Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC. Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC. When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2. These effects are cell maturation- and time-dependent and metabolite-specific.     

Parasite infection and Japanese cedar pollinosis in monkeys

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-kappaB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-kappaB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-kappaB inactivation in neutrophils occurs in concert with inhibition of calcineurin.     

Activity of ubiquitin-dependent pathway in response to oxidative stress. Ubiquitin-activating enzyme is transiently up-regulated

Relations between the ubiquitin pathway and cellular stress have been noted, but data regarding responses of the ubiquitin pathway to oxidative stress are scanty. This paper documents the response of this pathway to oxidative stress in lens cells. A brief exposure of lens epithelial cells to physiologically relevant levels of H2O2 induces a transient increase in activity of the ubiquitin-dependent pathway. Ubiquitin conjugation activity was maximal and increased 3. 5-9.2-fold over the activity noted in untreated cells by 4 h after removal of H2O2. By 24 h after removal of H2O2, ubiquitin conjugation activity returned to the level noted in untreated cells. In parallel to the changes in ubiquitin conjugation activity, the activity of ubiquitin-activating enzyme (E1), as determined by thiol ester formation, increased 2-6.7-fold during recovery from oxidation. Addition of exogenous E1 resulted in an increase in ubiquitin conjugation activity and in the levels of ubiquitin carrier protein (E2)-ubiquitin thiol esters in both the untreated cells and the H2O2-treated cells. These data suggest that E1 is the rate-limiting enzyme in the ubiquitin conjugation process and that the increases in ubiquitin conjugation activity which are induced upon recovery from oxidation are primarily due to increased E1 activity. The oxidation- and recovery-induced up-regulation of E1 activity is primarily due to post-synthetic events. Substrate availability and up-regulation of E2 activities also appear to be related to the enhancement in ubiquitinylation upon recovery from oxidative stress. The oxidation-induced increases in ubiquitin conjugation activity were associated with an increase in intracellular proteolysis, suggesting that the transient increase in ubiquitinylation noted upon recovery from oxidative stress may play a role in removal of damaged proteins from the cells.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) reduces toxoplasmastatic activity of human monocytes via induction of prostaglandin E2 (PGE2)

In this study we investigated the effect of human GM-CSF on the toxoplasmastatic activity and release of H2O2 and PGE2 by human monocytes. Incubation of monocytes from healthy controls with GM-CSF resulted in a dose-dependent reduction of toxoplasmastatic activity and a decrease in H2O2 production. Furthermore GM-CSF-treated monocytes released more PGE2 than untreated cells. To investigate the role of PGE2 in the reduced toxoplasmastatic activity of GM-CSF-treated monocytes, these cells were incubated with indomethacin. This resulted in a reduction of PGE2 release and restoration of toxoplasmastatic activity of monocytes treated with GM-CSF. GM-CSF reduces the toxoplasmastatic activity of monocytes via production of PGE2.     

Peculiarities of the osmotic response in dehydrated erythrocytes

The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determine. Basal enzyme activities and basal substrate concentrations were not significantly different between the three group of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p < 0.05) and to PP2C activity (p = 0.005) (adjusted R2, 53%). GP independent activity was correlated to GS independent activity (p < 0.07) and to PKA fractional activity (p = 0.005) (adjusted R2, 64%). PP2C activity was correlated to GS fractional activity (p < 0.0005) and to PP1/2A activity (p < 0.0001) (adjusted R2, 83%). PKA fractional activity was correlated to GP total activity (p < 0.0005) and to age (p = 0.001) (adjusted R2, 82%). G6P content was correlated to glycogen content (p < 0.05) and to PP2C activity (p = 0.0005) (adjusted R2, 73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.     

Mechanical assistance in cardiogenic shock after cardiac surgery

With [14C]oleate-labeled phosphatidylcholine as a substrate for phospholipase D the hydrolytic activity was measured by phosphatidic acid formation and the transphosphatidylation activity was measured by the phosphatidylethanol formed in the presence of ethanol. The pH optimum was 6.5 with dimethylglutarate as the buffer. EGTA inhibited the transphosphatidylation activity to a greater extent than the hydrolytic activity. In contrast CaCl2, BaCl2, MgCl2 and SrCl2 stimulated the hydrolytic activity without effecting the transphosphatidylation activity. BeCl2 another member of the group IIa transition metals was a very potent inhibitor of both the hydrolytic and transphophatidylation activity. GTP gamma S, an activator of G protein-mediated events, was an inhibitor of both activities.     

Interruption of estradiol signal transduction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through disruption of the protein phosphorylation pathway in adipose tissues from immature and mature female rats

At doses of 10-115 microg/kg, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased body and adipose tissue weights of mature female rats. Doses below 10 microg TCDD/kg decreased body and adipose tissue weights of immature, but not mature females. Doses of 2 and 10 microg TCDD/kg decreased adipose tissue epidermal growth factor receptor (EGFR) binding activity 5 and 7 days later in immature and mature females, respectively. At these times, there was a decrease in the activities of tyrosine kinase (TK), mitogen-activated protein kinase (MAP2K), and protein kinase A (PKA). In mature females, estradiol (E2, 15 microg/kg) increased TK and PKA activities and decreased MAP2K activity. In immature females, E2 decreased TK and PKA activities but not MAP2K activity. TCDD abolished the stimulatory effect of E2 on TK and PKA in mature females, and in immature females TCDD potentiated the negative effect of E2 on all three kinases. TCDD decreased binding of [3H]E2 to cytosolic and nuclear estrogen receptors (ERs) of mature and immature females, and antagonized the stimulatory effect of E2 on ER binding activity. E2 increased DNA binding activity of the estrogen response element (ERE) and activator protein-1, and TCDD antagonized this effect. Geldanamycin, an inhibitor of Src tyrosine kinase, reduced the effects of TCDD on body and adipose tissue weights. Geldanamycin antagonized the effects of TCDD on EGFR binding activity and TK activity. In cell-free preparations, TCDD antagonized E2 action on TK activity in mature females, as well as E2 action on PKA activity in immature females. We hypothesize that TCDD antagonizes E2 action in female adipose tissues through disruption of common cytosolic signal transduction pathways.     

A comparison of antioxidant enzyme activities in organ-cultured rhesus monkey lenses following peroxide challenge

PURPOSE: To analyze the activities of catalase, glutathione peroxidase and superoxide dismutase, three enzymes involved in the detoxification of reactive oxygen species in organ-cultured Rhesus monkey lenses. METHODS: Lenses freshly obtained from Rhesus monkeys were incubated at 37 degrees C for 2 h and assessed for lens integrity. Lenses were then oxidatively stressed by exposure to a bolus of hydrogen peroxide. The three enzyme activities were assayed 2, 4 and 24 h after exposure to the peroxide challenge. RESULTS: Freshly dissected lenses placed in organ culture exhibited a 20% decrease in catalase activity within 2 h. During the course of a 24 h incubation, catalase activity continued to decrease to a level 58% below that of freshly dissected monkey lenses. In contrast, the activity levels of both glutathione peroxidase and superoxide dismutase increased dramatically within the first 2 h of organ culture, with superoxide dismutase being most affected. Although glutathione peroxidase activity declined with incubation time, its level at the end of 24 h was still 36% greater than that of the fresh lenses. Superoxide dismutase activity remained elevated throughout the 24 h incubation period. The addition of a bolus of 0.25mM H2O2 to monkey lenses in culture had no effect on catalase activity. Two h after the peroxide insult, glutathione peroxidase activity decreased in comparison to control levels while the activity of superoxide dismutase increased by 43%. After 24 h, superoxide dismutase activity returned to values equivalent to the controls. In lenses challenged with 0.50mM H2O2, catalase and glutathione peroxidase activities decreased at 2 h, while superoxide dismutase activity increased 67% above control levels. At subsequent timepoints, catalase activity increased and reached control levels. In contrast, glutathione peroxidase activity continued to decrease with time eventually reaching fresh lens levels. Superoxide dismutase activity levels remained elevated and were equivalent to control values at 24 h. CONCLUSIONS: The data indicate that placement of monkey lenses into an organ culture system represents an environmental change sufficient to cause a response in antioxidant enzyme levels. The addition of H2O2 to this environment caused only superoxide dismutase to be stimulated above control lens levels.     

Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)-like activity

We have previously shown that caspase-2 (Nedd-2) is required for apoptosis induced by withdrawal of trophic support from PC12 cells and sympathetic neurons. Here, we examine the relationship of caspase-2 processing and cell death to induction of caspase-3 (CPP32)-like activity in PC12 cells. Caspase-2 processing, at a site tentatively identified as D333, led to the formation of an N-terminal 37 kDa product. This processing correlated temporally with induction of caspase-3-like activity. Agents previously shown to inhibit caspase-3-like activation, such as bcl-2 and the Cdk inhibitor flavopiridol, also acted upstream of caspase-2 processing. The general caspase inhibitors BAF and zVAD-FMK inhibited N-terminal caspase-2 processing. In contrast, the more selective caspase inhibitor DEVD-FMK inhibited the induction of caspase-3-like activity but did not affect caspase-2 processing or significantly suppress death in PC12 cells or sympathetic neurons. This indicates that caspase-3-like activity is not required for either caspase-2 processing or apoptosis in this paradigm. An antisense oligonucleotide to caspase-2 inhibited cell death but did not affect caspase-3-like activity, indicating that caspase-2 is not upstream of this activity and that activation of caspase-3-like caspases is not sufficient for death. Thus, in our paradigm, caspase-2 processing and caspase-3-like activity are induced independently of each other. Moreover, although death requires caspase-2, caspase-3-like activity is neither necessary nor sufficient for death.     

Protein phosphatase-1 and -2a activities in cultured fetal chick neurons: differential regulation by insulin and insulin-like growth factor-I

In this study, we examined the developmental expression and regulation by insulin and insulin-like growth factor-I (IGF-I) of protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) in cultured fetal chick neurons. Protein phosphatase activities were measured using 32P-labeled phosphorylase-a or 32P-labeled S6 kinase substrate peptide. In cell extracts from day 1-5 cultures, 40-45% of spontaneous protein phosphatase activity was due to PP-1. PP-2A accounted for the remaining 55-60% of enzyme activity. Spontaneous PP-1 activity increased by 100% in day 2 cultures and remained constant thereafter. PP-2A activity increased by 48% in day 2 cultures, with minimal increases in enzyme activity in later cultures. Under the assay conditions employed, at all times in culture a significant proportion (45-50%) of PP-1 was in an inactive form that could be reactivated by trypsin. PP-2A activity was not influenced by trypsin. Insulin stimulated neuronal PP-1 activity in day 4 and 5 cultures, but had no effect in earlier cultures. The activation of PP-1 by insulin was rapid, with a maximal effect (30-40% increase over basal levels) at 5 min with 10 ng/ml insulin. Insulin did not alter total (trypsin-released) PP-1 activity, the content of PP-1 catalytic subunit, or PP-2A activity at any time in culture. In contrast to insulin, IGF-I had no effect on PP-1 activity at any time in culture, but significantly increased PP-2A activity in day 5 cultures. Maximal stimulation of PP-2A activity by IGF-I was observed at 10 min, with an EC50 of 5 ng/ml. These results indicate that chick forebrain neurons contain both PP-1 and PP-2A activities and that neuronal PP-1 and PP-2A activities are differentially regulated by insulin and IGF-I. We conclude that although insulin and IGF-I share many steps in signal transduction, these growth factors have distinct actions on neuronal phosphatase activity that may impact on differences in their neurotropic actions.     

Elevated prostaglandin E2 production by monocytes is responsible for the depressed levels of natural killer and lymphokine-activated killer cell function in patients with breast cancer

BACKGROUND: Human natural killer (NK) cells mediate spontaneous cytotoxicity against tumor cells and represent the main precursors of lymphokine-activated killer (LAK) cell activity. A comparison of some aspects of NK and LAK cell activity was undertaken in 85 preoperative patients with breast cancer and 75 healthy donors. METHODS: NK cell activity (tested in 18-hour cultures of effector peripheral blood mononuclear cells [PBMC] with K562 or MOLT-4 tumor target cells) was significantly diminished in these patients as it was the fully mature LAK cell activity (i.e., interleukin-2 (IL-2)-induced cytotoxicity in PBMC) against NK resistant target cells. Using immunoenzymatic methods we showed that the reduced NK cell activity was due to abnormally high levels of prostaglandin E2 (PGE2) produced by monocytes in culture. RESULTS: PGE2 was found to suppress the production of IL-2 in these cultures. Removal of monocytes from PBMC restored to almost normal levels the deficient NK and LAK cell activity in patients with breast cancer and was also associated with a normalization in the levels of PGE2 and IL-2. Indomethacin and gamma-interferon (IFN-gamma) increased the NK and LAK cell activity in these patients up to the levels of healthy donors. When highly purified CD56+ cells (obtained by an immunomagnetic isolation technique) were used as effector cells, no differences in LAK cell activity could be noticed between healthy donors and patients with cancer. FACS and northern blot analyses demonstrated a PGE2-mediated down-regulation of IL-2 receptor (IL-2R) expression on CD56+ cells that correlated with reduced LAK cell activity. This inhibitory effect of PGE2 was noticeable in long-term LAK cultures and was abrogated in the presence of IFN-gamma or indomethacin. CONCLUSION: This study may have important implications in the potentiation of NK and LAK cell activity for immunotherapeutic protocols in patients with breast cancer.     

Determinants of the inhibitory action of purified 14-kDa phospholipases A2 on cell-free prothrombinase complex

It has been suggested (Kini, R. R., and Evans, H. J. (1987) J. Biol. Chem. 262, 14402-14407) that the anticoagulant activity of members of the 14-kDa phospholipase A2 (PLA2) family depends on the presence of basic residues within a variable surface region (residues 54-77) distinct from both the conserved catalytic machinery and surface sites mediating the antibacterial action of these enzymes (see Weiss, J., Inada, M., Elsbach, P., and Crowl, R. M. (1994) J. Biol. Chem. 269, 26331-26337). To further define the determinants of the anticoagulant activity of PLA2, we have analyzed the inhibitory effects of purified native and recombinant PLA2 on cell-free prothrombinase. Both native and recombinant wild-type pig pancreas (net charge -1) and human "secretory" PLA2 (net charge +15) produced similar dose-dependent inhibition of prothrombinase activity that was significantly less potent than a toxic PLA2 purified from snake venom. Site-specific mutations that either increased or decreased PLA2 activity toward bactericidal/permeability-increasing protein-treated Escherichia coli by up to 50-fold had no effect on antiprothrombinase activity. In contrast, substitution of Arg for Asp-59/Gly for Ser-60 in the pig PLA2 increased antiprothrombinase activity by 5-10-fold without affecting catalytic activity toward a range of phospholipid substrates or antibacterial activity. Comparison of antiprothrombinase activity of catalytically active and inactive forms of the PLA2 and under a range of phospholipid conditions revealed that the potent antiprothrombinase activity of native toxic venom PLA2 and of the D59R.S60G mutant pancreatic PLA2 reflect combined catalytic and noncatalytic actions, the latter apparently dependent on basic residues at discrete surface sites in the enzyme.     

The role and proposed mechanism by which oestradiol 17 beta-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations

Synthesis of the biologically active oestrogen, oestradiol, within breast tumours makes an important contribution to the high concentrations of oestrogens which are present in malignant breast tissues. In breast tumours, oestrone is preferentially converted to oestradiol by the Type I oestradiol 17 beta-hydroxysteroid dehydrogenase (E2DH). Several growth factors, such as insulin-like growth factor Type I, and cytokines, such as Tumour Necrosis Factor alpha (TNF alpha), have been shown to stimulate E2DH activity in MCF-7 breast cancer cells. As little is known about the regulation of Type I E2DH expression and activity in other breast cancer cell lines, the expression and activity of this enzyme was examined in other oestrogen receptor positive and also oestrogen receptor negative breast cancer cell lines. As it is possible that E2DH activity may be limited by co-factor availability, the effects of exogenous co-factors on enzyme activity in these cell lines was also investigated. For T47D and BT20 breast cancer cells, the addition of exogenous co-factors was found to enhance enzyme activity. TNF alpha, in addition to stimulating E2DH activity in MCF-7 cells, also increased activity in T47D and MDA-MB-231 cells, although to a lesser extent than in MCF-7 cells. An investigation of signalling pathways involved in the regulation of E2DH activity revealed that stimulation of both the protein kinase C (PKC) and PKA pathways may be involved in regulation of E2DH activity. As several growth factors and cytokines have now been found to be involved in regulating E2DH activity, the role that macrophages and lymphocytes have in supplying these factors and the mechanism by which these factors may stimulate tumour growth, is also reviewed.     

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model

OBJECTIVE: The purpose of this study was to determine whether increased cytosolic phospholipase A2 activity mediated arachidonic acid mobilization for prostaglandin synthesis in amnion at parturition. STUDY DESIGN: Amnion was collected immediately after delivery from four groups of patients: preterm (<37 weeks) with no labor or labor and term (>37 weeks) with no labor or labor and stored at -70 degrees C. Tissues were homogenized and centrifuged for 1 hour at 100,000 g, and cytosol was assayed for cytosolic phospholipase A2 activity with use of carbon 14-labeled 1-stearoyl-2 arachidonyl phosphatidylcholine plus 10 micromol/L unlabeled substrate and 5 mmol/L calcium in 10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.4. Incubations were performed in duplicate +/- 10 micromol/L arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic phospholipase A2 activity, at 30 degrees C for 45 minutes. RESULTS: Total cytosolic phospholipase A2 activity (in picomoles of arachidonic acid per minute per milligram of protein) calculated as the difference between the activity in the presence and absence of arachidonyl trifluoromethyl ketone was (mean +/- SE) as follows: preterm no labor (n = 7) 8.94 +/- 3.08, preterm with labor (n = 6) 6.79 +/- 2.31, term no labor (n = 7) 14.85 +/- 1.66, and term with labor (n = 5) 5.51 +/- 1.52. Enzyme activity increased with gestational age and was highest in the term no labor group. A significant decrease in cytosolic phospholipase A2 activity occurred with labor (p < 0.05). The greatest decrease in activity was in the term group (p < 0.05). CONCLUSION: Total cellular cytosolic phospholipase A2 activity in amnion is highest in anticipation of labor but during labor total activity is depleted, resulting in the low activity measured after delivery of the placenta. The substrate specificity and changes in amnion total cytosolic phospholipase A2 activity with labor strongly suggests a role in mediation of arachidonic acid mobilization and prostaglandin synthesis at labor.     

CaO-SiO2-Al2O3-MgO-FeO渣系FeO活度的计算模型

利用熔渣结构共存理论建立了CaO-SiO2-Al2O3-MgO-FeO渣系FeO活度的计算模型,并分析了1400℃时炉渣碱度、MgO和FeO含量对该渣系FeO活度的影响规律。结果表明：当CaO-SiO2-Al2O3-MgO-FeO渣系三元碱度为1.3,Al2O3含量为12wt%,FeO含量为2wt%条件下,随MgO含量的增加,炉渣FeO活度增大;当二元碱度为1.1,Al2O3含量为12wt%,MgO含量为10wt%时,FeO活度随随渣中FeO含量的增加呈线性增加;当渣中Al2O3、MgO和FeO含量分别为12wt%、10wt%和2wt%固定不变时,随着二元碱度的提高,炉渣FeO活度迅速增加。计算得到的上述规律和实测规律一致,说明了本模型用于分析FeO活度的正确性。     

Calcineurin protein phosphatase activity in peripheral blood lymphocytes

The protein phosphatase activity of peripheral blood T lymphocytes (PBLs) was examined to quantify the contribution of calcineurin and other members of the family of serine/threonine protein phosphatases. Using selective phosphatase inhibitors, the fractional phosphatase activities of calcineurin, protein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C) were determined. Okadaic acid was used to inhibit the activity of both PP1 and PP2A while cyclosporin A/cyclophilin or trifluoperazine were used as a specific inhibitors of the calmodulin-dependent phosphatase calcineurin. Using a [32P]labeled 19-residue phosphopeptide substrate, RII peptide, it was found that PP1 and PP2A comprise the majority of the total phosphatase activity in PBLs with okadaic acid inhibiting 80% of the phosphatase activity. The remaining 20% of the phosphatase activity can be attributed primarily to calcineurin since it was Ca2+ dependent, sensitive to inhibition by the calmodulin antagonist trifluoperazine, and inhibited by the complex of cyclosporin A (CsA) and cyclophilin. These results indicate that PBL extracts contain little PP2C activity. In addition, PBLs treated with CsA had measurably lower calcineurin activity in cell lysates. The measurement of calcineurin activity may provide a useful means of assessing the extent of immunosuppression during drug therapy.     

Interfacial activation, lysophospholipase and transacylase activity of group VI Ca2+-independent phospholipase A2

The Group VI 80-kDa Ca2+-independent phospholipase A2 (iPLA2) has been purified from murine P388D1 macrophages and Chinese hamster ovary (CHO) cells. The amino acid sequence of the iPLA2 has been determined and shown to contain a lipase consensus sequence and eight ankyrin repeats, which makes it distinct from Group I-V PLA2s. This enzyme appears to play a key role in mediating basal phospholipid remodeling. We now report that the Group VI iPLA2 displays interfacial activation toward short chain phospholipids, 1-octanoyl-2-heptanoyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine, and 1,2-dihexanoyl-sn-glycero-3-phosphocholine micelles. ATP protects the iPLA2 from a loss in activity as a result of prolonged incubation during the assay. Hence higher enzyme activity is observed in the presence than in the absence of ATP. Similar protection was obtained with glycerol. In addition, the iPLA2 exhibits multiple activities which are strongly dependent on substrate presentation. The lysophospholipase activity of this enzyme was diminished by Triton X-100 and stimulated by glycerol. With the combination of 50 microM Triton X-100 and 50% glycerol, the enzyme's lysophospholipase activity achieved equivalent activity to its PLA2 activity. The iPLA2 displayed both lysophospholipid/transacylase and phospholipid/transacylase activity, supporting the conclusion that the mechanism of action of iPLA2 proceeds through an acyl-enzyme intermediate as proposed for the Group IV cPLA2.     

Role of antioxidant enzymes in the induction of increased experimental metastasis by hydroxyurea

BACKGROUND: Treatment of tumor cells with hydroxyurea and other DNA-damaging agents has been shown to increase the experimental metastatic potential of these cells. PURPOSE: We sought to elucidate some of the biochemical and genetic changes that promote tumor cell metastasis in hydroxyurea-treated cells. We hypothesized that drug treatment induces resistance to oxidative damage and that elimination of this resistance reverses the drug-induced experimental metastatic capabilities of tumor cells. METHODS: We examined the effect of hydroxyurea treatment on B16 melanoma cells with respect to experimental metastatic potential, resistance to hydrogen peroxide (H2O2), glutathione peroxidase activity and messenger RNA (mRNA) level, glutathione reductase activity, glutathione levels, glutathione-S-transferase activity, and catalase activity and mRNA level. RESULTS: Hydroxyurea-treated cells were transiently more metastatic following intravenous injection in syngeneic mice and transiently more resistant than untreated cells to exogenous H2O2. Hydroxyurea-induced experimental metastases and H2O2 resistance were eliminated by depletion of intracellular glutathione with buthionine sulfoximine. Glutathione peroxidase activity and mRNA level, glutathione reductase activity, and reduced glutathione levels were all transiently increased in hydroxyurea-treated cells, whereas the increase in glutathione-S-transferase activity was sustained. Catalase activity was modestly increased with no increase in its mRNA levels. CONCLUSIONS: In B16 melanoma cells, experimental metastasis induced by hydroxyurea appears to depend on a process that requires glutathione. Hydroxyurea treatment also induces resistance to exogenous H2O2, which may be due to induction of glutathione and antioxidant enzyme activity. IMPLICATIONS: The role of antioxidants in B16 melanoma cells offers new insights into the metastatic process and the cellular response to chemotherapy.     

Type 2 iodothyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes

The goal of these studies was to define the rate-limiting steps in the inactivation of type 2 iodothyronine deiodinase (D2). We examined the effects of ATP depletion, a lysosomal protease inhibitor, and an inhibitor of actin polymerization on D2 activity in the presence or absence of cycloheximide or 3,3', 5'-triiodothyronine (reverse T3, rT3) in rat pituitary tumor cells (GH4C1). We also analyzed the effects of the proteasomal proteolysis inhibitor carbobenzoxy- L-leucyl-L-leucyl-L-leucinal (MG132). The half-life of D2 activity in hypothyroid cells was 47 min after cycloheximide and 60 min with rT3 (3 nM). rT3 and cycloheximide were additive, reducing D2 half-life to 20 min. D2 degradation was partially inhibited by ATP depletion, but not by cytochalasin B or chloroquine. Incubation with MG132 alone increased D2 activity by 30-40% for several hours, and completely blocked the cycloheximide- or rT3-induced decrease in D2 activity. These results suggest that D2 is inactivated by proteasomal uptake and that substrate reduces D2 activity by accelerating degradation through this pathway. This is the first demonstration of a critical role for proteasomes in the post-translational regulation of D2 activity.