Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.     

Differential enzyme activities in human esterase D phenotypes

Summary Red cell ESD activities have been determined in 78 individuals with ESD-1 phenotype, in 94 with ESD2-1 and in 28 with ESD-2 phenotype. The mean activities of these three groups were 276.7, 216.6 and 171.5 expressed as 10^-7 M methyl-umbelliferone produced/h/g Hb, respectively. The activities associated with a single ESD ^* 1 gene are estimated to be 60% higher than ESD ^* 2.     

Increase in esterase activity during maturation of Malus pumila fruit

In a range of dessert cultivars of Malus pumila, the esterase in the fruit showed a rise in activity during the period of fresh weight gain. This was the case when activity was expressed on a protein or a fresh weight basis. Both fruit which matured at 100 days after anthesis, and fruit which matured later, showed a similar rate of increase. However, a cultivar used for processing rather than eating showed a much reduced rate of esterase increase.     

Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes

We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun. 101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-¹⁴C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process.     

Glutathione S-transferase in human lymphoid cell lines and fractionated peripheral leucocytes.

Glutathione S-transferase activity was identified in cytosol from human lymphoid-cell lines and peripheral leucocytes (polymorphonuclear-leucocyte/monocyte and small-lymphocyte fractions) and compared with human liver enzyme. The findings of closely similar elution volume in gel filtration, substrate (1-chloro-2,4-dinitrobenzene) and inhibitory (probenecid) kinetics indicate that the liver, leucocyte and lymphoid-cell transferases are closely related. The interaction of reduced glutathione and 1-chloro-2,4-dinitrobenzene was shown to occur in intact-lymphoid-cell culture, to be linear with time and quantity of cells and to have kinetics similar to those of the enzyme reaction catalysed by cytosol.     

Polymorphism and modulation of cell wall esterase enzyme activities in the chicory root during the growing season

Pectins are major components of the primary plant cell wall. They can be both methylesterified and acetylesterified and de-esterification occurs by specific esterases. Proteins extracted by NaCl treatment from root cell walls of two chicory varieties (Cichorium intybus L. cv. Nausica and Arancha) sampled in an experimental field every 2 weeks between July 2002 and January 2003 were analysed by isoelectrofocalization, semi-denaturing SDS-PAGE, and quantitative assays for their esterase activity. Zymograms showed that chicory root pectin methylesterases belong to a multigene family. The isoelectric points of the pectin methylesterase isoforms ranged from pI 3.8 to pI 9.0. Concerning acetylesterases, only acidic isoforms between pI 4.1 and pI 5.2 were observed, but a large polymorphism of this class of enzymes could be identified in one variety. The results indicate that the root pectin methylesterase activity of the Nausica variety was correlated with ambient temperature, while no significant effect of temperature could be detected on any acetylesterase isoform.     

Characterization of Sertoli cell RNA synthetic activities in vitro at selected times during sexual maturation

In this study, Sertoli cell RNA synthetic activity in vitro was characterized at selected times during sexual maturation. Sertoli cells, isolated from rat testes undergoing the first wave of spermatogenesis and placed in culture for 4 days, exhibited 2-fold increases in soluble ribonucleotide pools and in total RNA concentrations over the age span of 18-35 days. High performance liquid chromatographic analysis of the ribonucleotide pools in Sertoli cells cultured from 18- and 33- to 34-day-old rats revealed that, in addition to the overall age-related doubling of concentrations, uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools were disproportionately increased 4- and 6-fold, respectively. In general, Sertoli cell contained relatively small amounts of UTP in comparison to several other cell types, but exhibited a high ADP:ATP ratio. A uniform 2-fold increase in the base composition of Sertoli cell RNA per mg DNA was observed over the age span of 18-35 days, with no preferential increase in any one specific nucleotide. There was no change in [3H]uridine incorporation (2 h) into RNA per cell (pmol/mg DNA), but decreased specific activity of the RNA (pmol/mg RNA) in Sertoli cells cultured from 35-day-old rats as compared to those from 18- to 19-day-old rats. Similar differences were noted in the specific activity of label incorporated into specific RNA bases. In contrast, the specific activity of the UTP-CTP soluble pool/mg DNA was only slightly increased. These data indicate that processes related to RNA synthesis in the Sertoli cell undergo a number of changes during the period of sexual maturation.     

Changes in proteolytic activities of human leukemic promyelocytes (HL-60 cells) during maturation

Proteolytic activity was measured in human leukemic promyelocytic cell line (HL-60) grown in culture, before and after the addition of agents which promote differentiation. The 36000 X g soluble fraction of the cells degraded [14C]globin with maximal activity at pH 3.6, while the insoluble fraction had a pH optimum at 8.0. This pattern did not change upon differentiation. The acid protease activity of the soluble fraction increased following differentiation. After 4 days in the presence of dimethyl sulfoxide, the differentiated cells exhibited 4-fold higher specific activity as compared with 4 day-old control cells. In contrast, the alkaline activity of the insoluble fraction of the differentiated cells was 4-fold lower than that of the undifferentiated cells. It is suggested that the changes in enzyme activities may serve the new functions acquired by the mature granulocytes.     

Protein kinase activities during maturation in xenopus laevis oocytes

Protein kinase activities have been compared in ovarian oocytes and in ovulated eggs of $\text{Xenopus laeyis}$.In ovaries and ovarian oocytes, we have detected, in addition to an already known (1) cyclic AMP stimulated phosphoprotein kinase, a second very active phosphoprotein kinase which is cAMP-independent.Besides these two activities, a third protein kinase activity becomes detectable after maturation and ovulation: it is a cAMP and cGMP-dependent histone kinase.     

Secretagogue-induced phosphoinositide metabolism in human leucocytes.

The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.     

Role of peroxidase and esterase activities during cotton fiber development

Cytoplasmic and salt-extracted wall peroxidase and nonspecific esterase activities along with growth analysis were investigated during the entire period of cotton fiber development. Both the peroxidase fractions, when assayed with chlorogenic and ferulic acids as substrates, recorded low levels during the fiber elongation phase, and a close relationship between cessation of elongation growth and increase in peroxidase activity was discernible. Nonspecific esterase activity in both cytoplasmic and salt-extracted fractions, on the other hand, showed higher activity during the elongation phase, whereas during the secondary thickening phase it decreased. The role of cytoplasmic peroxidase in IAA oxidation is discussed. It is suggested that esterases and peroxidases associated with wall fractions may well be involved in turnover of phenolic acids that are cross-linked to wall polysaccharides.     

Glucocorticoids affect human dendritic cell differentiation and maturation.

Because dendritic cells (DC) play a major role in the initiation of T cell-mediated immunity, we studied the effects of glucocorticoids, well-known inhibitors of the immune and inflammatory response, on the differentiation and maturation of human DC. DC were differentiated from human monocytes by culture with GM-CSF and IL-4 for 7 days with and without dexamethasone (Dex). Cells treated with Dex (10-8 M) (Dex-DC) developed a characteristic dendritic morphology; however, membrane phenotype analysis demonstrated that they were not fully differentiated. Dex-DC expressed low levels of CD1a and, unlike untreated cells, high levels of CD14 and CD16. Molecules involved in Ag presentation (CD40, CD86, CD54) were also impaired. In contrast, molecules involved in Ag uptake (mannose receptor, CD32) and cell adhesion (CD11/CD18, CD54) were up-regulated. After exposure to TNF-alpha or CD40 ligand, Dex-DC expressed lower levels of CD83 and CD86 than untreated cells. Dex-DC showed a higher endocytic activity, a lower APC function, and a lower capacity to secrete cytokines than untreated cells. Overall, these results indicate that DC differentiated in the presence of Dex are at a more immature stage. Moreover, Dex also partially blocked terminal maturation of already differentiated DC. In conclusion, our data suggest that glucocorticoids may act at the very first step of the immune response by modulating DC differentiation, maturation, and function.     

Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P₂X and P₂Y and PA₁. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre pubertal, mid pubertal and young adult (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.     

Naphthol-AS-D-chloroacetate esterase activity in globule leucocytes in the tracheal epithelium of rats

The enzyme naphthol-AS-D-chloroacetate esterase is histochemically demonstrated in the granules of globule leucocytes in the tracheal epithelium of rats. The enzyme reactivity may be used as a cytochemical marker of these cells. The previously postulated mast cell origin of globule leucocytes is doubted, and the possibility that globule leucocytes belong to the group of natural killer cells is discussed. The biological role of the located esterase and the function of the globule leucocytes are also briefly discussed.     

The isolation and properties of granulocytic colony-stimulating activities from medium conditioned by human peripheral leucocytes.

The colony-stimulating activity detected by its ability to promote colony formation by human granulopoietic progenitor cells was partially purified from medium conditioned by human peripheral leucocytes. The purification procedure utilized (NH4)2SO4 precipitation, DEAE-cellulose chromatography, hydroxyapatite chromatography and gel filtration on Sephadex G-150 and yielded a purification of about 1000-fold. The medium from cultures of non-leukaemic cells contained three molecular species of colony-stimulating activity with approximate molecular weights of 93000, 36500 and 14700. On the basis of their sensitivity to enzymes, these species of activity appeared to be proteins. In contrast, medium from cultures of leukaemic cells contained only one detectable molecular species with colony-stimulating activity, usually with an approximate molecular weight of 36500. The results are discussed in relation to concurrent studies on the association of the different species of colony-stimulating activity with the cell surface membrane.