Alfons Lawen

The mitochondrial membrane potential (Δψm) 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 (Δψm), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of Δψm 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 Δψm 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 Δψm has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of Δψm 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 Δψm 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 Δψm. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in Δψm.

Voltage-dependent anion-selective channel (VDAC) in the plasma membrane

Voltage‐dependent anion channels (VDACs) have originally been characterized as mitochondrial porins. Starting in the late 1980s, however, evidence began to accumulate that VDACs can also be expressed in plasma membranes. In this review, we briefly revisit the historical milestones in the discovery of plasma membrane‐bound VDAC, and we critically analyze the evidence for VDAC plasma membrane localization obtained from various purification strategies and recently from plasma membrane proteomics studies. We discuss the possible biological function and relevance of VDAC in the plasma membrane and finally discuss a hypothetical model of how VDAC may be targeted to the plasma membrane.

Mammalian Iron Homeostasis in Health and Disease: Uptake, Storage, Transport, and Molecular Mechanisms of Action

Iron is a crucial factor for life. However, it also has the potential to cause the formation of noxious free radicals. These double-edged sword characteristics demand a tight regulation of cellular iron metabolism. In this review, we discuss the various pathways of cellular iron uptake, cellular iron storage, and transport. Recent advances in understanding the reduction and uptake of non-transferrin-bound iron are discussed. We also discuss the recent progress in the understanding of transcriptional and translational regulation by iron. Furthermore, we discuss recent advances in the understanding of the regulation of cellular and systemic iron homeostasis and several key diseases resulting from iron deficiency and overload. We also discuss the knockout mice available for studying iron metabolism and the related human conditions.

Two routes of iron accumulation in astrocytes: ascorbate-dependent ferrous iron uptake via the divalent metal transporter (DMT1) plus an independent route for ferric iron

Astrocytes are central to iron and ascorbate homoeostasis within the brain. Although NTBI (non-transferrin-bound iron) may be a major form of iron imported by astrocytes in vivo, the mechanisms responsible remain unclear. The present study examines NTBI uptake by cultured astrocytes and the involvement of ascorbate and DMT1 (divalent metal transporter 1). We demonstrate that iron accumulation by ascorbate-deficient astrocytes is insensitive to both membrane-impermeant Fe(II) chelators and to the addition of the ferroxidase caeruloplasmin. However, when astrocytes are ascorbate-replete, as occurs in vivo, their rate of iron accumulation is doubled. The acquisition of this additional iron depends on effluxed ascorbate and can be blocked by the DMT1 inhibitor ferristatin/NSC306711. Furthermore, the calcein-accessible component of intracellular labile iron, which appears during iron uptake, appears to consist of only Fe(III) in ascorbate-deficient astrocytes, whereas that of ascorbate-replete astrocytes comprises both valencies. Our data suggest that an Fe(III)-uptake pathway predominates when astrocytes are ascorbate-deficient, but that in ascorbate-replete astrocytes, at least half of the accumulated iron is initially reduced by effluxed ascorbate and then imported by DMT1. These results suggest that ascorbate is intimately involved in iron accumulation by astrocytes, and is thus an important contributor to iron homoeostasis in the mammalian brain.

Transplasma membrane electron transport: enzymes involved and biological function.

Abstract The notion of transmembrane electron transport is usually associated with mitochondria and chloroplasts. However, since the early 1970s, it has been known that this phenomenon also occurs at the level of the plasma membrane. Ever since, evidence has accumulated for the existence of a plethora of transplasma membrane electron transport enzymes. In this review, we discuss the various enzymes known, their molecular characteristics and their biological functions.

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, specificin 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 or 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.

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 cyclophilins peptidyl-prolyl-cis/transisomerase activity is also canvassed.

Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells

K562 erythroleukemia cells import non-transferrin-bound iron (NTBI) by an incompletely understood process that requires initial iron reduction. The mechanism of NTBI ferrireduction remains unknown but probably involves transplasma membrane electron transport. We here provide evidence for a novel mechanism of NTBI reduction and uptake by K562 cells that utilizes transplasma membrane ascorbate cycling. Incubation of cells with dehydroascorbic acid, but not ascorbate, resulted in (i) accumulation of intracellular ascorbate that was blocked by the glucose transporter inhibitor, cytochalasin B, and (ii) subsequent release of micromolar concentrations of ascorbate into the external medium via a route that was sensitive to the anion channel inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonate. Ascorbate-deficient control cells demonstrated low levels of ferric citrate reduction. However, incubation of the cells with dehydroascorbic acid resulted in a dose-dependent stimulation of both iron reduction and uptake from radiolabeled [55Fe]ferric citrate. This stimulation was abrogated by ascorbate oxidase treatment, suggesting dependence on direct chemical reduction by ascorbate. These results support a novel model of NTBI reduction and uptake by K562 cells in which uptake is preceded by reduction of iron by extracellular ascorbate, the latter of which is subsequently regenerated by transplasma membrane ascorbate cycling.

Involvement of reactive oxygen species in capsaicinoid-induced apoptosis in transformed cells

Some varieties of sweet pepper accumulate non-pungent isosters of capsaicin, a type of compounds exemplified by capsiate. The only structural difference between capsaicin and capsiate is the link between the vanillyl and the acyl moieties, via an amide bond in the former and via an ester bond in the latter. By flow cytometry analyses we have determined that nor-dihydrocapsiate, a simplified analogue of capsiate, is a pro-oxidant compound that induces apoptosis in the Jurkat tumor cell line. The nuclear DNA fragmentation induced by nor-dihydrocapsiate is preceded by an increase in the production of reactive oxygen species and by a subsequent disruption of mitochondria transmembrane potential. Capsiate-induced apoptosis is initiated at the S phase of the cell cycle and is mediated by a caspase-3-dependent pathway. The accumulation of intracellular reactive oxygen species in capsiate-treated cells is greatly prevented by the presence of ferricyanide, suggesting that capsiates target a cellular redox system distinct from the one involved in the mitochondrial electron-chain transport. Methylation of the phenolic hydroxyl of nor-dihydrocapsiate completely abrogated the ability to induce reactive oxygen species and apoptosis, highlighting the relevance of the presence of a free phenolic hydroxyl for the pro-oxidant properties of capsaicinoids.

Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide Re‐evaluation of the molecular mass of various peptide synthetases

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re‐evaluated by SDS‐PAGE and CsCl density gradient centrifugation. In SDS‐PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremly low protein concentrations, such as 10–50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re‐evaluated in SDS‐PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.