Showing papers in "Iubmb Life in 2001"

The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology.

Abstract: Mitochondria are major sources of reactive oxygen species (ROS); the main sites of superoxide radical production in the respiratory chain are Complexes III and I; however, other mitochondrial enzymes, such as Complex II, glycerol-1-phosphate dehydrogenase, and dihydroorotate dehydrogenase, are also involved in production of ROS. ROS appear to be released both in the matrix and in the intermembrane space; however, their appearance outside the mitochondria may not be physiologically relevant. ROS production is increased in State 4 and in all conditions when the respiratory components are substantially in the reduced form. Accordingly, defects inducing decrease of electron transfer in the respiratory chain, as in many pathological conditions, are bound to enhance ROS production.

Translocation of proteins into mitochondria.

Abstract: The translocase of the outer mitochondrial membrane (TOM) is composed of receptors, a channel protein, and its modulators that function together to import proteins into mitochondria. Although the import pathway of proteins directed to the mitochondrial matrix has been well characterized, recent studies into the import pathway taken by proteins into the other submitochondrial compartments have broadened our understanding into the way the TOM machinery recognizes, interacts, and translocates proteins.

Ketone bodies, potential therapeutic uses.

Abstract: Ketosis, meaning elevation of D- β-hydroxybutyrate ( R -3-hydroxybutyrate) and acetoacetate, has been central to starving man's survival by providing nonglucose substrate to his evolutionarily hypertrophied brain, sparing muscle from destruction for glucose synthesis. Surprisingly, D- β-hydroxybutyrate (abbreviated “βOHB”) may also provide a more efficient source of energy for brain per unit oxygen, supported by the same phenomenon noted in the isolated working perfused rat heart and in sperm. It has also been shown to decrease cell death in two human neuronal cultures, one a model of Alzheimer's and the other of Parkinson's disease. These observations raise the possibility that a number of neurologic disorders, genetic and acquired, might benefit by ketosis. Other beneficial effects from βOHB include an increased energy of ATP hydrolysis ( ΔG') and its linked ionic gradients. This may be significant in drug-resistant epilepsy and in injury and anoxic states. The ability of βOHB to oxidize co-enzyme Q and reduce NADP + may also be important in decreasing free radical damage. Clinical maneuvers for increasing blood levels of βOHB to 2-5 mmol may require synthetic esters or polymers of βOHB taken orally, probably 100 to 150 g or more daily. This necessitates advances in food-science technology to provide at least enough orally acceptable synthetic material for animal and possibly subsequent clinical testing. The other major need is to bring the technology for the analysis of multiple metabolic “phenotypes” up to the level of sophistication of the instrumentation used, for example, in gene science or in structural biology. This technical strategy will be critical to the characterization of polygenic disorders by enhancing the knowledge gained from gene analysis and from the subsequent steps and modifications of the protein products themselves.

Complex I and Parkinson's disease.

Abstract: Complex I of the mammalian electron transfer chain is composed of at least 43 protein subunits, of which 7 are encoded by mtDNA. It catalyzes the transfer of electrons from NADH to ubiquinone and translocates protons from the mitochondrial matrix to the intermembrane space. It may also play direct roles in the mitochondrial permeability transition and in cell death pathways. Despite the limitations of current complex I assays, biochemical studies have suggested the presence of a mild, systemic defect of complex I in Parkinson's disease (PD). Recent experimental work has modeled this abnormality using rotenone to systemically inhibit complex I. Chronic rotenone exposure accurately recapitulated the pathological, biochemical, and behavioral features of PD. Thus, relatively subtle complex I abnormalities--either genetic or acquired--may be central to the pathogenesis of PD.

Reactive Oxygen Species and Signal Transduction

Abstract: Increasing evidence suggests a role for intracellular reactive oxygen species (ROS) as mediators of normal and pathological signal transduction pathways. In particular, a growing list of recent reports have demonstrated a rapid and significant increases in intracellular ROS following growth factor or cytokine stimulation. These ROS appear essential for a host of downstream signaling events. Biochemical characterization of this ligand-activated ROS production has revealed important information regarding the molecular composition of the cellular oxidases and the regulation of their activity by small GTPases. Work is proceeding on identifying strategies to identify how ROS might specifically regulate signaling pathways by altering the activity of direct target molecules. This review will focus on the progress in the rapid emerging area of oxidant or redox-dependent signal transduction and speculate how these insights might alter our view and treatment of diseases thought to be caused by oxidative stress.

Topology of the mitochondrial inner membrane: dynamics and bioenergetic implications.

Abstract: Electron tomography indicates that the mitochondrial inner membrane is not normally comprised of baffle-like folds as depicted in textbooks. In actuality, this membrane is pleomorphic, with narrow tubular regions connecting the internal compartments (cristae) to each other and to the membrane periphery. The membrane topologies observed in condensed (matrix contracted) and orthodox (matrix expanded) mitochondria cannot be interconverted by passive folding and unfolding. Instead, transitions between these morphological states likely involve membrane fusion and fission. Formation of tubular junctions in the inner membrane appears to be energetically favored, because they form spontaneously in yeast mitochondria following large-amplitude swelling and recontraction. However, aberrant, unattached, vesicular cristae are also observed in these mitochondria, suggesting that formation of cristae junctions depends on factors (such as the distribution of key proteins and/or lipids) that are disrupted during extreme swelling. Computer modeling studies using the "Virtual Cell" program suggest that the shape of the inner membrane can influence mitochondrial function. Simulations indicate that narrow cristae junctions restrict diffusion between intracristal and external compartments, causing depletion of ADP and decreased ATP output inside the cristae.

Protein Import Into Mitochondria

Abstract: Most mitochondrial proteins are encoded by the nuclear genome and thus have to be imported into mitochondria from the cytosol. Protein translocation across and into the mitochondrial membranes is a multistep process facilitated by the coordinated action of at least four specialized translocation systems in the outer and inner membranes of mitochondria. The outer membrane contains one general translocase, the TOM complex, whereas three distinct translocases are located in the inner membrane, which facilitates translocation of different classes of preproteins. The TIM23 complex mediates import of matrix-targeted preproteins with N-terminal presequences, whereas hydrophobic preproteins with internal targeting signals are inserted into the inner membrane via the TIM22 complex. The OXA translocase mediates the insertion of preproteins from the matrix space into the inner membrane. This review focuses on the structural organization and function of the import machinery of the model organisms of Saccharomyces cerevisiae and Neurospora crassa. In addition, the molecular basis of a new human mitochondrial disorder is discussed, the Mohr-Tranebjaerg syndrome. This is the first known disease, which is caused by an impaired mitochondrial protein import machinery leading to progressive neurodegeneration.

Respiratory chain supercomplexes.

Abstract: Respiratory chain supercomplexes have been isolated from mammalian and yeast mitochondria, and bacterial membranes. Functional roles of respiratory chain supercomplexes are catalytic enhancement, substrate channelling, and stabilization of complex I by complex III in mammalian cells. Bacterial supercomplexes are characterized by their relatively high detergent-stability compared to yeast or mammalian supercomplexes that are stable to sonication. The mobility of substrate cytochrome c increases in the order bacterial, yeast, and mammalian respiratory chain. In bacterial supercomplexes, the electron transfer between complexes III and IV involves movement of the mobile head of a tightly bound cytochrome c, whereas the yeast S. cerevisiae seems to use substrate channelling of a mobile cytochrome c, and mammalian respiratory chains have been described to use a cytochrome c pool. Dimeric ATP synthase seems to be specific for mitochondrial OXPHOS systems. Monomeric complex V was found in Acetobacterium woodii and Paracoccus denitrificans.

Hsp27: novel regulator of intracellular redox state.

Abstract: Small stress proteins are molecular chaperones that modulate the ability of cells to respond to several types of injuries. In this regard, a protection generated by the expression of mammalian small stress proteins against the cell death induced by oxidative stress has been described. This review summarizes the current knowledge of the protective mechanism generated by the expression of the major mammalian small stress protein Hsp27 in cells exposed to oxidative stress. A possible role of this chaperone protein in the presentation of oxidized proteins to the proteasome degradation machinery is proposed.

Nitric oxide, mitochondria, and cell death.

Abstract: NO or its derivatives (reactive nitrogen species: RNS) have three types of actions on mitochondria: 1) reversible inhibition of mitochondrial respiration at cytochrome oxidase by NO, and irreversible inhibition at multiple sites by RNS; 2) stimulation of mitochondrial production of superoxide, hydrogen peroxide, and peroxynitrite by NO; and 3) induction of mitochondrial permeability transition (MPT) by RNS. Similarly there are three main roles of mitochondria in NO-induced cell death: a) NO inhibition of respiration can induce necrosis (or excitotoxicity in neurons) and inhibit apoptosis if glycolysis is insufficient to compensate, b) RNS- or oxidant-induced signal transduction or DNA damage may activate the mitochondrial pathway to apoptosis, and c) RNS-induced MPT may induce apoptosis or necrosis.

Mitochondrial ROS metabolism: modulation by uncoupling proteins.

Abstract: Most of the oxygen consumed by aerobic organisms is reduced to water by the enzyme cytochrome c oxidase in the terminal reaction of the mitochondrial respiratory chain. A significant proportion of the oxygen molecules are converted to superoxide anion radicals by complexes I and III via a nonenzymatic process. A cascade of enzymes, some of them inside the mitochondria themselves, scavenges superoxide anions in order to protect cells from oxidative damage induced by reactive oxygen species (ROS). Unfortunately, the quantification of the fluxes of mitochondrial ROS inside living cells is currently almost impossible, and this in turn limits our knowledge. Presently, the involvement of mitochondrial ROS can only be demonstrated by indirect strategies and among them knockout techniques are the most convincing. The yield of superoxide generation and subsequently ROS production depend mostly on oxygen concentration but can be efficiently modulated by mitochondrial uncoupling. This role could be assumed in part by one of the Uncoupling Proteins (UCPs). These proteins have coenzyme Q as an obligatory partner and we present here the hypothesis of UCPs as a crucial element of the respiratory chain. ROS have been mostly involved in degenerative processes including ageing. More recently, numerous studies point out the role of ROS as true intracellular second messengers. A putative role of mitochondrial ROS as the sensing element of energy metabolism is discussed here. We propose that UCPs could play a central role in modulation of ROS-dependent signalling pathways and metabolic sensing via the modulation of ROS generation.

VEGF receptor signaling and endothelial function.

Abstract: Angiogenesis, the formation of new blood vessels from preexisting ones, is a central process during normal development and during pathological repair. Vascular endothelial growth factor-A (VEGF-A) can stimulate both physiological and pathological angiogensis. VEGF-A is a ligand for the two receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). Most biological functions of VEGF-A are mediated via VEGFR-2, whereas the role of VEGFR-1 is largely unknown. Activation of mitogen-activated kinase, stress-activated kinase, protein kinase C, and the Akt pathway are implicated in VEGF-A-dependent endothelial function, including cell survival, proliferation, generation of nitric oxide, and the induction of angiogenesis. Induction of metalloproteinases, activation of focal adhesion kinase and of PI3-kinase are implicated in VEGF-A-induced endothelial cell migration. The important role of nitric oxide as a mediator of endothelial function in vivo links the receptor signaling network to other biological effects.

Redox regulation by thioredoxin and thioredoxin-binding proteins.

Abstract: Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys- and constitutes a major thiol reducing system, the thioredoxin system. Thioredoxin plays multiple roles in cellular processes such as proliferation or apoptosis. It also promotes DNA binding of transcription factors such as NF-kappaB, AP-1, p53, and PEBP2. Overexpression of thioredoxin suppresses the degradation of IkappaB and the transactivation of NF-kappaB, whereas overexpression of nuclear-targeted thioredoxin exhibits the enhancement of NF-kappaB-dependent transactivation. ASK1, a MAP kinase kinase kinase mediating the TNF-alpha signal has been identified as a thioredoxin binding protein. Thioredoxin shows an inhibitory effect on the TNF-alpha induced activation of ASK1 and p38 MAP kinase pathway. We identified p40phox as the thioredoxin binding protein-1 (TBP-1) and vitamin D3 up-regulated protein 1 (VDUP1) as the thioredoxin binding protein-2 (TBP-2) by yeast two-hybrid system. TBP-2/VDUP1 negatively regulates the expression and reducing activity of thioredoxin. Thioredoxin interacting proteins may be involved in thioredoxin-mediating redox regulation.

Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation.

Abstract: Reactive oxygen species (ROS) production is a normal process of cell metabolism. In vitro environments usually increase cell production of ROS, which has been implicated as a main cause of cell damage. Nevertheless, the role of ROS in oocyte in vitro maturation (IVM) is controversial. In most cells, enzymatic antioxidant systems can attenuate the effect of oxidative stress by scavenging ROS. The aim of this work was to determine whether: (1) standard conditions of bovine oocyte IVM are responsible for oxidative stress; (2) cumulus cells participate in protection against oxidative stress of the oocyte; and (3) enzymatic antioxidant activity is present in oocytes and cumulus cells. Cumulus-oocyte complexes (COCs) were matured in TCM-199 10% steer serum for 24 h at 39 °C in 5% CO2:95% humidified air. Oxidative stress was determined by the 2', 7'-dichlorofluorescein diacetate assay. Superoxide dismutase (SOD), glutathione peroxidase, and catalase activities were measured spectrophotometrically.Under standard conditions of in vitro maturation, there was no increase in ROS production per COC (P > 0.05), but ROS level per cumulus cell diminished. There was no modification in ROS levels in oocytes matured in the presence versus the absence of their surrounding cumulus cells (P > 0.05). To the best of our knowledge, the presence of SOD, glutathione peroxidase and catalase activities were detected in oocytes and cumulus cells for the first time. Enzymatic units were lower in denuded oocytes with respect to cumulus (P 0.05) and increased in oocytes due to maturation (P > 0.05). The presence of activity of an enzymatic antioxidant system in the bovine oocyte would regulate in part ROS levels during IVM. Oocytes could be capable of controlling the increase in ROS because of the presence of their own enzymatic antioxidant system, SOD having the highest specific activity with respect to cumulus cells.

Multidimensional NMR methods for protein structure determination.

Abstract: Structural studies of proteins are critical for understanding biological processes at the molecular level. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for obtaining structural and dynamic information on proteins and protein-ligand complexes. In the present review, methodologies for NMR structure determination of proteins and macromolecular complexes are described. In addition, a number of recent advances that reduce the molecular weight limitations previously imposed on NMR studies of biomolecules are discussed, highlighting applications of these technologies to protein systems studied in our laboratories.

Regulation of HIF by the von Hippel-Lindau tumour suppressor: implications for cellular oxygen sensing.

Abstract: Hypoxia-inducible factor (HIF) is central in coordinating many of the transcriptional adaptations to hypoxia. Composed of a heterodimer of alpha and beta subunits, the alpha subunit is rapidly degraded in normoxia, leading to inactivation of the hypoxic response. Many models for a molecular oxygen sensor regulating this system have been proposed, but an important finding has been the ability to mimic hypoxia by chelation or substitution of iron. A key insight has been the recognition that HIF-alpha is targeted for degradation by the ubiquitin-proteasome pathway through binding to the von Hippel-Lindau tumour suppressor protein (pVHL), which forms the recognition component of an E3 ubiquitin ligase complex leading to ubiquitylation of HIF-alpha. Importantly, the classical features of regulation by iron and oxygen availability are reflected in regulation of the HIF-alpha/pVHL interaction. It has recently been shown that HIF-alpha undergoes an iron- and oxygen-dependent modification before it can interact with pVHL, and that this results in hydroxylation of at least one prolyl residue (HIF-1alpha, Pro 564). This modification is catalysed by an enzyme termed HIF-prolyl hydroxylase (HIF-PH), and compatible with all previously described prolyl-4-hydroxylases HIF-PH also requires 2-oxoglutarate as a cosubstrate. The key position of this hydroxylation in the degradation pathway of HIF-alpha, together with its requirement for molecular dioxygen as a co-substrate, provides the potential for HIF-PH to function directly as a cellular oxygen sensor. However, the ability of these enzyme(s) to account for the full range of physiological regulation displayed by the HIF system remains to be defined.

Signaling by the respiratory burst in macrophages.

Abstract: During phagocytosis or stimulation with a wide variety of agents, macrophages and other phagocytic cells produce reactive oxygen species (ROS) through activation of a multicomponent NADPH oxidase. ROS production through related NADPH oxidases has recently been demonstrated in several other cell types. Furthermore, the physiological generation of ROS production has now been clearly implicated in activating signaling pathways resulting in a broad array of physiological responses from cell proliferation to gene expression and apoptosis. This brief review suggests that: 1) hydrogen peroxide and superoxide, but not the hydroxyl radical, function as second messengers; 2) antioxidant enzymes function in the "turn-off" phase of signal transduction; 3) the chemistry of thiols is critical in redox signaling; and 4) the primary physiological role of the respiratory burst in macrophages may be in redox signaling rather than microbicidal activity.

Telethonin and other new proteins of the Z-disc of skeletal muscle.

Abstract: This brief review outlines some of the most relevant proteins of the Z-disc and the complex network of interactions that link them together in a stable structure. Apart from the well-known Z-disc proteins such as actin, cap-Z, titin, nebulin, and alpha-actinin 2, several other Z-disc proteins have been recently discovered, including telethonin and myotilin that have been linked to limb girdle muscular dystrophies. Some proteins including ALP and ZASP have known interaction domains (PDZ and LIM motifs), whereas others like FATZ have no canonical interaction domains, although they are known to bind several proteins. Another new Z-disc protein is gamma-filamin that could provide a link between the plasma membrane and myofibrils because it binds directly to gamma- and delta-sarcoglycans and indirectly to alpha-actinin 2 via FATZ and myotilin. A greater knowledge of Z-disc proteins and their interactions is essential for understanding their role in the structure and function of muscle.

HIF‐1 and AP‐1 Cooperate to Increase Gene Expression in Hypoxia: Role of MAP Kinases

Abstract: HIF-1 is the main transcription factor responsible for increased gene expression in hypoxia: VEGF, erythropoietin, GLUT-1, and glycolytic enzymes are such target genes and all participate in the adaptative response of cells to hypoxia. AP-1 activation by hypoxia has also been demonstrated in several cell lines and it cooperates with HIF-1 for increasing VEGF gene transcription in hypoxia. Both HIF-1 and AP-1 activation by hypoxia seems to involve members of the MAP kinase family. Here, we summarize the data indicating that ERK and JNK are needed for activation of HIF-1 and AP-1, respectively.

Uptake of calcium by mitochondria: transport and possible function.

Abstract: Vertebrate mitochondria contain a complex system for transport of Ca2+ and related ions, consisting of two saturable modes of Ca2+ influx and two separate, saturable mechanisms of Ca2+ efflux. The characteristics of the mechanisms of Ca2+ uptake, the uniporter and the RaM, are discussed here and suggestions are made about how the mechanisms may work together and separately to mediate the two physiological roles with which they are most commonly associated-control of the rate of cellular ATP production and induction of the permeability transition and apoptosis. It is argued that more subtlety of control of intramitochondrial free Ca2+ concentration ([Ca2+]m) must be used by the uniporter and the RaM to fulfill their physiological roles than has been commonly recognized. This is because an increase in [Ca2+]m is associated with both increased production of ATP which supports the continued life of the cell and with induction of the permeability transition and possibly apoptosis, which leads to cell death. The saturable mechanisms of mitochondrial Ca2+ efflux and the Ca2+-induced mitochondrial permeability transition, which can transport Ca2+ as well as other ions and molecules and is often considered as a Ca2+ transport mechanism, are being reviewed separately.

Multiple modes of transcriptional regulation by the HIV-1 Tat transactivator.

Abstract: Regulation of HIV-1 gene expression by the viral Tat transactivator is a critical step in the viral life cycle. Tat acts as a highly unusual transcription factor that interacts with a stem-loop RNA structure (TAR) found at the 5' end of all viral transcripts. There, it induces a modification of chromatin at the HIV-1 long terminal repeat (LTR) promoter and stimulates the recruitment of elongation-competent RNA polymerase II complexes capable of processive transcription. Increase of transcriptional elongation is the consequence of the interaction of Tat with cyclin T1, the cyclin component of CDK9, which phosphorylates the carboxy-terminal domain of RNA polymerase II to enhance its processivity. Tat-induced transcriptional activation of the LTR promoter is concomitant with recruitment of the transcriptional coactivators p300 and the highly homologue cAMP-responsive transcription factor binding protein (CBP). These large proteins act at the level of transcriptional initiation by bridging the basal transcription machinery with specific transcriptional activators. Furthermore, p300/CBP are histone acetyl-transferases capable of modulating the interaction of nucleosomes with DNA and with chromatin remodeling complexes. Besides histones, Tat itself is a substrate for the enzymatic activity of p300/CBP and of the associated factor P/CAF, suggesting a regulatory role of acetylation on the protein itself. Devising a unifying model for LTR activation that includes activities of Tat at the levels of both transcriptional initiation and transcriptional elongation is a challenging task at this moment. Nevertheless, protein localization studies indicate that both cyclin T1 and p300/CBP co-localize in specific subnuclear compartments, thus suggesting participation of both proteins in the formation of multimolecular complexes governing coordinated steps of transcriptional activation.

Evolutionary aspects of calmodulin.

Abstract: Calmodulin (CaM) is a major cellular sensor of calcium signaling, interacts with numerous proteins associated with cellular second messenger systems (e.g., cyclic AMP, nitric oxide), and is associated with neurosecretory activity. An identical CaM protein consisting of four helix-loop-helix regions that arose by gene duplication is encoded by three nonallelic mammalian genes that are some of the most highly conserved genes known. Differential tissue and cellular expression of each CaM suggest unique functions that promote strong selective preservation of these replicate, yet distinct, CaM genes in mammals. Each gene displays the same exon-intron arrangement but is characterized by distinct promoter elements and by unique 5'- and 3'-untranslated regions that are highly conserved among human, rat, and mouse. These distinct untranslated regions may permit regulation of CaM levels at discrete cellular sites during differentiation and in highly specialized cell types such as neurons.

Human Heart Cytosolic Reductases and Anthracycline Cardiotoxicity

Abstract: Anthracyclines are a class of antitumor drugs widely used for the treatment of a variety of malignancy, including leukemias, lymphomas, sarcomas, and carcinomas. Different mechanisms have been proposed for anthracycline antitumor effects including free-radical generation, DNA intercalation/binding, activation of signaling pathways, inhibition of topoisomerase II and apoptosis. A life-threatening form of cardiomyopathy hampers the clinical use of anthracyclines. According to the prevailing hypothesis, anthracyclines injure the heart by generating damaging free radicals through iron-catalyzed redox cycling. Although the "iron and free-radical hypothesis" can explain some aspects of anthracycline acute toxicity, it is nonetheless disappointing when referred to chronic cardiomyopathy. An alternative hypothesis implicates C-13 alcohol metabolites of anthracyclines as mediators of myocardial contractile dysfunction ("metabolite hypothesis"). Hydroxy metabolites are formed upon two-electron reduction of the C-13 carbonyl group in the side chain of anthracyclines by cytosolic NADPH-dependent reductases. Anthracycline alcohol metabolites can affect myocardial energy metabolism, ionic gradients, and Ca2+ movements, ultimately impairing cardiac contraction and relaxation. In addition, alcohol metabolites can impair cardiac intracellular iron handling and homeostasis, by delocalizing iron from the [4Fe-4S] cluster of cytoplasmic aconitase. Chronic cardiotoxicity induced by C-13 alcohol metabolite might be primed by oxidative stress generated by anthracycline redox cycling ("unifying hypothesis"). Putative cardioprotective strategies should be aimed at decreasing C-13 alcohol metabolite production by means of efficient inhibitors of anthracycline reductases, as short-chain coenzyme Q analogs and chalcones that compete with anthracyclines for the enzyme active site, or by developing novel anthracyclines less susceptible to reductive metabolism.

BCL-2 Proteins: Regulators of the Mitochondrial Apoptotic Program

Abstract: BCL-2 family members are pivotal regulators of the apoptotic process. Mitochondria seem to be a major site-of-action for these proteins. Several prominent alterations occur to mitochondria during apoptosis that include the release of intermembrane space molecules, changes in the membrane potential, ionic changes, and more. All these changes seem to be part of the mitochondrial apoptotic process. The BCL-2 family members are believed to be the major regulators of this program; however, their exact mechanism of action still remains a mystery. In addition, the exact contribution of mitochondria to the apoptotic process is still unclear. This review summarizes and examines the current knowledge regarding these two issues.

Casein kinase I: from obscurity to center stage.

Abstract: The casein kinase I (CKI) family of protein kinases is a group of highly related, ubiquitously expressed serine/threonine kinases found in all eukaryotic organisms from protozoa to man. Recent advances in diverse fields, including developmental biology and chronobiology, have elucidated roles for CKI in regulating critical processes such as Wnt signaling, circadian rhythm, nuclear import, and Alzheimer's disease progression.

Mitochondrial Calcium Homeostasis: Mechanisms and Molecules

Abstract: Despite the early characterisation of Ca2+ fluxes in isolated mitochondria and the ability of this ion to up-regulate dehydrogenases of the Krebs cycle, the low affinity of the organelle uptake pathways was a long-standing obstacle to the recognition of a physiological role for mitochondrial Ca2+ uptake. This review begins with a historical account of the main results that proved the contrary and provides a brief description of mitochondrial Ca2+ signals. Then, it discusses the characteristics of Ca2+ regulation of mitochondrial function. Finally, it summarizes recent discoveries on structural aspects of mitochondrial reticulum and its connections to signalling partners such as the endoplasmic reticulum or the plasma membrane.

The peroxisome in oxidative stress.

Abstract: Peroxisomes are one of the main sites in the cell where oxygen free radicals are both generated and scavenged. The balance between these two processes is believed to be of great importance for proper functioning of cells and has been implicated in aging and carcinogenesis. We will give an overview of the peroxisomal processes involved in the oxygen radical homeostasis and its implications for the cell.

Transfer of cationic antibacterial agents berberine, palmatine, and benzalkonium through bimolecular planar phospholipid film and Staphylococcus aureus membrane

Abstract: Some organic cations are known to be electrophoretically imported into bacterial cells and actively extruded from these cells by multidrug resistance (MDR) pumps. We have studied penetration of plant antimicrobial agents berberine and palmatine and synthetic antiseptic benzalkonium chloride through black planar phospholipid membrane (BLM) and membrane of Staphylococcus aureus cells. Gradients of these cations across BLM generated an electric potential difference. Penetrating anion tetraphenyl borate and phloretin (a plant substance decreasing membrane dipole potential) stimulated this effect. Under optimal conditions, the magnitude of the electric potential was close to theoretical, that is, 60 mV/10-fold cation gradient. Berberine accumulated in S. aureus cells as shown by direct measurement of berberine with a berberine-sensitive electrode. The berberine accumulation was prevented by protonophore CCCP and was stimulated by mutation in the MDR pump NorA. It is concluded that the plant alkaloids and benzalkonium are penetrating cations and substrates of an MDR pump.