Role of hepatocyte wounding on " Plasmodium " liver-stage development and survival

RESUME La première étape primordiale au cycle de vie du Plasmodium dans un hôte mammifère est l'invasion des hepatocytes par des sporozoites. L'infection finale des hepatocytes est précédée de la traversée de plusieurs cellules hôtes, rompant les membranes plasmiques et ayant comme résultat la sécrétion des facteurs cytotoliques dans le micro-environnement. Ce matériel endogène libéré est fortement stimulant/immunogène et peut servir de signal de danger initiant des réponses distinctes dans diverses cellules. De nos jours, le caractère essentiel et salutaire de la migration des sporozoites comme étape d'infection du Plasmodium est vivement controversée. Ainsi, notre étude a visé à caractériser l'effet de l'interaction du parasite avec ses cellules hôtes d'un point de vue immunologique. En particulier, nous avons voulu évaluer l'effet de la perte de matériel cellulaire pendant l'infection de Plasmodium sur les hepatocytes primaires de souris et sur des cultures cellulaires HepG2. Nous avons observé que les facteurs cytotoxiques dérivés des cellules endommagés activent NF-κB - un important régulateur de réponse inflammatoires -dans des cellules voisines des cellules endommagés, qui sont des cellules hôtes potentielles pour l'infection finale du parasite. Cette activation de NF-κB s'est produite peu de temps après l'infection et a mené in vitro et in vivo à une réduction d'infection de façon dépendante du temps, un effet qui a pu être compensé par l'addition de BAY11-7082, un inhibiteur spécifique de NF-κB. De plus, aucune activation de NF-κB avec des parasites SPECT-/-, incapables de traverser les hepatocytes, n'a été observée. Nous avons montré parla suite que l'activation de NF-κB induit l'expression de l'enzyme iNOS dans les hepatocytes, qui est responsable d'une diminution des hepatocytes infectés. En outre, les hepatocytes primaires des souris MyD88-/- n'ont montré ni activation de NF-κB, ni expression d'iNOS lors de l'infection, ce qui suggère la participation des membres de famille du Toll/IL-1 récepteur dans la reconnaissance des facteurs cytosoxiques. En effet, le manque de MyD88 a augmenté significativement l'infection in vitro et in vivo. D'autre part, un rôle bénéfique pour l'activation de NF-κB a été évalué. Les cellules infectées étaient plus résistantes contre l'apoptose induite par Fas (CD95/Apo-1) que les cellules non infectées ou les cellules infectées dans lesquelles NF-κB a été bloqué par BAY11-7082 in vitro. Paradoxalement, l'expression d'iNOS contribue à la protection des cellules infectées contre l'apoptose pax Fas, puisque le traitement avec l'inhibiteur spécifique SMT (S-methylisothiourea) a rendu les cellules infectées plus susceptibles à l'apoptose. Un effet bénéfique additionnel pour le parasite est que la plupart des cellules hôtes traversées présentent des peptides du parasite aux cellules T cytotoxiques spécifiques et peuvent donc réorienter la réaction immune spécifique sur les cellules non infectées. Nous montrons que les cellules hôtes endommagés par la migration du parasite induit l'inflammation, qui limite l'ampleur de l'infection. D'autre part, nos données soutiennent que la survie du parasite Plasmodium dans le foie est assurée par une augmentation de la résistance des hepatocytes contre l'apoptose. SUMMARY The first obligatory step of the Plasmodium life cycle in the mammalian host is the invasion of hepatocytes by sporozoites. Final hepatocyte infection involves the penetration of several host cells, whose plasma membranes are ruptured in the process, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory / immunogenic and can serve as a danger signal initiating distinct responses in various cells. To date, it is highly controversial whether sporozoite migration through hepatocytes is an essential and beneficial step for Plasmodium infection. Thus, our study aimed at characterizing the effect of the interaction of the parasite with its host cells from an immunological point of view In particular, we wanted to evaluate the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-κB - a main regulator of host inflammatory responses - in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-κB occurred shortly after infection and led to a reduction of infection load in a time dependent manner in vitro and in viva, an effect that could be reverted by addition of the specific NF-κB inhibitor BAY11-7082. In addition, no NF-κB activation was observed when SPECT-/- parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-κB activation causes the induction of inducible nitric oxide synthase (iNOS) expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88-/- mice showed no NF-κB activation and iNOS expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. In a further complementary series of experiments, we assessed a possible beneficial role for the activation of NF-κB. Infected cells were more resistant to Fas (CD95/Apo-1)-mediated apoptosis than uninfected cells or infected cells in which NF-κB was blocked by BAYl1-7082 in vitro. Paradoxically, iNOS expression contributes to the protection of infected cells from Fas-induced apoptosis, since treatment with the specific iNOS inhibitor SMT (S-Methylisothiourea Sulfate) rendered the infected cells more susceptible to apoptosis. An additional beneficial effect of host cell traversal for the parasite is the fact that mainly traversed cells present parasite-derived peptides to specific cytotoxic T cells and therefore may redirect the specific immune response to uninfected cells. In summary, we have shown that host cells wounded by parasite migration induce inflammation, which limits the extent of parasite infection. In addition, our data support the notion that survival of Plasmodium parasites in the liver is mediated by increasing the resistance of hepatocytes to Fas-induced apoptosis.

Consequences of modified lipid and glucose metabolism on peripheral nervous system structure and function

284 million people worldwide suffered from type 2 diabetes mellitus (T2DM) in 2010, which will, in approximately half of them, lead to the development of diabetic peripheral neuropathy (DPN). Although DPN is the most common complication of diabetes mellitus and the leading cause of non-traumatic amputations its pathophysiology is still poorly understood. To get more insight into the molecular mechanism underlying DPN in T2DM, I used a rodent model of T2DM, the db/db mice.¦ln vivo electrophysiological recordings of diabetic animals indicated that in addition to reduced nerve conduction velocity db/db mice also present increased nerve excitability. Further ex vivo evaluation of the electrophysiological properties of db/db nerves clearly established a presence of the peripheral nerve hyperexcitability (PNH) phenotype in diabetic animals. Using pharmacological inhibitors we demonstrated that PNH is mostly mediated by the decreased activity of Kv1 channels. ln agreement with these data 1 observed that the diabetic condition led to a reduced presence of the Kv1.2 subunits in juxtaparanodal regions of db/db peripheral nerves whereas its mANA and protein expression levels were not affected. Lmportantly, I confirmed a loss of juxtaparanodal Kv1.2 subunits in nerve biopsies from type 2 diabetic patients. Together these observations indicate that the type 2 diabetic condition leads to potassium-channel mediated changes of nerve excitability thus identifying them as potential drug targets to treat sorne of the DPN related symptoms.¦Schwann cells ensheath and isolate peripheral axons by the production of myelin, which consists of lipids and proteins in a ratio of 2:1. Peripheral myelin protein 2 (= P2, Pmp2 or FABP8) was originally described as one of the most abundant myelin proteins in the peripheral nervous system. P2, which is a member of the fatty acid binding protein (FABP) family, is a 14.8 kDa cytosolic protein expressed on the cytoplasmic side of compact myelin membranes. As indicated by their name, the principal role of FABPs is thought to be the binding and transport of fatty acids.¦To study its role in myelinating glial cells I have recently generated a complete P2 knockout mouse model (P2-/-). I confirmed the loss of P2 in the sciatic nerve of P2-/- mice at the mRNA and protein level. Electrophysiological analysis of the adult (P56) mutant mice revealed a mild but significant reduction in the motor nerve conduction velocity. lnterestingly, this functional change was not accompanied by any detectable alterations in general myelin structure. However, I have observed significant alterations in the mRNA expression level of other FABPs, predominantly FABP9, in the PNS of P2-/- mice as compared to age-matched P2+/+ mice indicating a role of P2 in the glial myelin lipid metabolism.¦Le diabète de type 2 touche 284 million de personnes dans le monde en 2010 et son évolution conduit dans la moitié des cas à une neuropathie périphérique diabétique. Bien que la neuropathie périphérique soit la complication la plus courante du diabète pouvant conduire jusqu'à l'amputation, sa physiopathologie est aujourd'hui encore mal comprise. Dans le but d'améliorer les connaissances moléculaires expliquant les mécanismes de la neuropathie liée au diabète de type 2, j'ai utilisé un modèle murin du diabète de type 2, les souris db/db.¦ln vivo, les enregistrements éléctrophysiologiques des animaux diabétiques montrent qu'en plus d'une diminution de la vitesse de conduction nerveuse, les souris db/db présentent également une augmentation de l'excitabilité nerveuse. Des mesures menées Ex­ vivo ont montré l'existence d'un phénotype d'hyperexcitabilité sur les nerfs périphériques isolés d'animaux diabétiques. Grâce à l'utilisation d'inhibiteurs pharmacologiques, nous avons pu démontrer que l'hyperexcitabilité démontrée était due à une réduction d'activité des canaux Kv1. En accord avec ces données, j'ai observé qu'une situation de diabète conduisait à une diminution des canaux Kv1.2 aux régions juxta-paranodales des nerfs périphériques db/db, alors que l'expression du transcrit et de la protéine restait stable. J'ai également confirmé l'absence de canaux Kv1.2 aux juxta-paranoeuds de biopsies de nerfs de patients diabétiques. L'ensemble de ces observations montrent que les nerfs périphériques chez les patients atteints de diabète de type 2 est due à une diminution des canaux potassiques rapides juxtaparanodaux les identifiant ainsi comme des cibles thérapeutiques potentielles.¦Les cellules de Schwann enveloppent et isolent les axones périphériques d'une membrane spécialisée, la myéline, composée de deux fois plus de lipides que de protéines. La protéine P2 (Pmp2 "peripheral myelin protein 2" ou FABP8 "fatty acid binding protein") est l'une des protéines les plus abondantes au système nerveux périphérique. P2 appartient à la famille de protéines FABP liant et transportant les acides gras et est une protéine cytosolique de 14,8 kDa exprimée du côté cytoplasmique de la myéline compacte.¦Afin d'étudier le rôle de P2 dans les cellules de Schwann myélinisantes, j'ai généré une souris knockout (P2-/-). Après avoir validé l'absence de transcrit et de protéine P2 dans les nerfs sciatiques P2-/-, des mesures électrophysiologiques ont montré une réduction modérée mais significative de la vitesse de conduction du nerf moteur périphérique. Il est important de noter que ces changements fonctionnels n'ont pas pu être associés à quelconque changement dans la structure de la myéline. Cependant, j'ai observé dans les nerfs périphériques P2-/-, une altération significative du niveau d'expression d'ARNm d'autres FABPs et en particulier FABP9. Ce dernier résultat démontre l'importance du rôle de la protéine P2 dans le métabolisme lipidique de la myéline.

Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity.

Eukaryotes contain inorganic polyphosphate (polyP) and acidocalcisomes, which sequester polyP and store amino acids and divalent cations. Why polyP is sequestered in dedicated organelles is not known. We show that polyP produced in the cytosol of yeast becomes toxic. Reconstitution of polyP translocation with purified vacuoles, the acidocalcisomes of yeast, shows that cytosolic polyP cannot be imported, whereas polyP produced by the vacuolar transporter chaperone (VTC) complex, an endogenous vacuolar polyP polymerase, is efficiently imported and does not interfere with growth. PolyP synthesis and import require an electrochemical gradient, probably as a driving force for polyP translocation. VTC exposes its catalytic domain to the cytosol and carries nine vacuolar transmembrane domains. Mutations in the VTC transmembrane regions, which are likely to constitute the translocation channel, block not only polyP translocation but also synthesis. Given that they are far from the cytosolic catalytic domain of VTC, this suggests that the VTC complex obligatorily couples synthesis of polyP to its import in order to avoid toxic intermediates in the cytosol. Sequestration of otherwise toxic polyP might be one reason for the existence of acidocalcisomes in eukaryotes.

Nuclear phytochrome a signaling promotes phototropism in Arabidopsis.

Phototropin photoreceptors (phot1 and phot2 in Arabidopsis thaliana) enable responses to directional light cues (e.g., positive phototropism in the hypocotyl). In Arabidopsis, phot1 is essential for phototropism in response to low light, a response that is also modulated by phytochrome A (phyA), representing a classical example of photoreceptor coaction. The molecular mechanisms underlying promotion of phototropism by phyA remain unclear. Most phyA responses require nuclear accumulation of the photoreceptor, but interestingly, it has been proposed that cytosolic phyA promotes phototropism. By comparing the kinetics of phototropism in seedlings with different subcellular localizations of phyA, we show that nuclear phyA accelerates the phototropic response, whereas in the fhy1 fhl mutant, in which phyA remains in the cytosol, phototropic bending is slower than in the wild type. Consistent with this data, we find that transcription factors needed for full phyA responses are needed for normal phototropism. Moreover, we show that phyA is the primary photoreceptor promoting the expression of phototropism regulators in low light (e.g., PHYTOCHROME KINASE SUBSTRATE1 [PKS1] and ROOT PHOTO TROPISM2 [RPT2]). Although phyA remains cytosolic in fhy1 fhl, induction of PKS1 and RPT2 expression still occurs in fhy1 fhl, indicating that a low level of nuclear phyA signaling is still present in fhy1 fhl.

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

Identification of GPx8 as a novel cellular substrate of the hepatitis C virus NS3-4A protease

Background: The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity (MAVS and TRIF) as well as a phosphatase involved in growth factor signaling (TC-PTP). The aim of this study was to identify novel cellular substrates of the NS3-4A protease and to investigate their role in the life cycle and pathogenesis of HCV. Methods: Cell lines inducibly expressing the NS3-4A protease were analyzed in basal as well as interferon- α -stimulated states by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. Candidates fulfilling strin- gent criteria for potential substrates or products of the NS3-4A protease were further investigated in different experimental sys- tems as well as in liver biopsies from patients with chronic hep- atitis C. Results: SILAC coupled with protein separation and mass spectrometry yielded > 5000 proteins of which 21 can- didates were selected for further analyses. These allowed us to identify GPx8, a membrane-associated peroxidase involved in disulfide bond formation in the endoplasmic reticulum, as a novel cellular substrate of the HCV NS3-4A protease. Cleavage occurs at cysteine in position 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic hepatitis C. Further functional studies, involving overexpression and RNA silencing, revealed that GPx8 is a proviral factor involved in viral particle production but not in HCV entry or RNA replica- tion. Conclusions: GPx8 is a proviral host factor cleaved by the HCV NS3-4A protease. Studies investigating the consequences of cleavage for GPx8 function are underway. The identification of novel cellular substrates of the HCV NS3-4A protease should yield new insights into the HCV life cycle and the pathogenesis of hepatitis C and may reveal novel angles for therapeutic inter- vention.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

Angiotensin-II mediates norepinephrine and neuropeptide-Y secretion in a human pheochromocytoma.

The potential role of angiotensin-II in mediating catecholamine and neuropeptide-Y release in a human pheochromocytoma has been investigated. Angiotensin-II type I receptors are transcribed and translated into functional proteins in a surgically removed pheochromocytoma. Primary cell culture of the tumor has been studied in a perfused system. Angiotensin-II increased the release of norepinephrine and neuropeptide-Y by the pheochromocytes. Activation of the angiotensin-II type I receptors by angiotensin-II was associated with a rise in cytosolic free calcium. The renin-angiotensin system may, therefore, contribute to the secretion of catecholamines and NPY occurring in patients with pheochromocytoma and when stimulated trigger hypertensive crisis.

Vesicular transport of d-serine in astrocytes

The concept of tripartite synapse suggests that astrocytes make up a functional synapse with pre- and postsynaptic neuronal elements to modulate synaptic transmission through the regulated release of neuromodulators called gliotransmitters. Release of gliotransmitters such as glutamate or D-serine has been shown to depend on Ca21-dependent exocytosis. However, the origin (cytosolic versus vesicular) of the released gliotransmitter is still a matter of debate. The existence of Ca21-regulated exocytosis in astrocytes has been questioned mostly because the nature of secretory organelles which are loaded with gliotransmitters is unknown. Here we show the existence of a population of vesicles that uptakes and stores glutamate and D-serine in astrocytes which are present in situ. Immunoisolated glial organelles expressing synaptobrevin 2 (Sb2) display morphological and biochemical features very similar to synaptic vesicles. We demonstrate that these organelles not only contain and uptake glutamate but also display a glia-specific transport activity for D-serine. Furthermore, we report that the uptake of D-serine is energized by a H1-ATPase present on the immunoisolated vesicles and that cytosolic chloride ions modulate the uptake of D-serine. Finally, we show that serine racemase (SR), the synthesizing enzyme for D-serine, is anchored to the membrane of glial organelles allowing a local and efficient concentration of the gliotransmitter to be transported. We conclude that vesicles in astrocytes do exist with the goal to store and release D-serine, glutamate and most likely other neuromodulators.

PPARalpha governs glycerol metabolism.

Glycerol, a product of adipose tissue lipolysis, is an important substrate for hepatic glucose synthesis. However, little is known about the regulation of hepatic glycerol metabolism. Here we show that several genes involved in the hepatic metabolism of glycerol, i.e., cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase (GPDH), glycerol kinase, and glycerol transporters aquaporin 3 and 9, are upregulated by fasting in wild-type mice but not in mice lacking PPARalpha. Furthermore, expression of these genes was induced by the PPARalpha agonist Wy14643 in wild-type but not PPARalpha-null mice. In adipocytes, which express high levels of PPARgamma, expression of cytosolic GPDH was enhanced by PPARgamma and beta/delta agonists, while expression was decreased in PPARgamma(+/-) and PPARbeta/delta(-/-) mice. Transactivation, gel shift, and chromatin immunoprecipitation experiments demonstrated that cytosolic GPDH is a direct PPAR target gene. In line with a stimulating role of PPARalpha in hepatic glycerol utilization, administration of synthetic PPARalpha agonists in mice and humans decreased plasma glycerol. Finally, hepatic glucose production was decreased in PPARalpha-null mice simultaneously fasted and exposed to Wy14643, suggesting that the stimulatory effect of PPARalpha on gluconeogenic gene expression was translated at the functional level. Overall, these data indicate that PPARalpha directly governs glycerol metabolism in liver, whereas PPARgamma regulates glycerol metabolism in adipose tissue.

Primordial neurosecretory apparatus identified in the choanoflagellate Monosiga brevicollis.

SNARE protein-driven secretion of neurotransmitters from synaptic vesicles is at the center of neuronal communication. In the absence of the cytosolic protein Munc18-1, synaptic secretion comes to a halt. Although it is believed that Munc18-1 orchestrates SNARE complexes, its mode of action is still a matter of debate. In particular, it has been challenging to clarify the role of a tight Munc18/syntaxin 1 complex, because this interaction interferes strongly with syntaxin's ability to form a SNARE complex. In this complex, two regions of syntaxin, the N-peptide and the remainder in closed conformation, bind to Munc18 simultaneously. Until now, this binary complex has been reported for neuronal tissues only, leading to the hypothesis that it might be a specialization of the neuronal secretion apparatus. Here we aimed, by comparing the core secretion machinery of the unicellular choanoflagellate Monosiga brevicollis with that of animals, to reconstruct the ancestral function of the Munc18/syntaxin1 complex. We found that the Munc18/syntaxin 1 complex from M. brevicollis is structurally and functionally highly similar to the vertebrate complex, suggesting that it constitutes a fundamental step in the reaction pathway toward SNARE assembly. We thus propose that the primordial secretion machinery of the common ancestor of choanoflagellates and animals has been co-opted for synaptic roles during the rise of animals.

In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles

Nanoparticles with pH-sensitive behavior may enhance the success of chemotherapy in many cancers by efficient intracellular drug delivery. Here, we investigated the effect of a bioactive surfactant with pH-sensitive properties on the antitumor activity and intracellular behavior of methotrexate-loaded chitosan nanoparticles (MTX-CS-NPs). NPs were prepared using a modified ionotropic complexation process, in which was included the surfactant derived from Nα,Nε-dioctanoyl lysine with an inorganic lithium counterion. The pH-sensitive behavior of NPs allowed accelerated release of MTX in an acidic medium, as well as membrane-lytic pH-dependent activity, which facilitated the cytosolic delivery of endocytosed materials. Moreover, our results clearly proved that MTX-CSNPs were more active against the tumor HeLa and MCF-7 cell lines than the free drug. The feasibilty of using NPs to target acidic tumor extracellular pH was also shown, as cytotoxicity against cancer cells was greater in a mildly acidic environment. Finally, the combined physicochemical and pH-sensitive properties of NPs generally allowed the entrapped drug to induce greater cell cycle arrest and apoptotic effects. Therefore, our overall results suggest that pH-sensitive MTX-CS-NPs could be potentially useful as a carrier system for tumor and intracellular drug delivery in cancer therapy.

Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, efflux, and response to incubation with ethanol.

Hepatocytes from rats that were fed ethanol chronically for 6-8 wk were found to have a modest decrease in cytosolic GSH (24%) and a marked decrease in mitochondrial GSH (65%) as compared with pair-fed controls. Incubation of hepatocytes from ethanol-fed rats for 4 h in modified Fisher's medium revealed a greater absolute and fractional GSH efflux rate than controls with maintenance of constant cellular GSH, indicating increased net GSH synthesis. Inhibition of gamma-glutamyltransferase had no effect on these results, which indicates that no degradation of GSH had occurred during these studies. Enhanced fractional efflux was also noted in the perfused livers from ethanol-fed rats. Incubation of hepatocytes in medium containing up to 50 mM ethanol had no effect on cellular GSH, accumulation of GSH in the medium, or cell viability. Thus, chronic ethanol feeding causes a modest fall in cytosolic and a marked fall in mitochondrial GSH. Fractional GSH efflux and therefore synthesis are increased under basal conditions by chronic ethanol feeding, whereas the cellular concentration of GSH drops to a lower steady state level. Incubation of hepatocytes with ethanol indicates that it has no direct, acute effect on hepatic GSH homeostasis.

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4¿dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.

Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway.

We previously reported that glucose can be released from GLUT2-null hepatocytes through a membrane traffic-based pathway issued from the endoplasmic reticulum. Here, we further characterized this glucose release mechanism using biosynthetic labeling protocols. In continuous pulse-labeling experiments, we determined that glucose secretion proceeded linearly and with the same kinetics in control and GLUT2-null hepatocytes. In GLUT2-deficient hepatocytes, however, a fraction of newly synthesized glucose accumulated intracellularly. The linear accumulation of glucose in the medium was inhibited in mutant, but not in control, hepatocytes by progesterone and low temperature, as previously reported, but, importantly, also by microtubule disruption. The intracellular pool of glucose was shown to be present in the cytosol, and, in pulse-chase experiments, it was shown to be released at a relatively slow rate. Release was not inhibited by S-4048 (an inhibitor of glucose-6-phosphate translocase), cytochalasin B, or progesterone. It was inhibited by phloretin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and low temperature. We conclude that the major release pathway segregates glucose away from the cytosol by use of a membrane traffic-based, microtubule-dependent mechanism and that the release of the cytosolic pool of newly synthesized glucose, through an as yet unidentified plasma membrane transport system, cannot account for the bulk of glucose release.