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Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

We present a detailed study of the valence and conduction bands of VO2 across the metal-insulator transition using bulk-sensitive photoelectron and O K x-ray absorption spectroscopies. We observe a giant transfer of spectral weight with distinct features that require an explanation which goes beyond the Peierls transition model as well as the standard single-band Hubbard model. Analysis of the symmetry and energies of the bands reveals the decisive role of the V 3d orbital degrees of freedom. Comparison to recent realistic many body calculations shows that much of the k dependence of the self-energy correction can be cast within a dimer model.

We found direct experimental evidence for an orbital switching in the V 3d states across the metal-insulator transition in VO2. We have used soft-x-ray absorption spectroscopy at the V L2,3 edges as a sensitive local probe and have determined quantitatively the orbital polarizations. These results strongly suggest that, in going from the metallic to the insulating state, the orbital occupation changes in a manner that charge fluctuations and effective bandwidths are reduced, that the system becomes more one dimensional and more susceptible to a Peierls-like transition, and that the required massive orbital switching can only be made if the system is close to a Mott insulating regime.

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer, but resistance to 5-FU remains a major obstacle to successful therapy. We generated 5-FU-resistant derivatives of the HCT116 human colon cancer cell line by serial passage of these cells in the presence of increasing 5-FU concentrations in an attempt to elucidate the biological mechanisms involved in resistance to 5-FU. Two resultant resistant derivatives, HCT116 ResB and ResD, were characterized for resistance phenotypes, genotypes, and gene expression using cells maintained long-term in 5-FU-free media. Compared to parental HCT116 cells that respond to 5-FU challenge by inducing high levels of apoptosis, ResB and ResD derivatives had significantly reduced apoptotic fractions when transiently challenged with 5-FU. ResB and ResD cells were respectively 27- and 121-fold more resistant to 5-FU, had increased doubling times, and significantly increased plating efficiencies compared to the parental cells. Both resistant derivatives retained the wild-type TP53 genotype, TP53 copy number and CGH profile characteristic of the parental line. Alterations in gene expression in the resistant derivatives compared to the parental line were assessed using oligonucleotide microarrays. Overall, the 5-FU-resistant derivatives were characterized by reduced apoptosis and a more aggressive growth phenotype, consistent with the observed up-regulation of apoptosis-inhibitory genes (e.g., IRAK1, MALT1, BIRC5), positive growth-regulatory genes (e.g., CCND3, CCNE2, CCNF, CYR61), and metastasis genes (e.g., LMNB1, F3, TMSNB), and down-regulation of apoptosis-promoting genes (e.g., BNIP3, BNIP3L, FOXO3A) and negative growth-regulatory genes (e.g., AREG, CCNG2, CDKN1A, CDKN1C, GADD45A). 5-FU metabolism-associated genes (e.g., TYMS, DTYMK, UP) and DNA repair genes (e.g., FEN1, FANCG, RAD23B) were also up-regulated in one or both resistant derivatives, suggesting that the resistant derivatives might be able to overcome both 5-FU inhibition of thymidylate synthase and the DNA damage caused by 5-FU, respectively. Development of 5-FU resistance thus appears to encompass deregulation of apoptosis-, proliferation-, DNA repair-, and metastasis-associated regulatory pathways.

At the cerebellar parallel fiber-Purkinje cell synapse, isolated presynaptic activity induces fast excitatory postsynaptic currents via ionotropic glutamate receptors while repetitive, high-frequency, presynaptic activity can also induce a slow excitatory postsynaptic current that is mediated by metabotropic glutamate receptors (mGluR1-EPSC). Here we investigated the involvement of glutamate uptake in the expression of the mGluR1-EPSC. Inhibitors of glutamate uptake led to a large increase of the mGluR1-EPSC. D-aspartate (0.4 mM) and L(-)-threo-3-hydroxyaspartate (0.4 mM) increased the mGluR1-EPSC approximately 4.5 and approximately 9-fold, respectively, while dihydrokainic acid (1 mM), had no significant effect on the mGluR1-EPSC. D-aspartate (0.4 mM) shifted the concentration-response curve of the depression of the mGluR1-EPSC by the low-affinity mGluR1 antagonist (S)-a-Methyl-4-carboxyphenylglycine [(S)-MCPG] to higher concentrations and decreased the stimulus intensity and the number of necessary stimuli to evoke an mGluR1-EPSC. Depression of the mGluR1-EPSC by rapid pressure application of (S)-MCPG at varying time intervals after tetanic stimulation of the parallel fibers indicated that the glutamate concentration in the peri- and extrasynaptic space decayed with time constants of 36 and 316 ms under control conditions and with inhibition of glutamate uptake, respectively. These results show that expression of the slow mGluR-mediated excitatory postsynaptic current is controlled by glutamate transporter activity. Thus in contrast to fast glutamatergic synaptic transmission, metabotropic glutamate receptor-mediated transmission is critically dependent on the activity and capacity of glutamate uptake.

CuMoO4 crystals reversibly change their color from green to brown upon heating, accompanied by a loss in transmittance. UV/VIS spectroscopic analysis revealed that these changes are due to particular electronic properties of the crystal instead of its chemical decomposition or structural change. Investigations were carried out in the temperature range 23-400 degrees C. The intensive green color of the crystal at room temperature is caused by a small transmission window between two absorption bands, the band gap of the crystal in the blue and the 3d9-->4p absorption of the Cu2+ ions in the red. With increasing temperature the band gap shifts towards longer wavelengths, and the crystal changes both color and transmittance. Spectroscopic features of the crystal are discussed together with the temperature dependence of its electrical resistance. Resistance measurements were performed simultaneously to the optical measurements.

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.

Purpose: To determine the electrophysiological properties of Müller (glial) cells from experimentally detached rabbit retinas., Methods: A stable local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Müller cells were acutely dissociated and studied by the whole-cell voltage-clamp technique., Results: The cell membranes of Müller cells from normal retinas were dominated by a large inwardly rectifying potassium ion (K+) conductance that caused a low-input resistance (<100 M(Omega)) and a high resting membrane potential (-82 +/- 6 mV). During the first week after detachment, the Müller cells became reactive as shown by glial fibrillary acidic protein (GFAP) immunoreactivity, and their inward currents were markedly reduced, accompanied by an increased input resistance (>200 M(Omega)). After 3 weeks of detachment, the input resistance increased further (>300 M(Omega)), and some cells displayed significantly depolarized membrane potentials (mean -69 +/- 18 mV). When PVR developed (in 20% of the cases) the inward K+ currents were virtually completely eliminated. The input resistance increased dramatically (>1000 MOmega), and almost all cells displayed strongly depolarized membrane potentials (-44 +/- 16 mV)., Conclusions: Reactive Müller cells are characterized by a severe reduction of their K+ inward conductance, accompanied by depolarized membrane potentials. These changes must impair physiological glial functions, such as neurotransmitter recycling and K+ ion clearance. Furthermore, the open probability of certain types of voltage-dependent ion channels (e.g., Ca2+-dependent K+ maxi channels) increases that may be a precondition for Müller cell proliferation, particularly in PVR when a dramatic downregulation of both inward current density and resting membrane potential occurs.

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate-cystine antiporter (system X(c)(-)), whereas, others suggest that it is taken up by the glutamate transporter (system X(AG)). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b(o,+) and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-beta-hydroxamate (AbetaH) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-alpha-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 microM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.

Müller glial cells from human and guinea-pig retinae were investigated using the whole-cell patch-clamp technique. Human Müller cells from eyes with different diseases were characterized by diminished inwardly-rectifying K(+) currents. A comparable reduction of these currents was achieved in guinea pig Müller cells by treatment with iodoacetate to generate ischemia-like conditions. Consequently, the membrane potentials were reduced significantly in both diseased human and iodoacetate-treated guinea-pig Müller cells as compared to normal controls. However, the potentials were still clearly negative. Delayed rectifier currents could still be recorded under these conditions. Application of quinine blocked the delayed rectifier K(+) channels, and resulted in a total breakdown of the membrane potentials. Thus, it becomes apparent that the glial delayed rectifier K(+) channels are necessary to maintain an 'auxiliary' membrane potential under certain pathological conditions that are characterized by an almost total loss of inward rectifier conductance. Therefore, the delayed rectifier K(+) channels of glial cells may become crucial for the support of basic glial functions.

Maintenance of isolated retinal Müller (glial) cells in glutamate-free solutions over 7 h causes a significant loss of their initial glutathione content; this loss is largely prevented by the blockade of glutamine synthesis using methionine sulfoximine (5 mM). Anoxia does not reduce the glutathione content of Müller cells when glucose (11 mM), glutamate and cystine (0.1 mM each) are present. In contrast, simulation of total ischemia (i.e., anoxia plus removal of glucose) decreases the glutathione levels dramatically, even in the presence of glutamate and cystine. Less severe effects are caused by high extracellular K+ (40 mM). Reactive oxygen species are generated in the retina under various conditions, such as anoxia, ischemia, and reperfusion. One of the crucial substances protecting the retina against reactive oxygen species is glutathione, a tripeptide constituted of glutamate, cysteine and glycine. It was recently shown that glutathione can be synthesized in retinal Müller glial cells and that glutamate is the rate-limiting substance. In this study, glutathione levels were determined in acutely isolated guinea-pig Müller cells using the glutathione-sensitive fluorescent dye monochlorobimane. The purpose was to find out how the glial glutathione content is affected by anoxia/ischemia and accompanying pathophysiological events such as depolarization of the cell membrane. Our results further strengthen the view that glutamate is rate-limiting for the glutathione synthesis in glial cells. During glutamate deficiency, as caused by e.g., impaired glutamate uptake, this amino acid is preferentially delivered to the glutamate-glutamine pathway, at the expense of glutathione. This mechanism may contribute to the finding that total ischemia (but not anoxia) causes a depletion of glial glutathione. In situ depletion may be accelerated by the ischemia-induced increase of extracellular K+, decreasing the driving force for glutamate uptake. The ischemia-induced lack of glutathione is particularly fatal considering the increased production of reactive oxygen species under this condition. Therefore the therapeutic application of exogenous free radical scavengers is greatly recommended.

The electrophysiological properties of Müller cells, the principal glial cells of the retina, are determined by several types of K(+) conductances. Both the absolute and the relative activities of the individual types of K(+) channels undergo important changes in the course of ontogenetic development and during gliosis. Although immature Müller cells express inwardly rectifying K(+) (K(IR)) currents at a very low density, the membrane of normal mature Müller cells is predominated by the K(IR) conductance. The K(IR) channels mediate spatial buffering K(+) currents and maintain a stable hyperpolarized membrane potential necessary for various glial-neuronal interactions. During "conservative" (i.e., non-proliferative) reactive gliosis, the K(IR) conductance of Müller cells is moderately reduced and the cell membrane is slightly depolarized; however, when gliotic Müller cells become proliferative, their K(IR) conductances are dramatically down-regulated; this is accompanied by an increased activity of Ca(2+)-activated K(+) channels and by a conspicuous unstability of their membrane potential. The resultant variations of the membrane potential may increase the activity of depolarization-activated K(+), Na(+) and Ca(2+) channels. It is concluded that in respect to their K(+) current pattern, mature Müller cells pass through a process of dedifferentiation before proliferative activity is initiated., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: To determine differences of K+ channel activity between Müller glial cells obtained from retinas of healthy human donors and of patients with retinal detachment and proliferative vitreoretinopathy., Methods: Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients. The whole-cell and the cell-attached configurations of the patch-clamp technique were used to characterize the current densities of different K+ channel types and the activity of single Ca2+ -activated K+ channels of big conductance (BK)., Results: Cells from patients displayed a less negative mean membrane potential (-52.8 mV) than cells from healthy donors (-80.6 mV). However, the membrane potentials in cells from patients scattered largely between -6 and -99 mV. The inwardly rectifying K+ permeability in cells from patients was strongly reduced (0.3 pA/pF) when compared with cells from healthy donors (6.0 pA/pF). At the resting membrane potential, single BK channels displayed a higher mean activity (open probability, Po, and channel current amplitude) in cells from patients (Po, 0.30) than in cells from healthy donors (Po: 0.03). The variations of BK current amplitudes were correlated with the variations of the membrane potential., Conclusions: The dominant expression of inwardly rectifying channels in cells from healthy donors is thought to support important glial cell functions such as the spatial buffering of extracellular K+. The downregulation of these channels and the less negative mean membrane potential in cells from patients should impair spatial buffering currents and neurotransmitter clearance. The increased activity of BK channels may support the proliferative activity of gliotic cells via feedback regulation of Ca2+ entry and membrane potential.

The cell-attached configuration of the patch-clamp technique was used to characterize the outward currents in acutely isolated Müller cells from the guinea pig retina. Sixty-five of 353 patches displayed macroscopic, outwardly rectifying currents due to depolarizing voltage steps. Single channel transitions were found in only two patches. The remaining patches did not reveal any voltage-dependent currents. Tail current analysis revealed a reversal potential close to the resting membrane potential. The currents disappeared if internal K+ was replaced by Cs+ in inside-out patches. From these results we conclude that guinea pig Müller cells possess voltage-dependent K+ channels that are distributed in clusters.

Adrenoceptors were among the first neurotransmitter receptors identified in glial cells, but it is not known whether these receptors meditate glial responses during neuronal activity. We show that repetitive nerve activity evoked a rise of intracellular calcium in Bergmann glia and neighboring Purkinje neurons of cerebellar slices of mice. The glial but not the neuronal calcium transient persisted during block of ionotropic and metabotropic glutamate receptors. In contrast, the glial calcium response was abolished by cyclopiazonic acid and prazosin; however, prazosin affected neither the inward current nor the resulting depolarization that accompanied the stimulus-induced glial calcium transients. The glial depolarization was attenuated by 38% by the mixture of glutamate receptor blockers, which abolished the evoked neuronal depolarization and afterhyperpolarization. Ba(2+) reduced the glial currents by 66% without affecting the concomitant calcium transients. In the presence of Ba(2+), the mixture of glutamate receptor blockers exerted no effect on the glial inward current or calcium rise. Furthermore, Ba(2+) greatly potentiated both the activity-related Purkinje cell inward current and the accompanying neuronal calcium rises. The results indicate that release of noradrenaline from afferent fibers activates a glial alpha(1) adrenoceptor that promotes calcium release from intracellular stores. Glial calcium rises are known to stimulate a diversity of processes such as transmitter release, energy metabolism, or proliferation. Thus the adrenoceptor-mediated mechanism described here is well suited for feedback modulation of neuronal function that is independent of glutamate.

Selective antibodies were used to assess the cellular and subcellular localization of glutathione, and the glutathione precursors gamma-glutamylcysteine, glutamate, and cysteine, in neuronal (photoreceptors) and non-neuronal (pigment epithelial cells and Müller cells) cell types in the outer retina of the guinea pig. In each cell type the highest level of glutathione immunoreactivity occurred in the mitochondria. The labeling density in the cytoplasmic matrix was higher (and the mitochondrial-cytoplasmic gold particle ratio lower) in pigment epithelial cells than in Müller cells and photoreceptors. The latter two cell types showed a mitochondrial-cytoplasmic gold particle ratio of 15.5 and 21.7, respectively. In contrast to glutathione, gamma-glutamylcysteine seemed to be enriched in the cytoplasmic matrix relative to the mitochondria. The immunogold labeling for this dipeptide was stronger in the pigment epithelial cells than in Müller cells and photoreceptors. Glutamate immunoreactivity was high in photoreceptors, intermediate in pigment epithelial cells, and low in Müller cells, while the cysteine immunogold signal was low in each cell type and cell compartment. The present results suggest that glutathione is concentrated in mitochondria but to different degrees in different cells. The low mitochondrial content of gamma-glutamylcysteine (the direct precursor of glutathione) is consistent with biochemical data indicating that glutathione is synthesized extramitochondrially and transported into the mitochondrial matrix. Judged from the immunocytochemical data, cysteine may be a rate-limiting factor in glutathione synthesis in each cell type while glutamate can be rate limiting only in Müller cells.

Objective: The aim of this study was to investigate the incidence of cardiopulmonary resuscitation (CPR) after cardiac surgery and to find predictors of survival., Design: A retrospective study with data obtained by chart review., Setting: A university hospital 24-bed cardiac surgical intensive care unit (ICU)., Participants: Between 1993 and 1994, 4,968 consecutive adult patients who underwent cardiac surgery at the authors' hospital were studied., Interventions: None., Main Results: One hundred thirteen of these patients (2.3%) were resuscitated. Seventy-nine patients (70%) survived to be discharged from the hospital. Significant predictors of survival were the time between admission to the ICU and initiation of CPR, CPR time, and creatine kinase (CK) and CK-MB values., Conclusions: The incidence of CPR after cardiac surgery was 2.3% with no difference between valve surgery and CABG. Best results were achieved when arrhythmias or bleeding were the predisposing causes. Further studies have to be undertaken concerning long-term results and quality of life of the discharged patients.