Inactivation of astrocytic glutamine synthetase by hydrogen peroxide requires iron.

The specific activity of brain glutamine synthetase (GS) is lowered in several neurodegenerative diseases that involve iron-mediated oxidative stress. The present study has investigated whether H2O2 directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H2O2 reacts with ferrous iron. Exposure of purified sheep brain GS to supraphysiological concentrations of H2O2 (1 mM for 30 min) reduced its specific activity by only 41%, indicating that the enzyme is fairly resistant to oxidation by peroxide. However, the enzyme was completely inactivated when co-incubated with H2O2, iron and ascorbate, indicating a vulnerability to oxidation by conditions that favour the production of hydroxyl radicals. Similarly, specific GS activity in cultured mouse astrocytes was resistant to supraphysiological concentrations of H2O2, with approximately 37% of activity remaining 3h after incubation with 1mM H2O2. This inactivation was prevented by the iron chelators 2,2'-dipyridyl or 1,10-phenanthroline, but not by their non-chelating analogues. These data suggest that inactivation of astrocytic GS is caused by H2O2 indirectly via the Fenton reaction as it required the presence of chelatable intracellular iron.

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update.

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (deltapsi(m)), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of deltapsi(m) may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of deltapsi(m) may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of deltapsi(m) has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of deltapsi(m) early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of deltapsi(m) may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect deltapsi(m). Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in deltapsi(m).

Didemnin B induces apoptosis in proliferating but not resting peripheral blood mononuclear cells.

We have previously shown that didemnin B, a branched cyclic depsipeptide composed of seven amino acids and two hydroxy acids, can induce rapid and complete apoptosis in HL-60 cells (Grubb, D.R. et al. (1995) Biochem. Biophys. Res. Commun. 215, 1130-1136). We now report that didemnin B can induce apoptosis in a wide range of transformed cell lines. Resting normal lymphocytes, however, are apparently unaffected by exposure to the drug. To investigate whether cell transformation, and/or cell proliferation is necessary for didemnin B to induce apoptosis, we examined the effect of didemnin B on freshly harvested human lymphocytes before and after stimulation with concanavalin A. Didemnin B induced apoptosis in normal lymphocytes only after mitogenic stimulation and therefore warrants further examination for its potential as a chemotherapeutic agent, especially for treatment of leukemia.

Mitochondrial cytochrome c release is caspase-dependent and does not involve mitochondrial permeability transition in didemnin B-induced apoptosis.

Permeability transition, and a subsequent drop in mitochondrial membrane potential (DeltaPsi(m)), have been suggested to be mechanisms by which cytochrome c is released from the mitochondria into the cytosol during apoptosis. Furthermore, a drop in DeltaPsi(m) has been suggested to be an obligate early step in the apoptotic pathway. Didemnin B, a branched cyclic peptolide described to have immunosuppressive, anti-tumour, and anti-viral properties, induces rapid apoptosis in a range of mammalian cell lines. Induction of apoptosis by didemnin B in cultured human pro-myeloid HL-60 cells is the fastest and most complete ever described with all cells being apoptotic after 3 h of treatment. By utilizing the system of didemnin B-induced apoptosis in HL-60 cells, and the potent inhibitors of mitochondrial permeability transition, cyclosporin A and bongkrekic acid, we show that permeability transition as determined by changes in DeltaPsi(m) and mitochondrial Ca2+ fluxing, is not a requirement for apoptosis or cytochrome c release. In this system, changes in mitochondrial membrane potential and cytochrome c release are shown to be dependent on caspase activation, and to occur concurrently with the release of caspase-9 from mitochondria, genomic DNA fragmentation and apoptotic body formation.

Plasma membrane NADH-oxidoreductase system: a critical review of the structural and functional data.

The observation in the early 1970s that ferricyanide can replace transferrin as a growth factor highlighted the major role plasma membrane proteins can play within a mammalian cell. Ferricyanide, being impermeant to the cell, was assumed to act at the level of the plasma membrane. Since that time, several enzymes isolated from the plasma membrane have been described, which, using NADH as the intracellular electron donor, are capable of reducing ferricyanide. However, their exact modes of action, and their physiological substrates and functions have not been solved to date. Numerous hypotheses have been proposed for the role of such redox enzymes within the plasma membrane. Examples include the regulation of cell signaling, cell growth, apoptosis, proton pumping, and ion channels. All of these roles may be a result of the function of these enzymes as cellular redox sensors. The emergence of many diverse roles for ferricyanide utilizing redox enzymes present in the plasma membrane might also, in part, be due to the numerous redox enzymes present within the membrane; the poor molecular characterization of the enzymes may be the reason for some of the diverging results reported in the literature as various researchers may be working on different enzymes. Here we review the diverse proposals given for structure and function to the plasma membrane NADH-oxidoreductase system(s) with a specific focus on those enzyme activities which can couple ferricyanide and NADH. Although they are still ill-defined enzymes, evidence is rising that they are of utmost significance for cellular regulation.

Rapamycin inhibits didemnin B-induced apoptosis in human HL-60 cells: evidence for the possible involvement of FK506-binding protein 25.

In the present paper we show that the immunosuppressant rapamycin inhibits the induction of apoptosis by didemnin B in human promyeloid HL-60 cells. The mechanism of this inhibition is investigated using FK506, which competes with rapamycin for binding to their common target FK506-binding protein (FKBP)12. The lack of competition for rapamycin-mediated inhibition of didemnin B-induced apoptosis by FK506 suggests that rapamycin inhibits apoptosis through some mechanism other than inhibition of p70 S6 kinase activation. The lack of inhibition of didemnin B-induced apoptosis by inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein (MAP) kinase kinase further supports the conclusion that rapamycin does not inhibit didemnin B-induced apoptosis through inhibition of the MAP kinase pathway. Furthermore, didemnin B-induced apoptosis is not inhibited by the inhibitors of cyclin-dependent kinase, roscovitine and olomoucine. This indicates that rapamycin does not act through inhibition of cyclin-dependent kinases. Together with the lack of competition for the effect of rapamycin by FK506, our data suggest the possible involvement of the FK506-binding protein, FKBP25, which is localized in the nucleus. This interpretation of our data gains support from the fact that didemnin B does not induce apoptosis in enucleated HL-60 cells, which supports the possible involvement of FKBP25 in the inhibition of apoptosis by rapamycin.

Changes in phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in processing of short-term and long-term memories after passive avoidance learning.

Characteristic autophosphorylation of calcium/ calmodulin-dependent protein kinase II (CaMKII) and its consequences have made this kinase an interesting target in studying the molecular pathway for important neuronal functions including learning and memory formation. In this article, we use immunoprecipitation and immunoblotting methods to detect changes in phosphorylation of CaMKII during memory formation in 1-day-old chicks trained in a single trial passive avoidance task. A 60-kDa protein has been immunoprecipitated from the chick brain with an anti-rabbit CaMKII antibody. This protein shows strong and specific immunoactivities with antibodies against the calmodulin binding site of CaMKII, and the N and C terminals of beta-CaMKII. Commercially available anti-phosphoserine and anti-phosphothreonine antibodies are shown to sensitively detect phosphorylation of purified CaMKII. The basal phosphorylation of CaMKII from the intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) regions of the chick brain is shown to be largely right hemisphere-lateralized. When chicks are subjected to a passive avoidance training experience, a specific increase in CaMKII phosphorylation is induced in the IMHV and LPO of the left hemisphere from those chicks whose memory for the training experience is successfully retrieved. While this specific increase in CaMKII phosphorylation is seen in both the left IMHV and left LPO in short-term memory, it is detectable only in the left LPO associated with long-term memory retrieval. The present results provide evidence that in vivo changes in phosphorylation of CaMKII are associated specifically with processing of distinct memory stages, which take place in specific brain regions.

Unspecific activation of caspases during the induction of apoptosis by didemnin B in human cell lines.

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD-fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis.

Peptidyl-prolyl-cis/trans-isomerase activity may be necessary for memory formation.

At present, evidence for a plethora of physiological roles for the different classes of peptidyl-prolyl-cis/trans-isomerases (PPIases, EC 5.2.1.8) is emerging. Cyclosporin A (CyA) has been previously reported to disrupt memory formation in a temporally specific manner, when administered intracranially to day-old chicks trained on a single-trial, passive-avoidance task [Bennett, P.C., Zhao, W., Lawen, A. and Ng, K.T. (1996) Brain Res. 730, 107-1171. CyA is known to inhibit both the PPIase activity of cyclophilin and, indirectly, the protein phosphatase activity of calcineurin. Therefore to begin to distinguish between these two functions we studied the effects on memory formation of three non-immunosuppressive CyA analogues, in order to study the involvement of cyclophilins. These drugs retain the capacity to bind to and inhibit the PPIase activity of cyclophilin, but do not bind in the complex with cyclophilin to calcineurin and, therefore, do not inhibit its phosphatase activity. All three drugs exert effects on memory formation comparable to those induced by CyA, significantly inhibiting memory formation when injected intracranially (50 fmol per hemisphere) immediately following training. Brain extracts from chicks treated with [MeVal4]CyA show a strong inhibition of cyclophilin activity. These data show a requirement for the PPIase activity of a cyclophilin for successful memory formation and constitute the first set of data establishing a physiological role for a cyclophilin.

Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators.

In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specific in vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADH:ferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADH:ferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group of groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system.

Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin.

Activation of the plasma membrane NADH-oxidoreductase (PMOR) system by addition of growth factors or extracellular electron acceptors stimulates cellular proliferation. We now show that the vanilloids capsaicin, dihydrocapsaicin, and resiniferatoxin are inhibitors of the NADH-oxidase activity of the PMOR system and that both these and two previously identified PMOR inhibitors (chloroquine and retinoic acid) induce apoptosis in human B-cell and mouse myeloid cell lines. At the optimal concentration, PMOR inhibitors can induce between 50 and 70% of apoptosis in mouse myeloid and human B-cell lines within 8-12 h, provided these cell lines do not express Bcl-2. The immunosuppressants cyclosporin A and fujimycin (tacrolimus) inhibit PMOR inhibitor-induced apoptosis. By using combinations of these immunosuppressants and excess amounts of their nonimmunosuppressive analogues, we demonstrate that in human B-cell lines the Bcl-2-sensitive apoptotic pathway triggered by PMOR inhibitors involves signaling through the protein phosphatase calcineurin. We suggest that the PMOR system is a redox sensor that can, depending on the ambient redox environment and the availability of growth factors, regulate plasma membrane calcium fluxes and signal for apoptosis through calcineurin. Bcl-2, a protein that is thought to inhibit apoptosis by regulating reactive oxygen species and calcium fluxes in the cell, inhibits this apoptotic pathway.

Cyclosporin A, an inhibitor of calcineurin, impairs memory formation in day-old chicks.

Considerable evidence exists that changes in the phosphorylation state of neuronal proteins are correlated with learning and that inhibition of various protein kinases disrupts memory formation. Given the reversible nature of protein phosphorylation, a role for protein phosphatases in memory processing also seems likely. It has been shown recently that administration of the phosphatase inhibitor, okadaic acid, disrupts memory formation in day-old chicks, with retention deficits first appearing at approximately 40 min post-training [93]. In the present study the intracranial administration of the immunosuppressant cyclosporin A was also found to produce retention deficits in day-old chicks trained on a single-trial, passive-avoidance task, but the deficits were not significant until 85 min post-training. The difference could not be attributed to differences in the pharmacokinetics of the drugs. Since okadaic acid preferentially inhibits protein phosphatases 1 and 2A, while cyclosporin A is reported to inhibit only the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, it is possible that different phosphatases may be involved in distinct stages of memory formation, as has been reported previously for protein kinases. The possibility that cyclosporin A may, in addition, act through inhibition of cyclophilin's peptidyl-prolyl-cis/transisomerase activity is also canvassed.

Protein tyrosine kinase inhibitors prevent didemnin B-induced apoptosis in HL-60 cells.

Didemnin B induces rapid apoptosis in human promyeloid HL-60 cells with an optimal concentration of 1 microM (Grubb et al. (1995) Biochem. Biophys. Res. Commum. 215, 1130-1136), but little is known about how it does so. In order to determine whether protein tyrosine phosphorylation is involved in this rapid induction of apoptosis, HL-60 cells were pre-treated with tyrosine kinase inhibitors for 1 h before didemnin B treatment. Genistein, 2,5-dihydroxycinnamic acid methyl ester, and a range of tyrphostins inhibit didemnin B-induced apoptotic morphology in a concentration-dependent manner. DNA fragmentation induced by didemnin B is also inhibited by genistein, 2,5-dihydroxycinnamic acid methyl ester, and tyrphostins.

Reversible denaturation of cyclosporin synthetase by urea.

The reversible denaturation of the multifunctional polypeptide, cyclosporin synthetase, by urea was analyzed. It is possible to discriminate between at least two stages of enzyme denaturation. While at low urea concentration (up to 0.8M) cyclosporin A formation is inhibited, synthesis of the diketopiperazine cyclo-(D-alanyl-N-methylleucyl), a molecule representing a partial sequence of cyclosporin A is still detectable. At higher concentrations of urea the enzyme preparation is totally inactive. This inactivation is a consequence of conformational change(s) of cyclosporin synthetase as shown by fluorescence emission spectra of native and denatured enzyme. These data imply a consecutive folding/defolding mechanism for the different domains forming the multifuntional polypeptide.

Didemnin B induces cell death by apoptosis: the fastest induction of apoptosis ever described.

Didemnin B, a cyclic N-methylated peptolide induces apoptosis in human HL-60 cells. When incubated with 1 microM didemnin B, unsynchronized HL-60 cultures undergo apoptosis to 100% within 140 minutes. Apoptosis has been assessed by the typical apoptotic morphology, the presence of double-stranded DNA fragments within the cytosol and the generation of DNA ladders. None of these characteristics of apoptosis are seen when HL-60 cells are pretreated with 1mM Zn2+ immediately before treatment with didemnin B.

Reversal of multidrug resistance by novel cyclosporin A analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro.

It was shown that cyclopeptolide SDZ 214-103 (10 microM) is more active in rhodamine-123 accumulation in actinomycin-D-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 microM), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR. In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A. For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated. At equipotent modulating doses of verapamil (10 microM) and cyclosporin A (10 microM) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporin A was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used.

Up-regulation of the plasma membrane oxidoreductase as a prerequisite for the viability of human Namalwa rho 0 cells.

We have studied aspects of the regulatory interrelationship between the plasma membrane oxidoreductase (PMOR) system and the mitochondrial respiratory capacity of human Namalwa cells. Although the role of mitochondria in the maintenance of cellular redox and energetic states is well established, the PMOR system in comparison is a poorly characterized enzyme system whose functions, particularly in relation to cellular metabolism, have not been clearly elucidated. Therefore we compared the PMOR and mitochondrial respiratory activities of human Namalwa cells during the induction by ethidium bromide treatment of rho 0 cells, which lack a functional mitochondrial respiratory system. The plasma membrane NADH-ferricyanide reductase activity of the PMOR system was found to increase in a stepwise manner concomitant with a decline in cellular mitochondrial respiratory activity. Addition of p-chloromercuriphenylsulfonic acid to the culture medium, at a concentration totally inhibiting the plasma membrane NADH-ferricyanide reductase in vitro, leads to cell death of rho 0 but not of rho + cells. Thus, the up-regulation of a functional PMOR system is a necessary phenomenon in maintaining the viability of mammalian rho 0 cells.

In vitro biosynthesis of ring-extended cyclosporins.

Cyclosporin synthetase, a multifunctional polypeptide, catalyses the biosynthesis of the set of natural cyclosporins. We report that this enzyme is also capable of introducing a beta-alanine into position 7 or 8 of the ring instead of the alpha-alanines present at these positions in cyclosporin A. This leads to 34-membered rings in contrast to the 33-membered ring of the cyclo-undecapeptide cyclosporin A. Both [beta Ala7]CyA and [beta Ala8]CyA show immunosuppressive activity. The cyclosporin synthetase-related enzyme peptolide SDZ 214-103 synthetase, on the other hand, does not incorporate either beta-alanine into position 7 or beta-hydroxy acids into position 8, confirming the previously described higher substrate specificity of this enzyme compared with cyclosporin synthetase [Lawen and Traber (1993) J. Biol. Chem. 268, 20452-20465].