Decrease of acid phosphatase activity in murine peritoneal macrophages infected with Leishmania donovani

The effect of infection with Leishmania donovani on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with L. donovani were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages were decreased significantly by infection with L. donovani. An inverse relation existed between concentration of acid phosphatase activity and the number of intracellular L. donovani. Reduced concentrations of acid phosphatase activity were also observed in macrophages uninfected but exposed to L. donovani. The isoenzyme composition in macrophages did not change during the course of infection with L. donovani. These results demonstrate that L. donovani reduces the acid phosphatase activity of macrophages.     

Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa)

Octopus vulgaris infected with Aggregata octopiana were collected from an open-water culture system in the Ría of Aldán (NW Spain). Digestive tract infection values were determined with the use of a Neubauer chamber by counting the number of A. octopiana sporocysts. After determining enzyme activity values by the colorimetric Api-Zym system Biomerieux, one representative enzyme of glycosidases, peptid hydrolases and phosphoric hydrolases showing high activity was spectrophotometrically analysed. The enzymes were maltase and leucine-aminopeptidase (LAP) involved in the absorption process, and acid phosphatase, a lysosomic enzyme, respectively. Enzymatic activity of maltase and LAP decreased significantly, with increased sporocyst counts. However, acid phosphatase activity increased with severity of infection, indicating the presence of degradative enzymes from phagocytic cells in the infected area. A detrimental effect on gastrointestinal function may result from a decrease or malfunction of absorption enzymes. The results suggest a malabsorption syndrome resulting from parasitic infection.     

Change in the isoenzyme composition of acid phosphatase in murine peritoneal macrophages exposed to or infected with Trypanosoma cruzi

The effect of infection with Trypanosoma cruzi on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated by using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with T. cruzi were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages did not change significantly by infection with T. cruzi. Also, the concentration levels of acid phosphatase activity did not change in macrophages uninfected but exposed to T. cruzi. On the other hand, the isoenzyme composition of acid phosphatase did change in macrophages exposed to or infected with T. cruzi. These results demonstrate that Trypanosoma cruzi affects the acid phosphatase composition of macrophages.     

Alkaline phosphatase activation and collagen synthesis in human skin fibroblasts in culture

It has been noted in regenerating wounds that alkaline phosphatase activity in fibroblasts reaches a maximum when the collagen production is greatest. Tissue culture studies were carried out to show that prednisolone phosphate, while increasing the specific activity of alkaline phosphatase in human diploid skin fibroblasts, did not affect accumulation of collagen-hydroxyproline in monolayers or media. Addition of sodium ascorbate, resulted in rapid accumulation of hydroxyproline in the culture over a 13-day interval, while alkaline phosphatase activity increased only slightly over the last 6 days. When prednisolone and ascorbate were added in combination, alkaline phosphatase activity was significantly increased: but accumulation of hydroxyproline was no greater than in cultures to which ascorbate alone was added. Activation of alkaline phosphatase induced by prednisolone phosphate does not appear to be directly related to the biosynthesis of collagen in human skin fibroblasts in tissue culture.     

Endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein.

On the assumption that dephosphorylation of the neurotropic coronavirus JHM (JHMV) nucleocapsid protein (N) may be connected with initiation of the infectious cycle we searched for a relevant host enzyme activity. Analysis of subcellular fractions from L-2 murine fibroblasts, separated by dual Percoll density gradients, revealed the presence of a phosphoprotein phosphatase (PPPase), co-sedimenting with the endososomal/prelysosomal material, which possesses high activity against N. With purified [32P]N as substrate it was demonstrated that this PPPase, distinguishable from acid and alkaline phosphatases, acts optimally at neutral pH in the presence of Mn2+ following treatment with a detergent. Complete inhibition with okadaic acid at 0.9-4.5 microM but not at 1-10 nM relegates this PPPase to a type 1 protein phosphatase. Similar PPPase activity for N was present in the endosome fraction of a rat Roc-1 astrocytoma-oligodendrocyte cell line and in homogenates of brain and cultured oligodendrocytes. Our data suggest that the phosphorylated N of the inoculum may be modified by the endosomal PPPase in host cells, including those from the CNS so as to facilitate the JHMV infectious process.     

Study of lysosomal enzymes in chick embryo fibroblasts infected with microorganisms of family Halprowiaceae (Chlamydiaceae)

It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.     

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.     

Acid phosphatase activity of human gingival fibroblasts measured using a simultaneous coupling semi-permeable membrane technique

Summary Acid phosphatase activity has been measured in cultured human gingival fibroblasts using a validated histochemical simultaneous coupling semi-permeable membrane technique. The histochemical reaction was linear over a three hour incubation period and had a pH optimum of 5.0. The activity was not increased by prior exposure to hypotonic acetate buffer and was inhibited by fluoride and molybdate but not by formaldehyde. These results indicate that the semi-permeable membrane technique described may be used for observing and measuring acid phosphatase activity in cultured fibroblasts. From results obtained using inhibitors, it appears that in these cells most of the acid phosphatase observed is lysosomal. The absence of any activation of activity following pre-incubation with hypotonic buffer indicates that the method is not suitable for monitoring lysosomal membrane function.     

Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.     

Inhibition of protein tyrosine phosphatase activity by diamide is reversed by epidermal growth factor in fibroblasts.

Diamide (azodicarboxylic acid bis(dimethylamide] inhibits protein tyrosine phosphatase activity in fibroblasts without altering protein tyrosine kinase activity associated with the epidermal growth factor receptor. The loss of protein tyrosine phosphatase activity caused by diamide is reversed by 2-mercaptoethanol or epidermal growth factor.     

Cytophotometric evaluation of alkaline phosphatase activity in neutrophilic granulocytes and fibroblasts during wound healing in rats

The most informative indexes of alkaline phosphatase activity in neutrophil granulocytes from the peripheral blood and in neutrophil granulocytes and fibroblasts from the wound were defined in the experimental study carried out on 60 rats. These indexes were used to reveal the relationship between blood system reactions and inflammatory and regeneration processes in the wound tissue. The division of neutrophil granulocytes into three functional groups in accordance with alkaline phosphatase activity was demonstrated to be reasonable. The progress of inflammatory and regeneration processes in the wound tissues was shown to be adequately reflected in changes of both general and relative number of blood circulating neutrophil granulocytes of the third functional group characterized by high alkaline phosphatase activity. The results of the study demonstrate that the cytophotometric method is highly informative. It can be used in the clinical practice for an objective evaluation of the wound healing process as well as for an estimation of the treatment efficacy.     

Enzyme activity during the growth and aging of human cells in vitro

Acid phosphatase, alkaline phosphatase, and lactic dehydrogenase activities have been compared in normal human diploid cell strains and in SV₄₀-transformed heteroploid cell lines derived from them. A higher level of acid phosphatase activity was observed in diploid cultures derived from adult lung than in cultures derived from fetal lung of similar passage levels. The alkaline phosphatase activity of normal diploid fibroblasts was significantly higher than that of SV₄₀-transformed cell lines derived from them. Generally, the lactic dehydrogenase activities of all these cell cultures were similar. Human diploid cells in culture “age,” in the sense that their ability to proliferate decreases with time during serial subcultivation. Evaluation of the activities of these three enzymes during the “aging” process showed that, although alkaline phosphatase and lactic dehydrogenase activities were similar in “young” and “senescent” cells, acid phosphatase showed a small but significant increase in the senescent cells.     

Involvement of the tyrosine phosphorylation on GSH transport in NIH3T3 fibroblasts

This study demonstrates the involvement of phosphotyrosine phosphatases on the activity and regulation of GSH ATP-dependent transport system that we have previously identified in NIH3T3 fibroblasts. This is shown by the fact that increases of the initial rate of GSH uptake were measured in NIH3T3 overexpressing a synthetic gene coding for a low-Mr-phosphotyrosine protein phosphatase (LMW-PTP), while decreases were obtained in NIH3T3 overexpressing the phosphatase inactive mutant (LMW-C12SPTP), with respect to NIH3T3neo. Moreover, these results have been confirmed by experiments performed in the same cells by vanadate, and H2O2 treatment on both GSH transport and mediated passive transport of glucose. A possible regulation of this transport system by platelet-derived growth factor receptor (PDGFr) with tyrosine kinase activity is also demonstrated. Moreover, these data show a relationship among GSH, PDGFr and phosphotyrosine phosphatase activity, and suggest a role of GSH transport systems on the cell proliferation process.     

Modulation of queuine uptake and incorporation into tRNA by protein kinase C and protein phosphatase

It has been suggested that the rate of queuine uptake into cultured human fibroblasts is controlled by phosphorylation levels within the cell. We show that the uptake of queuine is stimulated by activators of protein kinase C (PKC) and inhibitors of protein phosphatase; while inhibitors of PKC, and down-regulation of PKC by chronic exposure to phorbol esters inhibit the uptake of queuine into cultured human fibroblasts. Activators of cAMP- and cGMP-dependent kinases exert no effect on the uptake of queuine into fibroblast cell cultures. These studies suggest that PKC directly supports the activity of the queuine uptake mechanism, and that protein phosphatase activity in the cell acts to reverse this. Regardless of the modulation of uptake rate, the level of intracellular queuine base saturates in 6 h. However, there is still an effect on the incorporation rate of queuine into tRNA of fibroblast cultures even after 24 h. We now show that the incorporation of queuine into tRNA in cultured human fibroblasts by tRNA-guanine ribosyltransferase (TGRase) is also stimulated by activators of PKC and inhibitors of protein phosphatase: while inhibitors of PKC decrease the activity of this enzyme. These studies suggest that PKC supports both the cellular transport of queuine and the activity of TGRase in cultured human fibroblasts, and that protein phosphatase activity in fibroblasts acts to reverse this phenomenon. A kinase-phosphatase control system, that is common to controlling both intracellular signal transduction and many enzyme systems, appears to be controlling the availability of the queuine substrate and the mechanism for its incorporation into tRNA. Since hypomodification of transfer RNA with queuine is commonly observed in undifferentiated, rapidly growing and neoplastically transformed cells, phosphorylation of the queuine modification system may be critical regulatory mechanism for the modification of tRNA and subsequent control of cell growth and differentiation.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by reversible phosphorylation: modulation of enzymatic activity by low density lipoprotein, sterols, and mevalonolactone

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase exists in interconvertible active and inactive forms in cultured fibroblasts from normal and familial hypercholesterolemic subjects. The inactive form can be activated by endogenous or added phosphoprotein phosphatase. Active or partially active HMG-CoA reductase in cell extracts was inactivated by a ATP-Mg-dependent reductase kinase. Incubation of phosphorylated (inactive) HMG-CoA reductase with purified phosphoprotein phosphatase was associated with dephosphorylation (reactivation) and complete restoration of HMG-CoA reductase activity. Low density lipoprotein, 25-hydroxycholesterol, 7-ketocholesterol, and mevalonolactone suppressed HMG-CoA reductase activity by a short-term mechanism involving reversible phosphorylation. 25-Hydroxycholesterol, which enters cells without the requirement of low density lipoprotein-receptor binding, inhibited the HMG-CoA reductase activity in familial hypercholesterolemic cells by reversible phosphorylation. Measurement of the short-term effects of inhibitors on the rate of cholesterol synthesis from radiolabeled acetate revealed that HMG-CoA reductase phosphorylation was responsible for rapid suppression of sterol synthesis. Reductase kinase activity of cultured fibroblasts was also affected by reversible phosphorylation. The active (phosphorylated) reductase kinase can be inactivated by dephosphorylation with phosphatase. Inactive reductase kinase can be reactivated by phosphorylation with ATP-Mg and a second protein kinase from rat liver, designated reductase kinase kinase. Reductase kinase kinase activity has been shown to be present in the extracts of cultured fibroblasts. The combined results represent the initial demonstration of a short-term regulation of HMG-CoA reductase activity and cholesterol synthesis in normal and receptor-negative cultured fibroblasts involving reversible phosphorylation of both HMG-CoA reductase and reductase kinase.     

Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus.

In chicken embryo fibroblasts, phosphorylation of the 40S ribosomal protein S6 increases during G1 but returns to basal level by mitosis. In contrast, in Rous sarcoma virus (RSV)-transformed fibroblasts, S6 remains highly phosphorylated throughout mitosis. This study investigated the mechanism by which RSV alters the pattern of S6 phosphorylation. Pulse-chase experiments demonstrate that phosphate turnover in S6 is rapid in normal cells and in cells infected with an RSV transformation-defective virus. In contrast, phosphate turnover in S6 is severely reduced in cells infected with temperature-sensitive RSV at a temperature permissive for transformation, indicating a diminished S6 phosphatase activity. Fractionation of cell lysates by DEAE chromatography showed an almost threefold lower S6 phosphatase activity in RSV-transformed versus normal cells. The S6 phosphatase was sensitive to inhibitor 2 and specifically recognized by an antibody to type 1 phosphatase (PP1). The S6 phosphatase activity recovered by immunoprecipitation of PP1 was threefold lower in transformed cells, but the steady-state level of expression and the rate of synthesis of PP1 were not altered by oncogenic transformation. Together, the results show that transformation by RSV reduced the S6-PP1 activity.     

Comparison of protein profiles and enzymes in non-mycorrhizal and mycorrhizal roots of Pennisetum pedicellatum

Pennisetum pedicellatum plants were inoculated with Glomus mosseae, G. aggregatum and Gigaspora margarita. There were both quantitative and qualitative changes in the protein pattern of inoculated plants. Gi. margarita induced increase in protein in the plants. Acid phosphatase, alkaline phosphatase, superoxide dismutase and chitinase activities were high at the beginning of infection, but declined as the infection advanced. Gi. margarita was an efficient fungus in enhancing enzyme activity and proteins in roots compared with G. mosseae and G. aggregatum. Protein profile revealed the presence of 12 peaks in mycorrhizal plants compared with 8 in nonmycorrhizal plants.     

Akaline phosphatase: activity and variation in human diploid fibroblasts.

A method is introduced for the assay of alkaline phosphatase in homogenates of cultured human skin fibroblasts. In a first group of 11 strains, a four- to fifteen-fold increase of enzyme activity is consistently observed following a period of starvation. In the remaining 31 cell-strains similar specific activities of alkaline phosphatase are found irrespective of medium changes. In regularly fed cultures, an inverse exponential correlation between the specific activity of alkaline phosphatase and the age of the donor has been detected.