Energy levels of few electron quantum dots imaged and characterized by atomic force microscopy

Strong confinement of charges in few electron systems such as in atoms,molecules and quantum dots leads to a spectrum of discrete energy levels thatare often shared by several degenerate quantum states. Since the electronicstructure is key to understanding their chemical properties, methods that probethese energy levels in situ are important. We show how electrostatic forcedetection using atomic force microscopy reveals the electronic structure ofindividual and coupled self-assembled quantum dots. An electron additionspectrum in the Coulomb blockade regime, resulting from a change in cantileverresonance frequency and dissipation during tunneling events, shows one by oneelectron charging of a dot. The spectra show clear level degeneracies inisolated quantum dots, supported by the first observation of predictedtemperature-dependent shifts of Coulomb blockade peaks. Further, by scanningthe surface we observe that several quantum dots may reside on whattopologically appears to be just one. These images of grouped weakly andstrongly coupled dots allow us to estimate their relative coupling strengths.

Ingredients of a Casimir analog computer

We present the basic ingredients of a technique to compute quantum Casimirforces at micrometer scales using antenna measurements at tabletop, e.g.centimeter, scales, forming a type of analog computer for the Casimir force.This technique relies on a correspondence that we derive between the contourintegration of the Casimir force in the complex frequency plane and theelectromagnetic response of a physical dissipative medium in a finite, realfrequency bandwidth.

The metabolome of Chlamydomonas reinhardtii following induction of anaerobic H2 production by sulfur depletion.

Occurrence of Mitochondria-targeted Late Embryogenesis Abundant (LEA) Gene in Animals Increases Organelle Resistance to Water Stress

Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species. The deduced molecular mass of the 307-amino acid polypeptide from the brine shrimp Artemia franciscana is 34 kDa. Bioinformatic analyses reveal features typical of a Group 3 LEA protein, and subcellular localization programs predict targeting of the mature peptide to the mitochondrial matrix, based on an N-terminal, amphipathic presequence. Real-time quantitative PCR shows that Afralea3m mRNA is expressed manyfold higher in desiccation-tolerant embryonic stages when compared with intolerant nauplius larvae. Mitochondrial localization of the protein was confirmed by transfection of human hepatoma cells (HepG2/C3A) with a nucleotide construct encoding the first 70 N-terminal amino acids of AfrLEA3m in-frame with the nucleotide sequence for green fluorescence protein. The chimeric protein was readily incorporated into mitochondria of these cells. Successful targeting of a protein to human mitochondria by use of an arthropod signaling sequence clearly reveals the highly conserved nature of such presequences, as well as of the import machinery. Finally, mitochondria isolated from A. franciscana embryos, which naturally contain AfrLEA3m and trehalose, exhibit resistance to water stress (freezing) as evidenced by an unchanged capacity for oxidative phosphorylation on succinate + rotenone, a resistance that is absent in mammalian mitochondria lacking AfrLEA3m.

Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine

Multiple myeloma, an incurable plasma cell malignancy, is characterized by altered cellular metabolism and resistance to apoptosis. Recent connections between glucose metabolism and resistance to apoptosis provide a compelling rationale for targeting metabolic changes in cancer. In this study, we have examined the ability of the purine analogue 8-aminoadenosine to acutely reduce glucose consumption by regulating localization and expression of key glucose transporters. Myeloma cells counteracted the metabolic stress by activating autophagy. Co-treatment with inhibitors of autophagy results in marked enhancement of cell death. Glucose consumption by drug-resistant myeloma cells was unaffected by 8-aminoadenosine, and accordingly, no activation of autophagy was observed. However, these cells can be sensitized to 8-aminoadenosine under glucose-limiting conditions. The prosurvival autophagic response of myeloma to nutrient deprivation or to nucleoside analogue treatment has not been described previously. This study establishes the potential of metabolic targeting as a broader means to kill and sensitize myeloma and identifies a compound that can achieve this goal.

Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-induced DNA Damage via Physical Interaction with Xeroderma Pigmentosum Group A

In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation.

The Metabolome of Chlamydomonas reinhardtii following Induction of Anaerobic H2 Production by Sulfur Depletion

The metabolome of the model species Chlamydomonas reinhardtii has been analyzed during 120 h of sulfur depletion to induce anaerobic hydrogen (H(2)) production, using NMR spectroscopy, gas chromatography coupled to mass spectrometry, and TLC. The results indicate that these unicellular green algae consume freshly supplied acetate in the medium to accumulate energy reserves during the first 24 h of sulfur depletion. In addition to the previously reported accumulation of starch, large amounts of triacylglycerides were deposited in the cells. During the early 24- to 72-h time period fermentative energy metabolism lowered the pH, H(2) was produced, and amino acid levels generally increased. In the final phase from 72 to 120 h, metabolism slowed down leading to a stabilization of pH, even though some starch and most triacylglycerides remained. We conclude that H(2) production does not slow down due to depletion of energy reserves but rather due to loss of essential functions resulting from sulfur depletion or due to a build-up of the toxic fermentative products formate and ethanol.

Investigating the Structural Plasticity of a Cytochrome P450

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.

Phosphatase and Tensin Homologue on Chromosome 10 (PTEN) Directs Prostaglandin E2-mediated Fibroblast Responses via Regulation of E Prostanoid 2 Receptor Expression

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite that counters transforming growth factor-beta-induced fibroblast activation via E prostanoid 2 (EP2) receptor binding. Phosphatase and tensin homologue on chromosome 10 (PTEN) is a lipid phosphatase that, by antagonizing the phosphoinositol 3-kinase (PI3K) pathway, also inhibits fibroblast activation. Here, we show that PTEN directly regulates PGE(2) inhibition of fibroblast activation by augmenting EP2 receptor expression. The increase in collagen production and alpha-smooth muscle actin expression observed in fibroblasts in which PTEN is deficient was resistant to the usual suppressive effects of PGE(2). This was due to marked down-regulation of EP2, a G(s) protein-coupled receptor (GPCR) that mediates the inhibitory actions of this prostanoid via cAMP. pten(-/-) or PTEN-inhibited fibroblasts in which the PI3K pathway was blocked demonstrated a restoration of EP2 receptor expression, due to augmented gene transcription and mRNA instability. Importantly, restoration of the balance between PI3K and PTEN reestablished the inhibitory effect of PGE(2) on fibroblast activation. No such influence of PTEN was observed on alternative E prostanoid GPCRs. Moreover, our studies identified a positive feedback loop in which cAMP signaling enhanced EP2 receptor expression, independent of PTEN. Therefore, our findings indicate that PTEN regulates the antifibrotic effects of PGE(2) by a specific and permissive effect on EP2 receptor expression. Further, our data imply that cAMP signaling circumvents EP2 down-regulation in pten-deficient cells to restore EP2 receptor expression. This is the first description, to our knowledge, of PI3K/PTEN balance directing GPCR expression, and provides a novel mechanism for cellular effects of PTEN.

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1beta and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K(+) on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K(+) was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K(+) has been suggested to trigger inflammasome activation, in neural cells, high extracellular K(+) activates caspase-1 probably through pannexin 1.