bluuebunny/crossref_metadata_2025 huggingface dataset:
Leseflüssigkeit im Grundschulalter: Entwicklungsverlauf und Effekte systematischer Leseförderung
Authors: Müller Bettina , Križan Ana , Hecht Teresa , Richter Tobias , Ennemoser Marco
Published: July 2013

Die Fähigkeit des flüssigen Lesens stellt im Leseprozess einen Mediator zwischen den hierarchieniedrigen Prozessen der Worterkennung und den hierarchiehohen Prozessen des sinnentnehmenden Lesens auf Satz- und Textebene dar. Im Deutschen findet die explizite Förderung der Leseflüssigkeit jedoch erst seit wenigen Jahren systematische Beachtung, was sich in einem Mangel an evaluierten Trainingsprogrammen niederschlägt. Vor diesem Hintergrund untersucht diese Studie in einem experimentellen Prä-Post-Design die Entwicklung der Leseflüssigkeit leseschwacher Kinder in Klasse 2 und 4 sowie die Wirksamkeit dreier Lesetrainings auf die Geschwindigkeit der Leseflüssigkeitsentwicklung. Dazu wurden 58 Kinder der zweiten Klasse und 51 Kinder der vierten Klasse, deren Leseleistung unter dem Mittelwert ihrer Schulklasse lag, ausgewählt und randomisiert einer der drei Fördermaßnahmen oder einer Wartekontrollgruppe zugewiesen. Als Kriterium für die Entwicklung der Leseflüssigkeit diente die Anzahl korrekt gelesener Silben und Wörter in einem curriculumsbasierten Speedtest. Die Ergebnisse latenter Wachstumskurvenmodelle zeigen, dass die Leseflüssigkeit im Rahmen der regulären Leseentwicklung über die Klassenstufen 2 bis 4 hinweg zunahm. Zudem konnte die Entwicklung der Leseflüssigkeit durch systematische Fördermaßnahmen positiv beeinflusst werden: Ein Leseflüssigkeitstraining zur expliziten Förderung des flüssigen und betonten Lesens sowie ein Lesestrategietraining zur Verbesserung des sinnentnehmenden Lesens konnten in den Klassen 2 und 4 zur Beschleunigung der Leseflüssigkeit beitragen. Ein Phonics-Training, konzipiert zur Verbesserung der Phonem-Graphem-Assoziationen, zeigte nur in Klasse 4 Effekte auf die Leseflüssigkeit. Die Relevanz dieser Effekte für eine Verbesserung des Leseverständnisses wird diskutiert.
Functional Deterioration of Endothelial Nitric Oxide Synthase after Focal Cerebral Ischemia
Authors: Yagita Yoshiki , Kitagawa Kazuo , Oyama Naoki , Yukami Toshiro , Watanabe Akihiro , Sasaki Tsutomu , Mochizuki Hideki
Published: July 2013

Endothelial nitric oxide synthase (eNOS) dysfunction is related to secondary injury and lesion expansion after cerebral ischemia. To date, there are few reports about postischemic alterations in the eNOS regulatory system. The purpose of the present study was to clarify eNOS expression, Ser1177 phosphorylation, and monomer formation after cerebral ischemia. Male Wistar rats were subjected to transient focal cerebral ischemia. Endothelial nitric oxide synthase messenger RNA (mRNA) and protein expression increased ~ 8-fold in the ischemic lesion. In the middle cerebral artery core, eNOS-Ser1177 phosphorylation increased 6 hours after ischemia; however, there was an approximately 90% decrease in eNOS-Ser1177 phosphorylation observed 24 hours after ischemia that continued until at least 7 days after ischemia. Endothelial nitric oxide synthase monomer formation also increased 24 and 48 hours after ischemia ( P<0.05), and protein nitration progressed in parallel with monomerization. To assess the effect of a neuroprotective agent on eNOS dysfunction, we evaluated the effect of fasudil, a Rho-kinase inhibitor, on eNOS phosphorylation and dimerization. Postischemic treatment with fasudil suppressed lesion expansion and dephosphorylation and monomer formation of eNOS. In conclusion, functional deterioration of eNOS progressed after cerebral ischemia. Rho-kinase inhibitors can reduce ischemic lesion expansion as well as eNOS dysfunction in the ischemic brain.
Demonstration of the spin solar cell and spin photodiode effect
Authors: Endres B. , Ciorga M. , Schmid M. , Utz M. , Bougeard D. , Weiss D. , Bayreuther G. , Back C.H.
Published: July 2013

Spin injection and extraction are at the core of semiconductor spintronics. Electrical injection is one method of choice for the creation of a sizeable spin polarization in a semiconductor, requiring especially tailored tunnel or Schottky barriers. Alternatively, optical orientation can be used to generate spins in semiconductors with significant spin-orbit interaction, if optical selection rules are obeyed, typically by using circularly polarized light at a well-defined wavelength. Here we introduce a novel concept for spin injection/extraction that combines the principle of a solar cell with the creation of spin accumulation. We demonstrate that efficient optical spin injection can be achieved with unpolarized light by illuminating a p-n junction where the p-type region consists of a ferromagnet. The discovered mechanism opens the window for the optical generation of a sizeable spin accumulation also in semiconductors without direct band gap such as Si or Ge.
Tutorials on the Foundations of Pharmacometrics and Systems Pharmacology
Authors: Friberg LE
Published: July 2013

CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e53; doi:10.1038/psp.2013.27; advance online publication 3 July 2013
Sustainability-Related Media Coverage and Socioeconomic Development: A Regional and North–South Perspective
Authors: Barkemeyer Ralf , Figge Frank , Holt Diane
Published: July 2013

With this paper we discuss the differences between sustainability-related media agendas across different countries and regions. Utilising a sample of 115 leading national newspapers covering forty-one countries, we show that typically no homogeneous global trends exist with regard to sustainability-related media agendas. Instead, significant differences exist regarding the national-level prioritisations of sustainability-related issues in the countries under review. To some extent, these observed differences can be attributed to different levels of socioeconomic development as measured by Human Development Index scores and gross domestic product per capita. Here, generic differences can be identified between newspapers from the Global North and South, with a range of issues such as climate change emerging as typically Northern issues, whereas issues such as corruption and poverty show significantly higher levels of coverage across newspapers from the Global South. We conclude with a discussion of the results in the context of global environmental governance.
Perceiving Space and Optical Cues via a Visuo-Tactile Sensory Substitution System: A Methodological Approach for Training of Blind Subjects for Navigation
Authors: Segond Hervé , Weiss Déborah , Kawalec Magdalena , Sampaio Eliana
Published: July 2013

A methodological approach to perceptual learning was used to allow both early blind subjects (experimental group) and blindfolded sighted subjects (control group) to experience optical information and spatial phenomena, on the basis of visuo-tactile information transmitted by a 64-taxel pneumatic sensory substitution device. The learning process allowed the subjects to develop abilities in spatial localisation, shape recognition (with generalisation to different points of view), and monocular depth cue interpretation. During the training phase, early blind people initially experienced more difficulties than blindfolded sighted subjects (having previous perceptual experience of perspective) with interpreting and using monocular depth cues. The amelioration of the performance for all blind subjects during training sessions and the quite similar level of performance reached by two groups in the final navigation tasks suggested that early blind people were able to develop and apply cognitive understanding of depth cues. Both groups showed generalisation of the learning from the initial phases to cue identification in the maze, and subjectively experienced shapes facing them. Subjects' performance depended not only on their perceptual experience but also on their previous spatial competencies.
Space and Time: An Impact of Spatial Separation, Apparent Motion, and Perceptual Grouping on TOJ Performance
Authors: Baruch Orit , Yeshurun Yaffa , Shore David I
Published: July 2013

This study explored the effects of spacing and objecthood (ie grouping based on closure) on temporal order judgment (TOJ) with displays that either involved successive onset of the target stimuli, resulting in apparent motion (experiments 1 and 2), or included simultaneous onset but successive shortening of the stimuli, and therefore did not result in apparent motion (experiment 3). We found a robust effect of spatial separation whose nature depended on whether or not the display allowed the emergence of illusory motion. Specifically, with apparent motion TOJ was best with the smallest spacing, but without it TOJ was worst with the smallest spacing. Moreover, overall accuracy was better with, than without, apparent motion. A small effect of objecthood—poorer TOJ performance when the elements formed an object—emerged only when spacing was not manipulated. These findings suggest that different mechanisms mediate temporal processing when we have access to motion information than when we do not.
Responses Contribute to Context Effects on Ratio-Setting Timing Tasks
Authors: Ryan Lawrence J , Havens Anthony
Published: July 2013

Short durations in a range of standard durations are typically overreproduced and long durations underreproduced (‘Vierordt's Law’). This contextual distortion may result from each trial assimilating the central tendency of the preceding series. We examine whether responses as well as standard durations contribute to this distortion. Two experiments using nonequal-setting ratio tasks are described. On equal-setting ratio tasks the means of the standards and responses are nearly equal, but in half-setting and double-setting standards and responses differ. In both experiments a central tendency of standards plus responses better predicted the indifference interval than did the mean of the standards alone. The coefficients of variation were larger for all double-setting conditions than for half-setting at the same response durations, suggesting pooling of the trial variance with the central tendency's variance. Longer equal-setting reproductions were generated by randomly intermixing equal-setting with double-setting than by intermixing with half-setting. Thus, assimilation of a central tendency that includes both standards and responses can account for the location of the indifference intervals (’Vierordt's Law’), for the response variation and for the additive effect we observed where two entire response curves (for equal-setting) were shifted relative to each other by the influence of nonequal-setting trials.
Face Contour is Crucial to the Fat Face Illusion
Authors: Sun Yu-Hao , Quinn Paul C , Wang Zhe , Shi Huimin , Zhong Ming , Jin Haiyang , Ge Liezhong , Pascalis Olivier , Tanaka James W , Lee Kang
Published: July 2013

In 2010 Thompson reported a “fat face thin” illusion that, when next to an inverted face, an upright face looks “fatter”. Sun et al (2012 Perception41 117–120) observed that one of the faces need not be inverted for the illusion to emerge: When two identical faces are presented one above the other, the face at the bottom appears “fatter” than the top one. Neither inverted faces nor clocks induced the illusion. Here we conducted three experiments probing the role that face contour plays in producing the fat face illusion. In experiment 1 line drawing faces were found to induce the illusion, suggesting that face contour is important for producing the illusion. In experiment 2 line drawing faces with scrambled internal features and empty line drawing faces devoid of internal features were found to induce the illusion. In experiment 3 internal face features arranged in their canonical face layout, but not in a scrambled layout, were found to induce the illusion. However, the magnitude of the effect was significantly weaker than the effect obtained for empty face contour in experiment 2. Collectively, these results suggest that a fat face illusion is obtained when there is sufficient information in the stimulus to activate an internal face schema.
Walking through Apertures: Do You Know What You are Doing during Body-Scaled Action?
Authors: Keizer Anouk , De Bruijn Suzanne E M , Smeets Monique A M , Dijkerman H Chris , Postma Albert
Published: July 2013

We investigated whether body-scaled action is influenced by awareness of task performance. Participants walked through apertures varying in size. Awareness of performing the action was manipulated by asking half of the participants to concurrently perform a haptic memory task. Distracted participants showed a smaller maximum amount of shoulder rotation at A/Scrit. Walking through apertures thus seems a rather automatic/uniform process regarding action planning, but being distracted from performing the action appears to influence how the action is performed, and may increase its efficiency.
Effects of Additional Visual Oscillation on Vection under Voluntary Eye Movement Conditions—Retinal Image Motion is Critical in Vection Facilitation
Authors: Nakamura Shinji
Published: July 2013

Horizontal oscillation introduced into a visual inducer can enhance an observer's illusory perception of vertical self-motion induced by a vertically moving optic flow pattern. To examine the effects of increased retinal image motion on the facilitation of vection, vection strength was analysed under various stimulus conditions where the oscillation of the retinal image was determined both by the motion of the visual stimulus and the observer's eye movements. The results indicated that the facilitation of vection was regulated by the oscillation of the visual stimulus on the observer's retina rather than the stimulus oscillation per se. Thus, the current experiment clearly favours the hypothesis that increased retinal image motion plays a critical role in the facilitation of vection.
Simple 3-D Stimulus for Motion Parallax and its Simulation
Authors: Ono Hiroshi , Chornenkyy Yevgen , D'Amour Sarah
Published: July 2013

Simulation of a given stimulus situation should produce the same perception as the original. Rogers et al (2009 Perception38 907–911) simulated Wheeler's (1982, PhD thesis, Rutgers University, NJ) motion parallax stimulus and obtained quite different perceptions. Wheeler's observers were unable to reliably report the correct direction of depth, whereas Rogers's were. With three experiments we explored the possible reasons for the discrepancy. Our results suggest that Rogers was able to see depth from the simulation partly due to his experience seeing depth with random dot surfaces.
Severing and end-to-end annealing of neurofilaments in neurons
Authors: Uchida Atsuko , Çolakoğlu Gülsen , Wang Lina , Monsma Paula C. , Brown Anthony
Published: July 2013

Significance

Neurofilaments, which are the intermediate filaments of neurons, are major components of nerve cells, but their assembly dynamics have not been determined. Here we demonstrate efficient end-to-end annealing of neurofilaments in nerve cells, and we show that there also is a mechanism that severs these polymers. Our efforts thus identify severing of neuronal intermediate filaments in vivo and suggest a mechanism for regulating intermediate filament length involving a dynamic cycle of severing and end-to-end annealing.
Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis
Authors: Masters Thomas A. , Pontes Bruno , Viasnoff Virgile , Li You , Gauthier Nils C.
Published: July 2013

Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3′-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.
Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome
c
Authors: Ban-Ishihara Reiko , Ishihara Takaya , Sasaki Narie , Mihara Katsuyoshi , Ishihara Naotada
Published: July 2013

Mammalian cells typically contain thousands of copies of mitochondrial DNA assembled into hundreds of nucleoids. Here we analyzed the dynamic features of nucleoids in terms of mitochondrial membrane dynamics involving balanced fusion and fission. In mitochondrial fission GTPase dynamin-related protein (Drp1)-deficient cells, nucleoids were enlarged by their clustering within hyperfused mitochondria. In normal cells, mitochondrial fission often occurred adjacent to nucleoids, since localization of Mff and Drp1 is dependent on the nucleoids. Thus, mitochondrial fission adjacent to nucleoids should prevent their clustering by maintaining small and fragmented nucleoids. The enhanced clustering of nucleoids resulted in the formation of highly stacked cristae structures in enlarged bulb-like mitochondria (mito-bulbs). Enclosure of proapoptotic factor cytochrome
c
, but not of Smac/DIABLO, into the highly stacked cristae suppressed its release from mitochondria under apoptotic stimuli. In the absence of nucleoids, Drp1 deficiency failed to form mito-bulbs and to protect against apoptosis. Thus, mitochondrial dynamics by fission and fusion play a critical role in controlling mitochondrial nucleoid structures, contributing to cristae reformation and the proapoptotic status of mitochondria.
Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel
Authors: Du Yuzhe , Nomura Yoshiko , Satar Gul , Hu Zhaonong , Nauen Ralf , He Sheng Yang , Zhorov Boris S. , Dong Ke
Published: July 2013

Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNa
v
1–1, from
Aedes aegypti
in
Xenopus
oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various
Ae. aegypti
and
Anopheles gambiae
populations around the world. Our analysis shows that five of the nine mutations reduce AaNa
v
1–1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors.
Duality of holomorphic function spaces and smoothing properties of the Bergman projection
Authors: Herbig A.-K. , McNeal J. , Straube E.
Published: July 2013

Let

Ω
⋐

C

n

\Omega \Subset \mathbb {C}^{n}

be a domain with smooth boundary, whose Bergman projection

B
B

maps the Sobolev space

H

k

1

(
Ω
)

H^{k_{1}}(\Omega )

(continuously) into

H

k

2

(
Ω
)

H^{k_{2}}(\Omega )

. We establish two smoothing results: (i) the full Sobolev norm

‖
B
f

‖

k

2

\|Bf\|_{k_{2}}

is controlled by

L
2

L^2

derivatives of

f
f

taken along a single, distinguished direction (of order

≤

k

1

\leq k_{1}

), and (ii) the projection of a conjugate holomorphic function in

L

2

(
Ω
)

L^{2}(\Omega )

is automatically in

H

k

2

(
Ω
)

H^{k_{2}}(\Omega )

. There are obvious corollaries for when

B
B

is globally regular.
The Export of Major Histocompatibility Complex Class I Molecules from the Endoplasmic Reticulum of Rat Brown Adipose Cells Is Acutely Stimulated by Insulin
Authors: Malide Daniela , Yewdell Jonathan W. , Bennink Jack R. , Cushman Samuel W.
Published: July 2013

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase–dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.
Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis
Authors: Yamashiro Shigeko , Chern Hueylan , Yamakita Yoshihiko , Matsumura Fumio
Published: July 2013

Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division ofXenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.
The Catastrophe-promoting Activity of Ectopic Op18/Stathmin Is Required for Disruption of Mitotic Spindles But Not Interphase Microtubules
Authors: Holmfeldt Per , Larsson Niklas , Segerman Bo , Howell Bonnie , Morabito Justin , Cassimeris Lynne , Gullberg Martin
Published: July 2013

Oncoprotein18/stathmin (Op18) is a microtubule (MT) destabilizing protein that is inactivated during mitosis by phosphorylation at four Ser-residues. Op18 has at least two functions; the N-terminal region is required for catastrophe-promotion (i.e., transition from elongation to shortening), while the C-terminal region is required to inhibit MT-polymerization rate in vitro. We show here that a “pseudophosphorylation” derivative of Op18 (i.e., four Ser- to Glu-substitutions at phosphorylation sites) exhibits a selective loss of catastrophe-promoting activity. This is contrasted to authentic phosphorylation, which efficiently attenuates all activities except tubulin binding. In intact cells, overexpression of pseudophosphorylated Op18, which is not phosphorylated by endogenous kinases, is shown to destabilize interphase MTs but to leave spindle formation untouched. To test if the mitotic spindle is sensitive only to the catastrophe-promoting activity of Op18 and resistant to C-terminally associated activities, N- and C-terminal truncations with defined activity-profiles were employed. The cell-cycle phenotypes of nonphosphorylatable mutants (i.e., four Ser- to Ala-substitutions) of these truncation derivatives demonstrated that catastrophe promotion is required for interference with the mitotic spindle, while the C-terminally associated activities are sufficient to destabilize interphase MTs. These results demonstrate that specific Op18 derivatives with defined activity-profiles can be used as probes to distinguish interphase and mitotic MTs.
Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis inCaenorhabditis elegans
Authors: Yoder John H. , Han Min
Published: July 2013

We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations indli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of thedli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-celldli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective indli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1mutant background, indicating this is not an essential domain for DLI-1 function.
Genomic Expression Responses to DNA-damaging Agents and the Regulatory Role of the Yeast ATR Homolog Mec1p
Authors: Gasch Audrey P. , Huang Mingxia , Metzner Sandra , Botstein David , Elledge Stephen J. , Brown Patrick O.
Published: July 2013

Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. The human ATR kinase and its homolog in yeast, MEC1, play central roles in transducing the damage signal. To characterize the role of the Mec1 pathway in modulating the cellular response to DNA damage, we used DNA microarrays to observe genomic expression inSaccharomyces cerevisiae responding to two different DNA-damaging agents. We compared the genome-wide expression patterns of wild-type cells and mutants defective in Mec1 signaling, includingmec1, dun1, and crt1 mutants, under normal growth conditions and in response to the methylating-agent methylmethane sulfonate (MMS) and ionizing radiation. Here, we present a comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents, and identify specific features of the gene expression responses that are dependent on the Mec1 pathway. Among the hundreds of genes whose expression was affected by Mec1p, one set of genes appears to represent an MEC1-dependent expression signature of DNA damage. Other aspects of the genomic responses were independent of Mec1p, and likely independent of DNA damage, suggesting the pleiotropic effects of MMS and ionizing radiation. The complete data set as well as supplemental materials is available at http://www-genome.stanford.edu/mec1 .
On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial Release of an Apoptosis-inducing Factor duringDictyostelium discoideumCell Death
Authors: Arnoult Damien , Tatischeff Irène , Estaquier Jérome , Girard Mathilde , Sureau Franck , Tissier Jean Pierre , Grodet Alain , Dellinger Marc , Traincard Francİois , Kahn Axel , Ameisen Jean-Claude , Petit Patrice Xavier
Published: July 2013

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochromec that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideuminvolves early disruption of mitochondrial transmembrane potential (ΔΨm) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyosteliumcells trigger the breakdown of isolated mammalian andDictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific forDictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation duringDictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.
Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Authors: Stuermer Claudia A.O. , Lang Dirk M. , Kirsch Friederike , Wiechers Marianne , Deininger Sören-Oliver , Plattner Helmut
Published: July 2013

Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in ≤0.1-μm plasma membrane microdomains of neurons and astrocytes. In astrocytes, reggie-1 and -2 do not occur in caveolae but clearly outside these structures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidyl inositol-anchored proteins Thy-1 and F3 that, when activated by antibody cross-linking, selectively copatch with reggie. Jurkat cells, after cross-linking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This, and the accumulation of reggie proteins in detergent-resistant membrane fractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 properties of raft-associated proteins. It also suggests that reggie-1 and -2 participate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 together with fyn and Thy-1 increase in endolysosomes concurrent with a decrease at the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomain signaling centers in neurons and T cells, and the protein complex involved in signaling becomes subject to degradation.
Association of Human DEAD Box Protein DDX1 with a Cleavage Stimulation Factor Involved in 3′-End Processing of Pre-mRNA
Authors: Bléoo Stacey , Sun Xuejun , Hendzel Michael J. , Rowe John M. , Packer Mary , Godbout Roseline
Published: July 2013

DEAD box proteins are putative RNA helicases that function in all aspects of RNA metabolism, including translation, ribosome biogenesis, and pre-mRNA splicing. Because many processes involving RNA metabolism are spatially organized within the cell, we examined the subcellular distribution of a human DEAD box protein, DDX1, to identify possible biological functions. Immunofluorescence labeling of DDX1 demonstrated that in addition to widespread punctate nucleoplasmic labeling, DDX1 is found in discrete nuclear foci ∼0.5 μm in diameter. Costaining with anti-Sm and anti-promyelocytic leukemia (PML) antibodies indicates that DDX1 foci are frequently located next to Cajal (coiled) bodies and less frequently, to PML bodies. Most importantly, costaining with anti-CstF-64 antibody indicates that DDX1 foci colocalize with cleavage bodies. By microscopic fluorescence resonance energy transfer, we show that labeled DDX1 resides within a Förster distance of 10 nm of labeled CstF-64 protein in both the nucleoplasm and within cleavage bodies. Coimmunoprecipitation analysis indicates that a proportion of CstF-64 protein resides in the same complex as DDX1. These studies are the first to identify a DEAD box protein associating with factors involved in 3′-end cleavage and polyadenylation of pre-mRNAs.
Rab3A Is Involved in Transport of Synaptic Vesicles to the Active Zone in Mouse Brain Nerve Terminals
Authors: Leenders A.G. Miriam , da Silva Fernando H. Lopes , Ghijsen Wim E.J.M. , Verhage Matthijs
Published: July 2013

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca2+induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.
p38-mediated Regulation of an Fas-associated Death Domain Protein-independent Pathway Leading to Caspase-8 Activation during TGFβ-induced Apoptosis in Human Burkitt Lymphoma B Cells BL41
Authors: Schrantz Nicolas , Bourgeade Marie-Françoise , Mouhamad Shahul , Leca Gérald , Sharma Surendra , Vazquez Aimé
Published: July 2013

On binding to its receptor, transforming growth factor β (TGFβ) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFβ-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFβ-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFβ-treated cells. TGFβ induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFβ induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFβ-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFβ-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFβ-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFβ induced an apoptotic pathway via FADD-independent activation of caspase-8.
Rapid Intermittent Movement of Axonal Neurofilaments Observed by Fluorescence Photobleaching
Authors: Wang Lei , Brown Anthony
Published: July 2013

Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move along axons in a rapid, intermittent, and highly asynchronous manner. In contrast, studies on axonal neurofilaments using laser photobleaching have not detected movement. Here, we describe a modified photobleaching strategy that does permit the direct observation of neurofilament movement. Axons of cultured neurons expressing GFP-tagged neurofilament protein were bleached by excitation with the mercury arc lamp of a conventional epifluorescence microscope for 12–60 s. The length of the bleached region ranged from 10 to 60 μm. By bleaching thin axons, which have relatively few neurofilaments, we were able to reduce the fluorescent intensity enough to allow the detection of neurofilaments that moved in from the surrounding unbleached regions. Time-lapse imaging at short intervals revealed rapid, intermittent, and highly asynchronous movement of fluorescent filaments through the bleached regions at peak rates of up to 2.8 μm/s. The kinetics of movement were very similar to our previous observations on neurofilaments moving through naturally occurring gaps, which indicates that the movement was not impaired by the photobleaching process. These results demonstrate that fluorescence photobleaching can be used to study the slow axonal transport of cytoskeletal polymers, but only if the experimental strategy is designed to ensure that rapid asynchronous movements can be detected. This may explain the failure of previous photobleaching studies to reveal the movement of neurofilament proteins and other cytoskeletal proteins in axons.
The GPI Transamidase Complex ofSaccharomyces cerevisiaeContains Gaa1p, Gpi8p, and Gpi16p
Authors: Fraering Patrick , Imhof Isabella , Meyer Urs , Strub Jean-Marc , van Dorsselaer Alain , Vionnet Christine , Conzelmann Andreas
Published: July 2013

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430–650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430–650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.
Yarrowia lipolyticaCells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes
Authors: Lambkin Gareth R. , Rachubinski Richard A.
Published: July 2013

PEX genes encode peroxins, which are proteins required for peroxisome assembly. The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strainpex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da). Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes. Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid–containing medium, the metabolism of which requires intact peroxisomes. pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes. In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels inpex19 cells compared with its levels in wild-type cells. Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density. Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes. Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y. lipolytica. Our results are consistent with a role for Y. lipolytica Pex19p in stabilizing the peroxisomal membrane.
Cell Cycle-dependent Degradation of theSaccharomyces cerevisiaeSpindle Motor Cin8p Requires APCCdh1and a Bipartite Destruction Sequence
Authors: Hildebrandt Emily R. , Hoyt M. Andrew
Published: July 2013

Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly. Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase. Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p. Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates. We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo. The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal. The bipartite destruction sequence appears in other BimC kinesins as well. Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles.
Pmel17 Initiates Premelanosome Morphogenesis within Multivesicular Bodies
Authors: Berson Joanne F. , Harper Dawn C. , Tenza Danielle , Raposo Graça , Marks Michael S.
Published: July 2013

Melanosomes are tissue-specific organelles within which melanin is synthesized and stored. The melanocyte-specific glycoprotein Pmel17 is enriched in the lumen of premelanosomes, where it associates with characteristic striations of unknown composition upon which melanin is deposited. However, Pmel17 is synthesized as an integral membrane protein. To clarify its physical linkage to premelanosomes, we analyzed the posttranslational processing of human Pmel17 in pigmented and transfected nonpigmented cells. We show that Pmel17 is cleaved in a post-Golgi compartment into two disulfide-linked subunits: a large lumenal subunit, Mα, and an integral membrane subunit, Mβ. The two subunits remain associated intracellularly, indicating that detectable Mα remains membrane bound. We have previously shown that Pmel17 accumulates on intralumenal membrane vesicles and striations of premelanosomes in pigmented cells. In transfected nonpigmented cells Pmel17 associates with the intralumenal membrane vesicles of multivesicular bodies; cells overexpressing Pmel17 also display structures resembling premelanosomal striations within these compartments. These results suggest that Pmel17 is sufficient to drive the formation of striations from within multivesicular bodies and is thus directly involved in the biogenesis of premelanosomes.
Insulin-regulated Release from the Endosomal Recycling Compartment Is Regulated by Budding of Specialized Vesicles
Authors: Lampson Michael A. , Schmoranzer Jan , Zeigerer Anja , Simon Sanford M. , McGraw Timothy E.
Published: July 2013

In several cell types, specific membrane proteins are retained intracellularly and rapidly redistributed to the surface in response to stimulation. In fat and muscle, the GLUT4 glucose transporter is dynamically retained because it is rapidly internalized and slowly recycled to the plasma membrane. Insulin increases the recycling of GLUT4, resulting in a net translocation to the surface. We have shown that fibroblasts also have an insulin-regulated recycling mechanism. Here we show that GLUT4 is retained within the transferrin receptor-containing general endosomal recycling compartment in Chinese hamster ovary (CHO) cells rather than being segregated to a specialized, GLUT4-recycling compartment. With the use of total internal reflection microscopy, we demonstrate that the TR and GLUT4 are transported from the pericentriolar recycling compartment in separate vesicles. These data provide the first functional evidence for the formation of distinct classes of vesicles from the recycling compartment. We propose that GLUT4 is dynamically retained within the endosomal recycling compartment in CHO cells because it is concentrated in vesicles that form more slowly than those that transport TR. In 3T3-L1 adipocytes, cells that naturally express GLUT4, we find that GLUT4 is partially segregated to a separate compartment that is inaccessible to the TR. We present a model for the formation of this specialized compartment in fat cells, based on the general mechanism described in CHO cells, which may explain the increased retention of GLUT4 and its insulin-induced translocation in fat cells.