bluuebunny/crossref_metadata_2025 huggingface dataset:
Lossless hybridization between photovoltaic and thermoelectric devices
Authors: Park Kwang-Tae , Shin Sun-Mi , Tazebay Abdullah S. , Um Han-Don , Jung Jin-Young , Jee Sang-Won , Oh Min-Wook , Park Su-Dong , Yoo Bongyoung , Yu Choongho , Lee Jung-Ho
Published: July 2013

The optimal hybridization of photovoltaic (PV) and thermoelectric (TE) devices has long been considered ideal for the efficient harnessing solar energy. Our hybrid approach uses full spectrum solar energy via lossless coupling between PV and TE devices while collecting waste energy from thermalization and transmission losses from PV devices. Achieving lossless coupling makes the power output from the hybrid device equal to the sum of the maximum power outputs produced separately from individual PV and TE devices. TE devices need to have low internal resistances enough to convey photo-generated currents without sacrificing the PV fill factor. Concomitantly, a large number of p-n legs are preferred to drive a high Seebeck voltage in TE. Our simple method of attaching a TE device to a PV device has greatly improved the conversion efficiency and power output of the PV device (~30% at a 15°C temperature gradient across a TE device).
Promising operational stability of high-efficiency organic light-emitting diodes based on thermally activated delayed fluorescence
Authors: Nakanotani Hajime , Masui Kensuke , Nishide Junichi , Shibata Takumi , Adachi Chihaya
Published: July 2013

Organic light-emitting diodes (OLEDs) are attractive for next-generation displays and lighting applications because of their potential for high electroluminescence (EL) efficiency, flexibility and low-cost manufacture. Although phosphorescent emitters containing rare metals such as iridium or platinum produce devices with high EL efficiency, these metals are expensive and their blue emission remains unreliable for practical applications. Recently, a new route to high EL efficiency using materials that emit through thermally activated delayed fluorescence (TADF) was demonstrated. However, it is unclear whether devices that emit through TADF, which originates from the contributions of triplet excitons, are reliable. Here we demonstrate highly efficient, stable OLEDs that emit via TADF by controlling the position of the carrier recombination zone, resulting in projected lifetimes comparable to those of tris(2-phenylpyridinato)iridium(III)-based reference OLEDs. Our results indicate that TADF is intrinsically stable under electrical excitation and optimization of the surrounding materials will enhance device reliability.
First example of a high-level correlated calculation of the indirect spin–spin coupling constants involving tellurium: tellurophene and divinyl telluride
Authors: Rusakov Yury Yu. , Krivdin Leonid B. , Østerstrøm Freja F. , Sauer Stephan P. A. , Potapov Vladimir A. , Amosova Svetlana V.
Published: July 2013

The125Te–1H spin–spin coupling constants of tellurophene and divinyl telluride were calculated at the SOPPA and DFT levels, in agreement with experimental data.
What Type of Knowledge Rules Where? Legally Regulated Participation in Large-Scale Mobile Infrastructure Planning in Sweden
Authors: Larsson Stefan
Published: July 2013

I investigate the field of tension between the national and the local level in spatial planning from a decision-making perspective. In doing so, I analyse the legal regulation for a large-scale 3G mobile infrastructure development in Sweden with a focus on how participation is expressed both in terms of the ‘law in books' as well as empirically, ‘in action’. Theoretically, a model of decision making is elaborated, based on two axes: one concerning the decision level on a central or national to local scale, and one concerning what type of knowledge is regarded as the most legitimate in terms of a calculating approach versus a communicative approach. These two issues or approaches to decision making— who decides and based on what knowledge—are of direct importance for understanding the frameworks as well as the practical outcomes of public participation. The case of 3G in Sweden demonstrates how different types of knowledge are perceived as legitimate at different levels in the planning system. For example, appeals against building permits rarely change the outcome of permits issued, and appeals based on fear of electromagnetic radiation are always rejected. The juridification of a given mast conflict meant a development from a deliberative approach, where any concern is heard, to a calculating and expert-based one, where the general stance on a particular topic (such as whether or not the electromagnetic radiation from mobile masts is hazardous) is applied. This means that what knowledge is legitimate depends on where in the permit process it is presented.
The Effect of Head Turn and Illumination on the Perceived Direction of Gaze
Authors: West Roger W
Published: July 2013

Thirty observers judged the direction of monocular and binocular gaze from a model's LCD-imaged head when the head, gaze, and illumination were either straight or turned 20.6° to the side. The judged direction of binocular eye contact from a turned head was judged to go past the direction of the observer, while judged monocular gaze from the eye toward the observer followed that for binocular gaze. Although judged monocular gaze from the eye away from the observer also passed the observer, it did so by a smaller amount. The judged direction of binocular gaze in the same direction as the head turn was judged to pass the direction of the head turn. The judged direction of monocular gaze from the eye away from the observer was nearly true, but the judged direction of monocular gaze from the eye toward the observer was judged as closer to the observer than true. Illumination of the model's head from the side made the direction of both head turn and gaze appear to move away from the direction of the light source. These results are discussed in connection with angle k and the perceived direction of head turn.
Alcohol Consumption Enhances Vection
Authors: Seno Takeharu , Nakamura Shinji
Published: July 2013

We assessed changes in forward vection following alcohol consumption, and found that alcohol consumption enhanced vection. This result indicates that alcohol can affect psychophysical processes responsible for self-motion perception.
Seeing Further Than Your Nose
Authors: van Tonder Gert , Zavagno Daniele , Sakurai Kenzo , Ono Hiroshi
Published: July 2013

Among monocular depth cues, ocular parallax (first described formally by Brewster in 1844) remains mostly unknown, its role in perception still not investigated scientifically. Given that every single eye movement induces ocular parallax, it is a potentially useful depth cue. This paper is an attempt to revive interest in the topic. As a monocular depth cue, ocular parallax naturally leads us to consider its benefit for a monocularly enucleated individual. Throughout history, numerous illustrious personalities coped with this fate in various ways. Here, we consider some historical insights into the visual life of the erstwhile duke of Urbino, Federico da Montefeltro (1422–1482), warlord and patron to the painter Piero della Francesca, and the Japanese warlord, Masamune Date (1567–636), a vocal patron of exploration and the arts.
Inverse boundary value problems for the perturbed polyharmonic operator
Authors: Krupchyk Katsiaryna , Lassas Matti , Uhlmann Gunther
Published: July 2013

We show that a first order perturbation

A
(
x
)
⋅
D
+
q
(
x
)

A(x)\cdot D+q(x)

of the polyharmonic operator

(
−
Δ

)
m

(-\Delta )^m

,

m
≥
2

m\ge 2

, can be determined uniquely from the set of the Cauchy data for the perturbed polyharmonic operator on a bounded domain in

R

n

\mathbb {R}^n

,

n
≥
3

n\ge 3

. Notice that the corresponding result does not hold in general when

m
=
1

m=1

.
Serine Phosphorylation of Focal Adhesion Kinase in Interphase and Mitosis: A Possible Role in Modulating Binding to p130Cas
Authors: Ma Amy , Richardson Alan , Schaefer Erik M. , Parsons J. Thomas
Published: July 2013

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130Cas.
Ric1p and the Ypt6p GTPase Function in a Common Pathway Required for Localization ofTrans-Golgi Network Membrane Proteins
Authors: Bensen Eric S. , Yeung Bonny G. , Payne Gregory S.
Published: July 2013

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Δ) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Δ cells also harboringend3Δ to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes.SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Δ. Further comparison of ric1Δ and ypt6Δ cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Δ andypt6Δ cells. SLY1–20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.
The Sda1 Protein Is Required for Passage through Start
Authors: Zimmerman Zachary A. , Kellogg Douglas R.
Published: July 2013

We have used affinity chromatography to identify proteins that interact with Nap1, a protein previously shown to play a role in mitosis. Our studies demonstrate that a highly conserved protein called Sda1 binds to Nap1 both in vitro and in vivo. Loss of Sda1 function causes cells to arrest uniformly as unbudded cells that do not increase significantly in size. Cells arrested by loss of Sda1 function have a 1N DNA content, fail to produce the G1 cyclin Cln2, and remain responsive to mating pheromone, indicating that they arrest in G1 before Start. Expression of CLN2 from a heterologous promoter in temperature-sensitive sda1 cells induces bud emergence and polarization of the actin cytoskeleton, but does not induce cell division, indicating that the sda1 cell cycle arrest phenotype is not due simply to a failure to produce the G1 cyclins. The Sda1 protein is absent from cells arrested in G0 and is expressed before Start when cells reenter the cell cycle, further suggesting that Sda1 functions before Start. Taken together, these findings reveal that Sda1 plays a critical role in G1 events. In addition, these findings suggest that Nap1 is likely to function during G1. Consistent with this, we have found that Nap1 is required for viability in cells lacking the redundant G1 cyclins Cln1 and Cln2. In contrast to a previous study, we have found no evidence that Sda1 is required for the assembly or function of the actin cytoskeleton. Further characterization of Sda1 is likely to provide important clues to the poorly understood mechanisms that control passage through G1.
Vps41p Function in the Alkaline Phosphatase Pathway Requires Homo-oligomerization and Interaction with AP-3 through Two Distinct Domains
Authors: Darsow Tamara , Katzmann David J. , Cowles Christopher R. , Emr Scott D.
Published: July 2013

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein–dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of theseVPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.
Mitochondrial Ca2+-induced Ca2+Release Mediated by the Ca2+Uniporter
Authors: Montero Mayte , Alonso Maria Teresa , Albillos Almudena , Garcı́a-Sancho Javier , Alvarez Javier
Published: July 2013

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca2+during cell stimulation. The present study focuses on the pathways for mitochondrial Ca2+efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca2+uptake and increased the cytosolic [Ca2+] ([Ca2+]c) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca2+]ckinetics and inhibited Ca2+release from Ca2+-loaded mitochondria. This effect was due to inhibition of mitochondrial Na+/Ca2+exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca2+]ctriggered fast Ca2+release from these depolarized Ca2+-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca2+-induced Ca2+release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca2+uniporter. This novel kind of mitochondrial Ca2+-induced Ca2+release might contribute to Ca2+clearance from mitochondria that become depolarized during Ca2+overload.
Lipid Rafts Act as Specialized Domains for Tetanus Toxin Binding and Internalization into Neurons
Authors: Herreros Judit , Ng Tony , Schiavo Giampietro
Published: July 2013

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.
Characterization of Human Palladin, a Microfilament-associated Protein
Authors: Mykkänen Olli-Matti , Grönholm Mikaela , Rönty Mikko , Lalowski Maciej , Salmikangas Paula , Suila Heli , Carpén Olli
Published: July 2013

Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, α-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the α-helical domain of ezrin and by Ig-domains 2–3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.
Dual Role of H-Ras in Regulation of Lymphocyte Function Antigen-1 Activity by Stromal Cell-derived Factor-1α: Implications for Leukocyte Transmigration
Authors: Weber Kim S.C. , Ostermann Georg , Zernecke Alma , Schröder Andreas , Klickstein Lloyd B. , Weber Christian
Published: July 2013

We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1α (SDF-1α) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH2-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1α-induced Akt phosphorylation. SDF-1α triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1–mediated transendothelial chemotaxis but not VLA-4–dependent transmigration induced by SDF-1α. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1α, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1α in leukocytes, establishing a complex and bimodal involvement of H-Ras.
Cooperative Control of Akt Phosphorylation, bcl-2 Expression, and Apoptosis by Cytoskeletal Microfilaments and Microtubules in Capillary Endothelial Cells
Authors: Flusberg Deborah A. , Numaguchi Yasushi , Ingber Donald E.
Published: July 2013

Capillary endothelial cells can be switched between growth and apoptosis by modulating their shape with the use of micropatterned adhesive islands. The present study was carried out to examine whether cytoskeletal filaments contribute to this response. Disruption of microfilaments or microtubules with the use of cytochalasin D or nocodazole, respectively, led to levels of apoptosis in capillary cells equivalent to that previously demonstrated by inducing cell rounding with the use of micropatterned culture surfaces containing small (<20 μm in diameter) circular adhesive islands coated with fibronectin. Simultaneous disruption of microfilaments and microtubules led to more pronounced cell rounding and to enhanced levels of apoptosis approaching that observed during anoikis in fully detached (suspended) cells, indicating that these two cytoskeletal filament systems can cooperate to promote cell survival. Western blot analysis revealed that the protein kinase Akt, which is known to be critical for control of cell survival became dephosphorylated during cell rounding induced by disruption of the cytoskeleton, and that this was accompanied by a decrease in bcl-2 expression as well as a subsequent increase in caspase activation. This ability of the cytoskeleton to control capillary endothelial cell survival may be important for understanding the relationship among extracellular matrix turnover, cell shape changes, and apoptosis during angiogenesis inhibition.
Serial Analysis of Gene Expression in Plasmodium falciparum Reveals the Global Expression Profile of Erythrocytic Stages and the Presence of Anti-Sense Transcripts in the Malarial Parasite
Authors: Patankar Swati , Munasinghe Anusha , Shoaibi Azadeh , Cummings Leda M. , Wirth Dyann F.
Published: July 2013

Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of ∼8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform “profiling” nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation ofPlasmodium gene expression.
Role for Telomere Cap Structure in Meiosis
Authors: Maddar Haggar , Ratzkovsky Nir , Krauskopf Anat
Published: July 2013

Telomeres, the natural ends of eukaryotic chromosomes, are essential for the protection of chromosomes from end-to-end fusions, recombination, and shortening. Here we explore their role in the process of meiotic division in the budding yeast, Kluyveromyces lactis. Telomerase RNA mutants that cause unusually long telomeres with deregulated structure led to severely defective meiosis. The severity of the meiotic phenotype of two mutants correlated with the degree of loss of binding of the telomere binding protein Rap1p. We show that telomere size and the extent of potential Rap1p binding to the entire telomere are irrelevant to the process of meiosis. Moreover, we demonstrate that extreme difference in telomere size between two homologous chromosomes is compatible with the normal function of telomeres during meiosis. In contrast, the structure of the most terminal telomeric repeats is critical for normal meiosis. Our results demonstrate that telomeres play a critical role during meiotic division and that their terminal cap structure is essential for this role.
Simultaneous Tyrosine and Serine Phosphorylation of STAT3 Transcription Factor Is Involved in Rho A GTPase Oncogenic Transformation
Authors: Aznar Salvador , Valerón Pilar F. , del Rincon Sonia Victoria , Pérez Leandro Fernández , Perona Rosario , Lacal Juan Carlos
Published: July 2013

Stats (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that on a specific stimulus migrate to the nucleus and exert their transcriptional activity. Here we report a novel signaling pathway whereby RhoA can efficiently modulate Stat3 transcriptional activity by inducing its simultaneous tyrosine and serine phosphorylation. Tyrosine phosphorylation is exerted via a member of the Src family of kinases (SrcFK) and JAK2, whereas the JNK pathway mediates serine phosphorylation. Furthermore, cooperation of both tyrosine as well as serine phosphorylation is necessary for full activation of Stat3. Induction of Stat3 activity depends on the effector domain of RhoA and correlates with induction of both Src Kinase-related and JNK activities. Activation of Stat3 has biological implications. Coexpression of an oncogenic version of RhoA along with the wild-type, nontransforming Stat3 gene, significantly enhances its oncogenic activity on human HEK cells, suggesting that Stat3 is an essential component of RhoA-mediated transformation. In keeping with this, dominant negative Stat3 mutants or inhibition of its tyrosine or serine phosphorylation completely abrogate RhoA oncogenic potential. Taken together, these results indicate that Stat3 is an important player in RhoA-mediated oncogenic transformation, which requires simultaneous phosphorylation at both tyrosine and serine residues by specific signaling events triggered by RhoA effectors.
GCP5 and GCP6: Two New Members of the Human γ-Tubulin Complex
Authors: Murphy Steven M. , Preble Andrea M. , Patel Urvashi K. , O'Connell Kathy L. , Dias D. Prabha , Moritz Michelle , Agard David , Stults John T. , Stearns Tim
Published: July 2013

The γ-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human γ-tubulin complex and the identification of its subunits. The human γ-tubulin complex is a ring of ∼25 nm, has a subunit structure similar to that reported for γ-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human γ-tubulin complex components confirmed the presence of four previously identified components (γ-tubulin and γ-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the γ-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire γ-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of γ-tubulin, GCP2, GCP3, and GCP4 within the γ-tubulin complex. Thus, the γ-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.
Lag1p and Lac1p Are Essential for the Acyl-CoA–dependent Ceramide Synthase Reaction inSaccharomyces cerevisae
Authors: Schorling Stefan , Vallée Béatrice , Barz Wolfgang P. , Riezman Howard , Oesterhelt Dieter
Published: July 2013

Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae.Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1Δlac1Δ cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA–dependent ceramide synthase reaction. The sphingolipid synthesis defect inlag1Δlac1Δ cells can be partially corrected by overexpression of YPC1 orYDC1, encoding ceramidases that have been reported to have acyl-CoA–independent ceramide synthesis activity. Quadruple mutant cells (lag1Δlac1Δypc1Δydc1Δ) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover,lag1Δlac1Δ cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA–dependent ceramide synthase reaction.
Increased Levels of Keratin 16 Alter Epithelialization Potential of Mouse Skin Keratinocytes In Vivo and Ex Vivo
Authors: Wawersik Matthew J. , Mazzalupo Stacy , Nguyen Diem , Coulombe Pierre A.
Published: July 2013

The process of wound repair in adult skin is complex, involving dermal contraction and epithelial migration to repair the lesion and restore the skin's barrier properties. At the wound edge, keratinocytes undergo many changes that engender an epithelialization behavior. The type II keratin 6 and type I keratins 16 and 17 are induced well before cell migration begins, but the role of these proteins is not understood. Forced expression of human K16 in skin epithelia of transgenic mice has been shown to cause dose-dependent skin lesions concomitant with alterations in keratin filament organization and in cell adhesion. Here we show, with the use of a quantitative assay, that these transgenic mice show a delay in the closure of full-thickness skin wounds in situ compared with wild-type and low-expressing K16 transgenic mice. We adapted and validated an ex vivo skin explant culture system to better assess epithelialization in a wound-like environment. Transgenic K16 explants exhibit a significant reduction of keratinocyte outgrowth in this setting. This delay is transgene dose-dependent, and is more severe when K16 is expressed in mitotic compared with post-mitotic keratinocytes. Various lines of evidence suggest that the mechanism(s) involved is complex and not strictly cell autonomous. These findings have important implications for the function of K16 in vivo.
Cell Cycle-dependent Expression and Nucleolar Localization of hCAP-H
Authors: Cabello Olga A. , Eliseeva Elena , He WeiGong , Youssoufian Hagop , Plon Sharon E. , Brinkley B. R. , Belmont John W.
Published: July 2013

Condensin is a conserved 13S heteropentamer composed of two nonidentical structural maintenance of chromosome (SMC) family proteins, in Xenopus XCAP-C and XCAP-E, and three regulatory subunits, XCAP-D2, XCAP-G, and XCAP-H. Both biochemical and genetic analyses have demonstrated an essential role for the 13S condensin complex in mitotic chromosome condensation. Further, a potential requirement for condensin in completion of chromatid arm separation in early anaphase is demonstrated by the mutational phenotypes of the Drosophila homologues ofXCAP-H, barren and XCAP-C,DmSMC4. In this study we have investigated the expression and subcellular distribution of hCAP-H, the human homolog of XCAP-H, in order to better understand its cellular functions. Transcription of hCAP-H was restricted to proliferating cells with highest expression during the G2 phase of the cell cycle. In contrast, cellular hCAP-H protein levels were constant throughout the cell cycle. hCAP-H was found to be associated with mitotic chromosomes exhibiting a nonuniform but symmetric distribution along sister chromatids. The symmetry of hCAP-H association with sister chromatids suggests that there are sequence-dependent domains of condensin aggregation. During interphase hCAP-H, -C, and -E, have distinct punctate nucleolar localization, suggesting that condensin may associate with and modulate the conformation and function of rDNA. hCAP-H association with condensed chromatin was not observed in the early phase of chromosome condensation when histone H3 phosphorylation has already taken place. This finding is consistent with the hypothesis that histone H3 phosphorylation precedes condensin-mediated condensation.
Signaling through Adenylyl Cyclase Is Essential for Hyphal Growth and Virulence in the Pathogenic FungusCandida albicans
Authors: Rocha Cintia R. C. , Schröppel Klaus , Harcus Doreen , Marcil Anne , Dignard Daniel , Taylor Brad N. , Thomas David Y. , Whiteway Malcolm , Leberer Ekkehard
Published: July 2013

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned theCaCDC35 gene encoding C. albicansadenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles ofCaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygouscacdc35Δ cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.
Novel Stress-responsive Genes EMG1 and NOP14 Encode Conserved, Interacting Proteins Required for 40S Ribosome Biogenesis
Authors: Liu Phillip C. C. , Thiele Dennis J.
Published: July 2013

Under stressful conditions organisms adjust the synthesis, processing, and trafficking of molecules to allow survival from and recovery after stress. In baker's yeast Saccharomyces cerevisiae, the cellular production of ribosomes is tightly matched with environmental conditions and nutrient availability through coordinate transcriptional regulation of genes involved in ribosome biogenesis. On the basis of stress-responsive gene expression and functional studies, we have identified a novel, evolutionarily conserved gene, EMG1, that has similar stress-responsive gene expression patterns as ribosomal protein genes and is required for the biogenesis of the 40S ribosomal subunit. The Emg1 protein is distributed throughout the cell; however, its nuclear localization depends on physical interaction with a newly characterized nucleolar protein, Nop14. Yeast depleted of Nop14 or harboring a temperature-sensitive allele of emg1 have selectively reduced levels of the 20S pre-rRNA and mature18S rRNA and diminished cellular levels of the 40S ribosomal subunit. Neither Emg1 nor Nop14 contain any characterized functional motifs; however, isolation and functional analyses of mammalian orthologues of Emg1 and Nop14 suggest that these proteins are functionally conserved among eukaryotes. We conclude that Emg1 and Nop14 are novel proteins whose interaction is required for the maturation of the 18S rRNA and for 40S ribosome production.
Rab4 Regulates Formation of Synaptic-like Microvesicles from Early Endosomes in PC12 Cells
Authors: de Wit Heidi , Lichtenstein Yael , Kelly Regis B. , Geuze Hans J. , Klumperman Judith , van der Sluijs Peter
Published: July 2013

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.
Interactions between Syntaxins Identify at Least Five SNARE Complexes within the Golgi/Prevacuolar System of the Arabidopsis Cell
Authors: Sanderfoot Anton A. , Kovaleva Valya , Bassham Diane C. , Raikhel Natasha V.
Published: July 2013

The syntaxin family of soluble N-ethyl maleimide sensitive factor adaptor protein receptors (SNAREs) is known to play an important role in the fusion of transport vesicles with specific organelles. Twenty-four syntaxins are encoded in the genome of the model plant Arabidopsis thaliana. These 24 genes are found in 10 gene families and have been reclassified as syntaxins of plants (SYPs). Some of these gene families have been previously characterized, with the SYP2-type syntaxins being found in the prevacuolar compartment (PVC) and the SYP4-type syntaxins on thetrans-Golgi network (TGN). Here we report on two previously uncharacterized syntaxin groups. The SYP5 group is encoded by a two-member gene family, whereas SYP61 is a single gene. Both types of syntaxins are localized to multiple compartments of the endomembrane system, including the TGN and the PVC. These two groups of syntaxins form SNARE complexes with each other, and with other Arabidopsis SNAREs. On the TGN, SYP61 forms complexes with the SNARE VTI12 and either SYP41 or SYP42. SYP51 and SYP61 interact with each other and with VTI12, most likely also on the TGN. On the PVC, a SYP5-type syntaxin interacts specifically with a SYP2-type syntaxin, as well as the SNARE VTI11, forming a SNARE complex likely involved in TGN-to-PVC trafficking.
Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s
Authors: Pfund Christine , Huang Peggy , Lopez-Hoyo Nelson , Craig Elizabeth A.
Published: July 2013

Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains. Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of “classic” Hsp70s. An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function. However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements. Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more “classical” Hsp70, Ssa. BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo. Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo. These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function. This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro. These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains.
Microtubule-dependent Organization of Vaccinia Virus Core–derivd Early mRNAs into Distinct Cytoplasmic Structures
Authors: Mallardo Massimo , Schleich Sibylle , Krijnse Locker Jacomine
Published: July 2013

Vaccinia virus (vv) early transcription can be reconstituted in vitro from purified virions; in this assay mRNAs are made inside the viral core and subsequently extruded. Although the in vitro process has been extensively characterized, relatively little is known about vv early transcription in vivo. In the present study the fate of vv early mRNAs in infected HeLa cells was followed by BrUTP transfection and confocal and electron microscopy. The extruded vv early mRNAs were found to be organized into unique granular cytoplasmic structures that reached a size up to 1 μm. By EM these structures appeared as amorphous electron-dense cytoplasmic aggregates that were surrounded by ribosomes. Confocal images showed that the RNA structures were located some distance away from intracellular cores and that both structures appeared to be aligned on microtubules (MTs), implying that MT tracks connected mRNAs and cores. Accordingly, intact MTs were found to be required for the typical punctate organization of viral mRNAs. Biochemical evidence supported the notion that vv mRNAs were MT associated and that MT depletion severely affected viral (but not cellular) mRNA synthesis and stability. By confocal microscopy the viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting away from intracellular cores to their sites of cytoplasmic accumulation.
Two Related Kinesins,klp5+andklp6+, Foster Microtubule Disassembly and Are Required for Meiosis in Fission Yeast
Authors: West Robert R. , Malmstrom Terra , Troxell Cynthia L. , McIntosh J. Richard
Published: July 2013

The kinesin superfamily of microtubule motor proteins is important in many cellular processes, including mitosis and meiosis, vesicle transport, and the establishment and maintenance of cell polarity. We have characterized two related kinesins in fission yeast,klp5+andklp6+, that are amino-terminal motors of the KIP3 subfamily. Analysis of null mutants demonstrates that neither klp5+norklp6+, individually or together, is essential for vegetative growth, although these mutants have altered microtubule behavior. klp5Δ and klp6Δ are resistant to high concentrations of the microtubule poison thiabendazole and have abnormally long cytoplasmic microtubules that can curl around the ends of the cell. This phenotype is greatly enhanced in the cell cycle mutant cdc25–22, leading to a bent, asymmetric cell morphology as cells elongate during cell cycle arrest. Klp5p-GFP and Klp6p-GFP both localize to cytoplasmic microtubules throughout the cell cycle and to spindles in mitosis, but their localizations are not interdependent. During the meiotic phase of the life cycle, both of these kinesins are essential. Spore viability is low in homozygous crosses of either null mutant. Heterozygous crosses of klp5Δ with klp6Δ have an intermediate viability, suggesting cooperation between these proteins in meiosis.
Distinct Roles of Frontal and Rear Cell-Substrate Adhesions in Fibroblast Migration
Authors: Munevar Steven , Wang Yu-li , Dembo Micah
Published: July 2013

Cell migration involves complex physical and chemical interactions with the substrate. To probe the mechanical interactions under different regions of migrating 3T3 fibroblasts, we have disrupted cell-substrate adhesions by local application of the GRGDTP peptide, while imaging stress distribution on the substrate with traction force microscopy. Both spontaneous and GRGDTP-induced detachment of the trailing edge caused extensive cell shortening, without changing the overall level of traction forces or the direction of migration. In contrast, disruption of frontal adhesions caused dramatic, global loss of traction forces before any significant shortening of the cell. Although traction forces and cell migration recovered within 10–20 min of transient frontal treatment, persistent treatment with GRGDTP caused the cell to develop traction forces elsewhere and reorient toward a new direction. We conclude that contractile forces of a fibroblast are transmitted to the substrate through two distinct types of adhesions. Leading edge adhesions are unique in their ability to transmit active propulsive forces. Their functions cannot be transferred directly to existing adhesions upon detachment. Trailing end adhesions create passive resistance during cell migration and readily redistribute their loads upon detachment. Our results indicate the distinct nature of mechanical interactions at the leading versus trailing edges, which together generate the mechanical interactions for fibroblast migration.
Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity
Authors: Matthies Heinrich J.G. , Baskin Ronald J. , Hawley R. Scott
Published: July 2013

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NODDTWand NOD“DR2”) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in theDrosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport.