bluuebunny/crossref_metadata_2025 huggingface dataset:
CdTe/Zn
1−x
Mg
x
Te
self-assembled quantum dots: Towards room temperature emission
Authors: Tinjod F. , Moehl S. , Kheng K. , Gilles B. , Mariette H.
Published: December 2003

We report the dependence of the growth and the optical properties of self-assembled CdTe/Zn1−xMgxTe quantum dots on the barrier Mg content x (0⩽x⩽0.3). Due to the decrease of the lattice mismatch between CdTe and Zn1−xMgxTe with increasing x, we use a technique for inducing dot formation, based on efficient reduction of the surface energy by deposition of amorphous Te, which is then desorbed. Mg incorporation in the barriers leads to a better heavy-hole confinement along the growth axis, which is manifested in photoluminescence (PL) studies by both an extension of the radiative regime temperature range (up to 150 K for 30% Mg) and a strong increase of the activation energy for the nonradiative recombination. However, the in-plane confinement is less enhanced, which allows observation of interdot carrier transfer with increasing temperature, as evidenced directly by the analysis of PL intensities for different single dots. Our temperature-dependent data (time-resolved and microphotoluminescence) suggest that this transfer consists of a thermally activated process via the two-dimensional wetting-layer states rather than a direct tunneling (hopping) process.
Role of internal stress on room-temperature permittivity of BaTiO3 ceramics and thin films
Authors: Lee Jung-Kun , Lee Young Hyun , Hong Kug Sun , Jang Jin-Wook
Published: December 2003

Anomalies observed in the room-temperature permittivity of BaTiO3 (BTO) ceramics and thin films with second phases were investigated. In BTO ceramics with slight Ba-excess composition, Ba2TiO4 particles were found and the overall sample permittivity increased, compared to stoichiometric BTO. For BTO films with Ti-excess composition, Ti-excess intergranular phase was found and the permittivity was significantly lower than that of the stoichiometric film. Transmission electron microscopy revealed that the BTO ceramics/films were under high stress due to the thermal mismatch with these second phases. To explain the different behaviors of permittivity in two cases, Landau–Devonshire theory was applied, based on a geometric model depicting the different state of stress between BTO matrixes and second phases. The calculation showed that the permittivity change predicted by the theory was consistent with experimental results. This study illustrates the effect of the geometry of the second phase and thermal stress on ferroelectric properties.
Interface and defect structures of Zn–ZnO core–shell heteronanobelts
Authors: Ding Y. , Kong X. Y. , Wang Z. L.
Published: December 2003

Interface and defect structures of Zn–ZnO core–shell nanobelts have been investigated using high-resolution transmission electron microscopy. Most of the nanobelts can be classified into two types from their growth directions: [21̄1̄0] and [0001], with the top/bottom surfaces being (0001) and (21̄1̄0), respectively. The Zn core and ZnO shell overlapped areas display a two-dimensional moiré pattern resulting from the lattice mismatch. In the 〈21̄1̄0〉 growth nanobelts, a network of three sets of misfit dislocations relaxes the mismatch strain in the top/bottom interfaces, and every set rotates 60° with respect to the other; there are two types of grains oriented in specific orientations that compose the side wall of the ZnO shell. In the [0001] growth nanobelts, a network containing a set of stacking faults in (0001) planes and a set of misfit dislocations in (011̄0) planes takes the main role in the misfit relaxation. Threading dislocations indicated by terminating moiré fringes are present in both of them, which are located at the small angle rotated boundary between adjacent misoriented ZnO grains.
Thermal wave scattering by spheres
Authors: Garrido F. , Salazar A.
Published: December 2003

In this article, the solution of the periodic temperature fluctuation of an opaque material with buried spheres as a result of a modulated illumination is presented. The model includes the multiple scattering of the plane thermal wave, generated at the sample surface, when interacting with the spheres and with the sample surfaces. A series solution to the heat diffusion equation and a matrix formulation to solve the scattering coefficients are used. The solution is general in the sense that there are neither restrictions on the number of spheres, their size, and position inside the material nor on the thermal properties of spheres and sample. Measurements on calibrated samples using an infrared radiometric microscope validate our model. This theory is specially suited to the thermal characterization and nondestructive evaluation of particulate composites using photothermal techniques.
Electrical and optical characterizations of self-assembled quantum dots formed by the atomic layer epitaxy technique
Authors: Park Young Min , Park Young Ju , Kim Kwang Moo , Shin Jae Cheol , Song Jin Dong , Lee Jung Il , Yoo Keon-Ho
Published: December 2003

We investigated the electrical and optical properties of InGaAs self-assembled quantum dots grown using the atomic layer epitaxy (ALE) technique. Dots–in–a–well structures were grown by alternately supplying InAs and GaAs sources on an InGaAs layer and covering with another InGaAs layer. Three samples produced with different numbers of cycles of alternate InAs/GaAs supply were characterized by capacitance-voltage and photoluminescence (PL) measurements. For the ten cycle dots–in–a–well structure, a strong zero-dimensional electron confinement was observed even at room temperature. On the other hand, for the five-cycle structure, the PL results indicate that the InGaAs quantum well structure coexists unstably with premature quantum dots. By comparing the results for samples with different numbers of cycles, we suggest that an ALE dots–in–a–well structure can be formed by the aggregation of In and Ga atoms incorporated into the InGaAs quantum well layer when the number of cycles exceeds the critical number of seven cycles.
Carrier thermalization, cooling and diffusion in InSb through a nonlinear pump-probe technique
Authors: Lobad Ahmed , Schlie L. A. (Vern)
Published: December 2003

We investigated the ultrafast carrier dynamics in InSb using a nonlinear pump-probe technique. The measurement involves two pump pulses chopped at different frequencies and the detection of the probe modulation at their sum frequency. We show that the nonlinear signature and dynamics of the transmission-bleaching signal enables the differentiation of carrier thermalization from cooling dynamics. We follow the evolution of the injected carrier distribution from a nonthermal to a thermal one and calculate its absolute temperature and energy decay rates as a function of time from a single transmission pump-probe measurement. The carrier scattering rate in the thermalization stage increases by (3 ps)−1 with doubling the injected carrier density. For an initial injected excess energy of 650 K, carrier thermalization is complete after 1.7 ps with a thermal distribution of 370 K. We also demonstrate the ability to follow carrier diffusion through the decay of the carrier grating decay in a transmission measurement.
Adhesion shear theory of ice friction at low sliding velocities, combined with ice sintering
Authors: Maeno Norikazu , Arakawa Masahiko
Published: December 2003

Adhesion and shear deformation of ice have been traditionally considered to be responsible for ice friction at sliding velocities lower than about 10−2 m/s, but the simple mechanism cannot explain the recent finding that the ice–ice friction coefficient increases with decreasing sliding velocity. This article proposes an improved adhesion shear theory, which takes account of junction growth of asperities at the sliding ice interface due to sintering. At lower sliding velocities and higher homologous temperatures, contacts of ice asperities develop resulting in the increase of friction force.
TGF-β receptor kinase inhibitor enhances growth and integrity of embryonic stem cell–derived endothelial cells
Authors: Watabe Tetsuro , Nishihara Ayako , Mishima Koichi , Yamashita Jun , Shimizu Kiyoshi , Miyazawa Keiji , Nishikawa Shin-Ichi , Miyazono Kohei
Published: December 2003

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However, the molecular mechanisms that regulate their differentiation, proliferation, and endothelial sheet formation remain to be elucidated. Here, we show that members of the transforming growth factor (TGF)-β superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5–days postcoitum embryos. TGF-β and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-β and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover, SB-431542 up-regulated the expression of claudin-5, an endothelial specific component of tight junctions. These results suggest that endogenous TGF-β/activin signals play important roles in regulating vascular growth and permeability.
PLA2 activity is required for nuclear shrinkage in caspase-independent cell death
Authors: Shinzawa Koei , Tsujimoto Yoshihide
Published: December 2003

Apoptosis is defined on the basis of morphological changes like nuclear fragmentation and chromatin condensation, which are dependent on caspases. Many forms of caspase-independent cell death have been reported, but the mechanisms are still poorly understood. We found that hypoxic cell death was independent of caspases and was associated with significant nuclear shrinkage. Neither Bcl-2 nor Apaf-1 deficiency prevented hypoxic nuclear shrinkage. To understand the molecular mechanism of the nuclear shrinkage, we developed an in vitro system using permeabilized cells, which allowed us to purify a novel member of the phospholipase A2 (PLA2) family that induced nuclear shrinkage. Purified PLA2 induced nuclear shrinkage in our permeabilized cell system. PLA2 inhibitors prevented hypoxic nuclear shrinkage in cells and cell death. Hypoxia caused elevation of PLA2 activity and translocation of intracellular PLA2s to the nucleus. Knockdown of the Ca2+-independent PLA2 delayed nuclear shrinkage and cell death. These results indicate that Ca2+-independent PLA2 is crucial for a caspase-independent cell death signaling pathway leading to nuclear shrinkage.
Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow
Authors: Setiadi Hendra , McEver Rodger P.
Published: December 2003

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.
Caspr regulates the processing of contactin and inhibits its binding to neurofascin
Authors: Gollan Leora , Salomon Daniela , Salzer James L. , Peles Elior
Published: December 2003

Three cell adhesion molecules are present at the axoglial junctions that form between the axon and myelinating glia on either side of nodes of Ranvier. These include an axonal complex of contacin-associated protein (Caspr) and contactin, which was proposed to bind NF155, an isoform of neurofascin located on the glial paranodal loops. Here, we show that NF155 binds directly to contactin and that surprisingly, coexpression of Caspr inhibits this interaction. This inhibition reflects the association of Caspr with contactin during biosynthesis and the resulting expression of a low molecular weight (LMw), endoglycosidase H–sensitive isoform of contactin at the cell membrane, which remains associated with Caspr but is unable to bind NF155. Accordingly, deletion of Caspr in mice by gene targeting results in a shift from the LMw- to a HMw-contactin glycoform. These results demonstrate that Caspr regulates the intracellular processing and transport of contactin to the cell surface, thereby affecting its ability to interact with other cell adhesion molecules.
Voltage-dependent Ca2+ Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis
Authors: Schuhmeier R.P. , Melzer W.
Published: December 2003

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the “removal model fit” approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 μM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at −16 and −20 mV, respectively, compared with −2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 μM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.
S4 Movement in a Mammalian HCN Channel
Authors: Vemana Sriharsha , Pandey Shilpi , Larsson H. Peter
Published: December 2003

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.
Changes in Local S4 Environment Provide a Voltage-sensing Mechanism for Mammalian Hyperpolarization–activated HCN Channels
Authors: Bell Damian C. , Yao Huan , Saenger Renee C. , Riley John H. , Siegelbaum Steven A.
Published: December 2003

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (∼3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.
Reconstituted TOM Core Complex and Tim9/Tim10 Complex of Mitochondria Are Sufficient for Translocation of the ADP/ATP Carrier across Membranes
Authors: Vasiljev Andreja , Ahting Uwe , Nargang Frank E. , Go Nancy E. , Habib Shukry J. , Kozany Christian , Panneels Valérie , Sinning Irmgard , Prokisch Holger , Neupert Walter , Nussberger Stephan , Rapaport Doron
Published: December 2003

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.
Rac GTPase Instructs Nuclear Factor-κB Activation by Conveying the SCF Complex and IkBα to the Ruffling Membranes
Authors: Boyer Laurent , Travaglione Sara , Falzano Loredana , Gauthier Nils C. , Popoff Michel R. , Lemichez Emmanuel , Fiorentini Carla , Fabbri Alessia
Published: December 2003

Nuclear factor-κB (NF-κB) is a ubiquitously expressed transcription factor that plays a central role in directing a vast range of cellular functions. Its activation is controlled by the Rac GTPase and relies on the coordinated cooperation of the E3–ligase complex SCFβTrCP, composed by Skp-1/Cullin-1, Rbx/Roc1, and the β-TrCP proteins. Recently, Cullin-1 has been reported to form a complex with the activated Rac GTPase. Here, we show that the specific activation of the Rac GTPase, besides directing its own positioning, induces the relocalization of the SCF component Cullin-1 to the ruffling membranes. This occurred only if the ruffles were stimulated by the Rac GTPase and was accompanied by the repositioning to the same intracellular compartment of the SCF protein Skp-1 and the ubiquitin-like molecule Nedd-8. The SCF substrate IkBα was also directed to the ruffling membranes in a Rac-dependent way. The novelty of these findings is in respect to the demonstration that the correct positioning at the ruffling membranes is crucial for the subsequent series of events that leads to IkBα proteasomal degradation and the resultant activation of NF-κB. Consequently, this points to the role of Rac as a docking molecule in NF-κB activation.
Mammalian Late Vacuole Protein Sorting Orthologues Participate in Early Endosomal Fusion and Interact with the Cytoskeleton
Authors: Richardson Simon C. W. , Winistorfer Stanley C. , Poupon Viviane , Luzio J. Paul , Piper Robert C.
Published: December 2003

In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to “tether” vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function.
γ-Tubulin Plays an Essential Role in the Coordination of Mitotic Events
Authors: Prigozhina Natalie L. , Oakley C. Elizabeth , Lewis Amanda M. , Nayak Tania , Osmani Stephen A. , Oakley Berl R.
Published: December 2003

Recent data from multiple organisms indicate that γ-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the γ-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (∼6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that γ-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.
Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells
Authors: Hara Takahiko , Ishida Hiroshi , Raziuddin Razi , Dorkhom Stephan , Kamijo Keiju , Miki Toru
Published: December 2003

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin–coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin–coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.
Regulation of Longevity inCaenorhabditis elegansby Heat Shock Factor and Molecular Chaperones
Authors: Morley James F. , Morimoto Richard I.
Published: December 2003

The correlation between longevity and stress resistance observed in long-lived mutant animals suggests that the ability to sense and respond to environmental challenges could be important for the regulation of life span. We therefore examined the role of heat shock factor (HSF-1), a master transcriptional regulator of stress-inducible gene expression and protein folding homeostasis, in the regulation of longevity. Down-regulation of hsf-1 by RNA interference suppressed longevity of mutants in an insulin-like signaling (ILS) pathway that functions in the nervous system of Caenorhabditis elegans to influence aging. hsf-1 was also required for temperature-induced dauer larvae formation in an ILS mutant. Using tissue-specific expression of wild-type or dominant negative HSF-1, we demonstrated that HSF-1 acts in multiple tissues to regulate longevity. Down-regulation of individual molecular chaperones, transcriptional targets of HSF-1, also decreased longevity of long-lived mutant but not wild-type animals. However, suppression by individual chaperones was to a lesser extent, suggesting an important role for networks of chaperones. The interaction of ILS with HSF-1 could represent an important molecular strategy to couple the regulation of longevity with an ancient genetic switch that governs the ability of cells to sense and respond to stress.
A Striking Quality Control Subcompartment inSaccharomyces cerevisiae: The Endoplasmic Reticulum-associated Compartment
Authors: Huyer Gregory , Longsworth Gaby L. , Mason Deborah L. , Mallampalli Monica P. , McCaffery J. Michael , Wright Robin L. , Michaelis Susan
Published: December 2003

The folding of nascent secretory and membrane proteins is monitored by the endoplasmic reticulum (ER) quality control system. Misfolded proteins are retained in the ER and can be removed by ER-associated degradation. As a model for the ER quality control of multispanning membrane proteins in yeast, we have been studying mutant forms of Ste6p. Here, we identify mislocalized mutant forms of Ste6p that induce the formation of, and localize to, prominent structures that are absent in normal cells. We have named these structures ER-associated compartments (ERACs), based on their juxtaposition to and connection with the ER, as observed by fluorescence and electron microscopy. ERACs comprise a network of tubulo-vesicular structures that seem to represent proliferated ER membranes. Resident ER lumenal and membrane proteins are present in ERACs in addition to their normal ER localization, suggesting there is no barrier for their entry into ERACs. However, the forms of Ste6p in ERACs are excluded from the ER and do not enter the secretory pathway; instead, they are ultimately targeted for ER-associated degradation. The presence of ERACs does not adversely affect secretory protein traffic through the ER and does not lead to induction of the unfolded protein response. We propose that ERACs may be holding sites to which misfolded membrane proteins are specifically diverted so as not to interfere with normal cellular functions. We discuss the likelihood that related ER membrane proliferations that form in response to certain other mutant or unassembled membrane proteins may be substantially similar to ERACs.
Sec61p Contributes to Signal Sequence Orientation According to the Positive-Inside Rule
Authors: Goder Veit , Junne Tina , Spiess Martin
Published: December 2003

Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated. Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the “positive-inside rule.” We tested the role of conserved charged residues of Sec61p, the major component of the translocon in Saccharomyces cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis by using diagnostic model proteins. Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences. These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal. For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides. Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.
Transcriptional Remodeling in Response to Iron Deprivation inSaccharomyces cerevisiae
Authors: Shakoury-Elizeh Minoo , Tiedeman John , Rashford Jared , Ferea Tracey , Demeter Janos , Garcia Emily , Rolfes Ronda , Brown Patrick O. , Botstein David , Philpott Caroline C.
Published: December 2003

The budding yeast Saccharomyces cerevisiae responds to depletion of iron in the environment by activating Aft1p, the major iron-dependent transcription factor, and by transcribing systems involved in the uptake of iron. Here, we have studied the transcriptional response to iron deprivation and have identified new Aft1p target genes. We find that other metabolic pathways are regulated by iron: biotin uptake and biosynthesis, nitrogen assimilation, and purine biosynthesis. Two enzymes active in these pathways, biotin synthase and glutamate synthase, require an iron-sulfur cluster for activity. Iron deprivation activates transcription of the biotin importer and simultaneously represses transcription of the entire biotin biosynthetic pathway. Multiple genes involved in nitrogen assimilation and amino acid metabolism are induced by iron deprivation, whereas glutamate synthase, a key enzyme in nitrogen assimilation, is repressed. A CGG palindrome within the promoter of glutamate synthase confers iron-regulated expression, suggesting control by a transcription factor of the binuclear zinc cluster family. We provide evidence that yeast subjected to iron deprivation undergo a transcriptional remodeling, resulting in a shift from iron-dependent to parallel, but iron-independent, metabolic pathways.
Phagocytosis of Necrotic Cells by Macrophages Is Phosphatidylserine Dependent and Does Not Induce Inflammatory Cytokine Production
Authors: Brouckaert Greet , Kalai Michael , Krysko Dmitri V. , Saelens Xavier , Vercammen Dominique , Ndlovu `Matladi , Haegeman Guy , D'Herde Katharina , Vandenabeele Peter
Published: December 2003

Apoptotic cells are cleared by phagocytosis during development, homeostasis, and pathology. However, it is still unclear how necrotic cells are removed. We compared the phagocytic uptake by macrophages of variants of L929sA murine fibrosarcoma cells induced to die by tumor necrosis factor-induced necrosis or by Fas-mediated apoptosis. We show that apoptotic and necrotic cells are recognized and phagocytosed by macrophages, whereas living cells are not. In both cases, phagocytosis occurred through a phosphatidylserine-dependent mechanism, suggesting that externalization of phosphatidylserine is a general trigger for clearance by macrophages. However, uptake of apoptotic cells was more efficient both quantitatively and kinetically than phagocytosis of necrotic cells. Electron microscopy showed clear morphological differences in the mechanisms used by macrophages to engulf necrotic and apoptotic cells. Apoptotic cells were taken up as condensed membrane-bound particles of various sizes rather than as whole cells, whereas necrotic cells were internalized only as small cellular particles after loss of membrane integrity. Uptake of neither apoptotic nor necrotic L929 cells by macrophages modulated the expression of proinflammatory cytokines by the phagocytes.
The Yeast Casein Kinase Yck3p Is Palmitoylated, then Sorted to the Vacuolar Membrane with AP-3-dependent Recognition of a YXXϕ Adaptin Sorting Signal
Authors: Sun Beimeng , Chen Linyi , Cao Wei , Roth Amy F. , Davis Nicholas G.
Published: December 2003

Our previous work found the two yeast plasma membrane-localized casein kinases Yck1p and Yck2p to be palmitoylated on C-terminal Cys-Cys sequences by the palmitoyl transferase Akr1p. The present work examines a third casein kinase, Yck3p, which ends with the C-terminal sequence Cys-Cys-Cys-Cys-Phe-Cys-Cys-Cys. Yck3p is palmitoylated and localized to the vacuolar membrane. While the C-terminal cysteines are required for this palmitoylation, Akr1p is not. Palmitoylation requires the C-terminal Yck3p residues 463-524, whereas information for vacuolar sorting maps to the 409-462 interval. Vacuolar sorting is disrupted in cis through deletion of the 409-462 sequences and in trans through mutation of the AP-3 adaptin complex; both cis- and trans-mutations result in Yck3p missorting to the plasma membrane. This missorted Yck3p restores 37°C viability to yck1Δ yck2-ts cells. yck1Δ yck2-ts suppressor mutations isolated within the YCK3 gene identify the Yck3p vacuolar sorting signal—the tetrapeptide YDSI, a perfect fit to the YXXϕ adaptin-binding consensus. Although YXXϕ signals have a well-appreciated role in the adaptin-mediated sorting of mammalian cells, this is the first signal of this class to be identified in yeast.
Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin Subunits
Authors: Werner Nicola Susann , Windoffer Reinhard , Strnad Pavel , Grund Christine , Leube Rudolf Eberhard , Magin Thomas Michael
Published: December 2003

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.
Role of Binding of Plectin to the Integrin β4 Subunit in the Assembly of Hemidesmosomes
Authors: Koster J. , van Wilpe S. , Kuikman I. , Litjens S.H.M. , Sonnenberg A.
Published: December 2003

We have previously shown that plectin is recruited into hemidesmosomes through association of its actin-binding domain (ABD) with the first pair of fibronectin type III (FNIII) repeats and a small part of the connecting segment (residues 1328–1355) of the integrin β4 subunit. Here, we show that two proline residues (P1330 and P1333) in this region of the connecting segment are critical for supporting β4-mediated recruitment of plectin. Additional binding sites for the plakin domain of plectin on β4 were identified in biochemical and yeast two-hybrid assays. These sites are located at the end of the connecting segment (residues 1383–1436) and in the region containing the fourth FNIII repeat and the C-tail (residues 1570–1752). However, in cells, these additional binding sites cannot induce the assembly of hemidesmosomes without the interaction of the plectin-ABD with β4. Because the additional plectin binding sites overlap with sequences that mediate an intramolecular association of the β4 cytoplasmic domain, we propose that they are not accessible for binding and need to become exposed as the result of the binding of the plectin-ABD to β4. Furthermore, these additional binding sites might be necessary to position the β4 cytoplasmic domain for an optimal interaction with other hemidesmosomal components, thereby increasing the efficiency of hemidesmosome assembly.
The Role of Heat Shock Transcription Factor 1 in the Genome-wide Regulation of the Mammalian Heat Shock Response
Authors: Trinklein Nathan D. , Murray John I. , Hartman Sara J. , Botstein David , Myers Richard M.
Published: December 2003

Previous work has implicated heat shock transcription factor 1 (HSF1) as the primary transcription factor responsible for the transcriptional response to heat stress in mammalian cells. We characterized the heat shock response of mammalian cells by measuring changes in transcript levels and assaying binding of HSF1 to promoter regions for candidate heat shock genes chosen by a combination of genome-wide computational and experimental methods. We found that many heat-inducible genes have HSF1 binding sites (heat shock elements, HSEs) in their promoters that are bound by HSF1. Surprisingly, for 24 heat-inducible genes, we detected no HSEs and no HSF1 binding. Furthermore, of 182 promoters with likely HSE sequences, we detected HSF1 binding at only 94 of these promoters. Also unexpectedly, we found 48 genes with HSEs in their promoters that are bound by HSF1 but that nevertheless did not show induction after heat shock in the cell types we examined. We also studied the transcriptional response to heat shock in fibroblasts from mice lacking the HSF1 gene. We found 36 genes in these cells that are induced by heat as well as they are in wild-type cells. These results provide evidence that HSF1 does not regulate the induction of every transcript that accumulates after heat shock, and our results suggest that an independent posttranscriptional mechanism regulates the accumulation of a significant number of transcripts.
Neuropeptide‐Y Y5 Receptors Modulate the Onset and Maintenance of Operant Ethanol Self‐Administration
Authors: Schroeder Jason P. , Iller Kimberly A. , Hodge Clyde W.
Published: December 2003

Background: Neuropeptide Y (NPY) is the most abundant and widely distributed peptide in the mammalian central nervous system and increases feeding behavior at NPY Y1 or Y5 receptor subtypes. Recent pharmacological and mutant mouse data indicate that NPY activity at its receptors can influence ethanol self‐administration, although the direction and strength of this influence are not clear.

Methods: Effects of the novel NPY Y5 receptor antagonist L‐152,804 on the onset and maintenance of operant self‐administration were examined in male C57BL/6J mice, which were trained to self‐administer ethanol (10% v/v) versus water via the sucrose substitution method during 16 hr overnight sessions. After 4 months of baseline responding, mice were injected with L‐152,804 (0, 10, 30, or 60 mg/kg, intraperitoneally) before operant sessions. Potential locomotor effects of L‐152,804 and possible interaction with the sedative properties of ethanol also were examined.

Results: All three doses of L‐152,804 significantly delayed the onset of ethanol‐reinforced responding relative to vehicle injection. L‐152,804 produced no effect on the total number of ethanol‐ or water‐reinforced responses per 16 hr session. However, L‐152,804 selectively modulated the temporal distribution of ethanol‐reinforced responding depending on the dose (10 and 60 mg/kg) and time point measured in a manner consistent with blockade of ethanol reinforcement. Additional experiments determined that L‐152,804 (10 or 60 mg/kg) did not alter spontaneous locomotor activity or influence the sedative effects of ethanol (4 g/kg).

Conclusions: These results indicate that blockade NPY Y5 receptor activity modulates the onset and maintenance of ethanol self‐administration. For this reason, NPY‐Y5 receptor antagonists may be useful in medical management of alcohol abuse and alcoholism.
Ethanol Sensitivity of Recombinant Homomeric and Heteromeric AMPA Receptor Subunits Expressed in Xenopus Oocytes
Authors: Akinshola B. Emmanuel , Yasuda Robert P. , Peoples Robert W. , Taylor Robert E.
Published: December 2003

Background: Ethanol is known to acutely inhibit AMPA receptor function, and sensitivity of AMPA receptors to ethanol is dependent on subunit composition in vivo and in vitro. A commonly used in vitro expression system for studying recombinant receptor subunits is the Xenopus laevis oocyte and two‐electrode voltage‐clamp electrophysiological recording. To date, ethanol sensitivity of injected receptor subunit complementary RNA (cRNA) has not been shown to be correlated with the actual expression of receptor subunits in oocytes. In this study, we compared ethanol sensitivity of homomeric and heteromeric AMPA receptor subunits microinjected into Xenopus oocytes and confirmed subunit expression in oocytes by immunoblot.

Methods: cRNAs coding for the “flop” type AMPA GluR1 or GluR3 (homomeric), GluR2/GluR3 (heteromeric combination), and GluR1/2/3 (heteromeric combination) were microinjected in equimolar amounts of 16 to 20 ng into oocytes, which were studied for their sensitivity to ethanol. Oocytes injected with cRNA for homomeric or heteromeric subunit combinations were homogenized and the expressed subunits quantified with anti‐GluR1, anti‐GluR2, and anti‐GluR2/3 antibodies.

Results: Ethanol concentrations of 10 to 500 mM consistently inhibited currents activated in oocytes by 200 μM kainic acid. The expressed homomeric GluR1 receptor and heteromeric GluR1/2/3 receptor combination currents showed similar sensitivity to ethanol inhibition with half‐maximal inhibition values of 170 ± 12 mM and 176 ± 8 mM, respectively. The expressed homomeric GluR3 receptor and heteromeric GluR2/3 receptor combination currents were differentially sensitive to ethanol inhibition with respective IC50 values of 238 ± 9 mM and 338 ± 16 mM.

Conclusion: The expressed homomeric and heteromeric “flop” type AMPA receptors were differentially sensitive to ethanol, which may in part explain differential ethanol sensitivity in native neurons.