Phosphatase Activity Research Articles

Antimutagenicity of supercritical CO2 extracts of Terminalia catappa leaves and cytotoxicity of the extracts to human hepatoma cells.

Natural antimutagens may prevent cancer and are therefore of great interest to oncologists and the public at large. Phytochemicals are potent antimutagen candidates. When the Ames test was applied to examine the antimutagenic potency of supercritical carbon dioxide (SC-CO(2)) extracts of Terminalia catappa leaves at a dose of 0.5 mg/plate, toxicity and mutagenicity were not detected. The antimutagenic activity of SC-CO(2) extracts increased with decreases of temperature (60, 50, and 40 degrees C) and pressure (4000, 3000, and 2000 psi) used for extraction. The most potent antimutagenicity was observed in extracts obtained at 40 degrees C and 2000 psi. At a dose of 0.5 mg of extract/plate, approximately 80% of the mutagenicity of benzo[a]pyrene (B[a]P, with S-9) and 46% of the mutagenicity of N-methyl-N '-nitroguanidine (MNNG, without S-9) were inhibited. Media supplemented with SC-CO(2) extracts at a range of 0-500 microg/mL were used to cultivate human hepatoma (Huh 7) and normal liver (Chang liver) cells. The viability of the cells was assayed by measuring cellular acid phosphatase activity. A dose-dependent growth inhibition of both types of cells was observed. The SC-CO(2) extracts were more cytotoxic to Huh 7 cells than to Chang liver cells. The observation that SC-CO(2) extracts of T. catappa leaves did not induce mutagenicity at the doses tested while exhibiting potent antimutagenicity and were more cytotoxic to human hepatoma cells than to normal liver cells is of merit and warrants further investigation.

Phosphatase activity and copper uptake during growth of Aspergillus niger

The fungus Aspergillus niger produced extracellular and cellular acid phosphatase activities during growth in the presence or absences of copper ions in the medium. The levels of both phosphatase activities depended on the copper concentrations in the medium. Enzyme activities were maximal at the mid-exponential growth phase, as well as at the higher copper concentration and decreased as growth time increased from 12 to 48 h. The total uptake of copper (II) by mycelia, growing at the presence of copper was highest when the levels of enzyme activities were maximal. Between 10 and 20% copper (II) was not removed by washing in 0.1 M H 2SO 4, suggesting that this copper (II) was bound intracellularly by mycelia of different age. On the other hand, copper ions slightly inhibited cellular acid phosphatase activity of Aspergillus niger with a K i of 0.89 mM. Acid phosphatase overproduction, as well as copper uptake of mycelia indicated a possible participation of acid phosphatases in the resistance of the fungal strain to the copper toxicity.

Characterization of glucocorticoid inhibition of antigen-induced inositolphosphate formation by rat basophilic leukemia cells: possible involvement of phosphatases

The suppress effect of glucocortocoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1–30 min enhanced production of [ 3 H]inositol monophosphate (IP 1), inositol 1,4-biphosphate (IP 2) and inositol 1,4,5-triphosphate (IP 3) by about 5–10-fold. Pretreatment with hydrocortisone (HC) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol 4,5-bisphosphate (PIP 2) by an average of 50%. Maximal inhibition of hydrolysis of PIP 2 and reduction in stimulation of IP 3 formation was reached after 4 h of preincubation with 2·10 −6 M of HC. Cycloheximide and RU486, a GC receptor antagonist, completely prevented the inhibitory effect of HC on IP formation. Other GC, dexamethasone (DEX) and triamcinolone (each at 2·10 −7 M) markedly suppressed antigen induced IP 3 production, while aldosterone and sex steroids such as estradiol and progesterone (each at 2·10 −6 M) were virtually inactive. Antigen-stimulated phosphorylation of a 18 kDa and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. DEX also doubled the activity of cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is specific, receptor-mediated, dependent on protein synthesis and possibly mediated by protein phosphatase activity.

Glucocorticoid inhibition of antigen-induced inositol phosphate formation: possible involvement of phosphatases.

The suppressive effect of glucocorticoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1-30 min enhanced production of [3H]inositol monophosphate (IP1), inositol 1,4-biphosphate (IP2) and inositol 1,4,5-triphosphate (IP3) by about 5-10 fold. Pretreatment with hydrocortisone (HC) and dexamethasone (DEX) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol-4-5-biphosphate (PIP2) by an average of 50% Antigen-stimulated phosphorylation of an 18 kDA and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. Moreover, DEX doubled cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is possibly mediated, among other things, by protein phosphatase activity.

Acid phosphatase activity in monocytes and sera of patients with Hodgkin's disease

Acid phosphatase (AP) levels have been found to be decreased in monocytes of 38 patients with Hodgkin's disease as compared to 16 healthy subjects. Low cellular AP activity was associated with the presence of active disease, stage IV and lymphocyte depletion type. Lactate dehydrogenase (LDH) was decreased in monocytes of patients but the difference did not reach statistical significance. In parallel, serum AP activities have been found to be increased in patients. There was an inverse correlation between the monocyte and serum AP levels which can be explained by assuming AP secretion from monocytes of patients in vivo.

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells.

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.

Cytochemical determination of acid phosphatase activity in isolated rat hepatocytes during starvation-induced proteolysis.

Acid phosphatase activity in isolated rat liver parenchymal cells has been investigated with quantitative histochemical means during short-term starvation, which leads to a considerable loss in protein mass in the parenchyma. Animals trained to a meal-feeding regime in which food was available during 1 h only per 24 h (using an automatic food dispensing machine), were sacrificed 6, 12, 18, 24 and 36 h after food had been withheld at the time point (23.00 h) of meal feeding. Acid phosphatase activity was analysed cytophotometrically in isolated hepatocytes incorporated into polyacrylamide gels before the enzyme reaction technique with the post-azo coupling was carried out. No indication could be found for any significant changes in the amount of acid phosphatase activity per individual hepatocyte during the entire period of fasting, as compared with two time points (11.00 and 23.00 h) before the theoretical onset of fasting. It is concluded that the considerable enhancement of protein degradation in the lysosomal apparatus during fasting is not reflected by changes in the cellular acid phosphatase activity.

Cellular acid phosphatase activity: Correlation of cytochemical and biochemical measurements

Methods for comparing results of cellular acid phosphatase activities obtained by quantitative cytospectrophotometry with those obtained by biochemical analysis are needed to express the cytospectrophotometric data in biochemical units. Since naturally occurring cells have differing amounts of acid phosphatase, enzyme activity was measured cytochemically and biochemically in polymorphonuclear leukocytes and peritoneal and alveolar macrophages from male rats to determine if these measurements permitted construction of a line correlating the two parameters. Cellular acid phosphatase activity, as measured cytospectrophotometrically and biochemically, increased proportionately with polymorphonuclear leukocytes having the lowest activities and alveolar macrophages the highest. These values when subjected to linear regression analysis fixed a line with a correlation coefficient of 0.95 demonstrating that cytochemical and biochemical activities of acid phosphatase activity can be correlated using naturally occurring cells.

The adaptive acid phosphatase of Tetrahymena pyriformis GL-9. Effect of endocytosis of various nutrients.

An adaptive increase in the cellular acid phosphatase activity ofTetrahymena pyriformis GL-9 was produced in response to ingestion of particulate materials such as yeast cells, bacteria or puff-ball spores. The particulate component of disrupted yeast cells also produced the response, whereas it was not initiated by the soluble cellular components of yeast. Macromolecules such as glycogen and ribonucleic acid evoked the response, whereas glucose, casein hydrolysate, albumin and latex particles failed to do so. It is suggested that this lysosomal enzyme increase is related to the quality of the material ingested and not to its particulate nature or to the endocytic uptake process itself; although a joint effect of endocytosis (apart from physically taking in the material) and quality of material cannot be excluded. The results also indicate that there is a relationship between ingestion of certain materials and cell division, even when taking place in a soluble growth medium which is entirely adequate nutritionally.

Endocytosis in Tetrahymena pyriformis: The selectivity of uptake of particles and the adaptive increase in cellular acid phosphatase activity

Summary The cellular acid phosphatase activity of Tetrahymena pyriformis , grown in peptone-yeast extract medium, is markedly increased after edocytosis of yeast suspension, but not by uptake of various sizes of polystyrene or polyvinyltoluene latex particles. This suggests that the adaptive enzyme increase in response to endocytosis and intracellular digestion is not due solely to the act of endocytosis itself and that the quality of the material taken up is itself involved. It is calculated that about ten latex particles (1.95 μ diameter) are taken up per average Tetrahymena per minute. This is equivalent to a volume uptake of 42 μm 3 /min/average cell. An area of membrane greater than the entire surface area of the cell is calculated to have been involved during the 150 min period of endocytosis. Starved Tetrahymena showed increased cellular acid phosphatase activity after endocytosis of yeast suspension or of peptone-yeast extract, but did not endocytize latex particles to any great extent. Microscopic examinations showed that fed Tetrahymena rapidly endocytized useful materials such as peptone, yeast suspension or washed living bacteria and also indigestible materials such as latex particles and ink suspensions. Starved cells on the other hand, took in only the useful materials. Latex particles presented to the starved cells at the same time as peptone were however rapidly taken up. Other results suggest that cells grown in peptone-yeast extract medium take up particles at rates proportional to their relative numerical concentrations and that this uptake is not markedly related to particle size. Digestible particles are not taken up in preference to indigestible ones under these conditions. The results are discused.

DNA-synthesis in peritoneal mononuclear leucocytes

DNA synthesis by mouse peritoneal mononuclear leucocytes was studied during their differentiation into macrophages in vivo and in vitro. A direct correlation was found between the rate of DNA synthesis, estimated by the incorporation of 3H-thymidine into the cells, and their degree of macrophage differentiation, measured as cellular acid phosphatase activity. Radioautographs of the cells showed that DNA synthesis is limited to morphologically well-differentiated macrophages.