bluuebunny/crossref_metadata_2025 huggingface dataset:
Coercivity of magnetic domain wall motion near the edge of a terrace
Authors: Hsieh Yung-Chieh , Mansuripur M.
Published: July 2002

Domain wall motion near the edges of terraces (e.g., grooves, pits, plateaus, etc.) is studied using analytical techniques based on the minimum energy principle and computer simulations based on the dynamic Landau–Lifshitz–Gilbert equation. One-dimensional lattices of magnetic dipoles with variations either of the easy axis direction (corresponding to a tilt of the anisotropy axis at the edge) or of the nearest-neighbor exchange force (corresponding to a changing film thickness) are considered. We show that the coercivity caused by the terrace edge could be as large as several kilo Oe.
Thermal stability of diamondlike carbon films
Authors: Akkerman Z. L. , Efstathiadis H. , Smith F. W.
Published: July 2002

IR and UV-VIS optical spectroscopy have been used to study the transformations of the properties of diamondlike carbon films following isothermal anneals from Ta=300 to 650 °C. Several processes due to the annealing have been observed: (1) the increase of the absorption due to vibrations of unsaturated bonds of C(sp2) atoms at ∼1600 cm−1 for Ta≥350 °C, (2) the decrease of the absorption due to C(sp3)–H bonds at Ta≥350 °C, and (3) the reduction of the optical energy gap. Analysis of the kinetics has shown that the dehydrogenation of the alloys and the formation of unsaturated bonds may proceed independently. The reduction of the energy gap is related to the formation of C(sp2) atoms with unsaturated bonding which occurs mostly in hydrogen-free regions. Intensive graphitization of the films occurs above Ta=650 °C. Transformations of C–H bonds are proposed to occur via fast rearrangement in stressed regions leading to formation of new C(sp2)–H bonds and formation of methane molecules as the most important product of the anneals inside the polymeric highly hydrogenated regions in the alloys. It appears that both bond removal and reordering have taken place as a result of annealing.
Lifetime and degradation effects in polymer light-emitting diodes
Authors: Parker I. D. , Cao Y. , Yang C. Y.
Published: July 2002

The operational lifetime of polymer light-emitting diodes was studied at several temperatures in the range from 25 to 85 °C. When operated in constant current mode, at luminances greater than 100 cd/m 2, lifetimes of around 20 000 h were noted. Two significant changes in performance were found during continuous operation: the luminance of the devices varied in a nonmonotonic fashion, and the operating voltage increased in a linear fashion. Both of these changes were thermally activated, with the changes accelerated at higher temperatures. These changes are also accelerated at higher current densities. We discuss possible mechanisms for these degradation processes.
Bottom giant magnetoresistance spin valves using a radio frequency reactive bias-sputtered α-Fe2O3 antiferromagnetic layer
Authors: Bae Seongtae , Judy Jack H. , Egelhoff W. F. , Chen P. J.
Published: July 2002

The giant magnetoresistance (GMR) characteristics were investigated for rf reactive bias-sputtered α-Fe2O3(50 nm) based bottom spin-valve structures. The GMR ratio of this structure was found to be affected by Cu interlayer thickness. When the Cu interlayer thickness was changed from 1.6 to 3.1 nm, the GMR ratio varied from 14.2%(tcu=1.8 nm) to 8.3%(tcu=3.1 nm). The MR sensitivity and MR ratio dependence were studied for Si/α-Fe2O3(50 nm)/(NiFe, or Co)/Cu(2.3 nm)/(NiFe, or Co) structures. The MR ratio of NiFe(5.0%) based spin valve was much smaller than that of Co(16.1%), but it (0.6%/Oe) showed much higher MR sensitivity than that of Co(0.3%/Oe). The α-Fe2O3 input sputtering power was also changed from 600 to 1000 W for Si/α-Fe2O3(50 nm)/Co(or NiFe)/Cu(2.3 nm)/Co(or NiFe) structures. With increasing input power, the MR ratio increased for both Co (up to 15%) and NiFe (up to 3.1%). The main reason for the increase of MR ratio was attributed to the smoother surface. The average surface roughness measured by ex-situ AFM had 1.38 nm (600 W), 0.9 nm (800 W), and 0.87 nm (1000 W), respectively.
General method for removing resonance singularities in quantum mechanical perturbation theory
Authors: Kuhler Kathleen M. , Truhlar Donald G. , Isaacson Alan D.
Published: July 2002

This paper presents a way of improving second-order perturbation theory calculations by summing contributions of uncoupled excitations to infinite order. For problems involving molecular vibrations, the new theory is shown to give similar results to conventional second-order perturbation theory when the system treated has no near resonances but also to give accurate and stable results even very close to resonance. The new theory is tested by comparison to converged variational calculations for vibrational energy levels of formaldehyde, formaldehyde-d2, and two two-dimensional model subsystems based on formaldehyde.
Mapping of rotational isomeric state chains with asymmetric torsional potential energy functions on a high coordination lattice: Application to polypropylene
Authors: Haliloǧlu Türkan , Mattice Wayne L.
Published: July 2002

A high coordination lattice model was recently introduced for simulating coarse-grained rotational isomeric state (RIS) chains in which the bonds have symmetric torsional potential energy functions, E(φ)=E(−φ). This symmetry was exploited in the coarse-graining and mapping onto the high coordination lattice, thereby making the procedure unsuitable (without modification) for application to chains where one or more bonds has an asymmetric torsion potential energy function, E(φ)≠E(−φ). The necessary modification is described here, and then documented by mapping previously described RIS models for isotactic and syndiotactic polypropylene onto the high coordination lattice. Each bead on the high coordination lattice represents a monomer unit, C2H4, of polypropylene. The conditional probabilities derived from the RIS model form the basis for the acceptance of the single bead moves used in the Monte Carlo simulations on the 2nnd lattice. The simulated chains have reasonable mean-square end-to-end distances and mean-square radii of gyration. The relaxation of the end-to-end vector follows the stretched exponential behavior, exp[−(t/τ)β], where β=0.5 and τ is the correlation time. The elaboration retains the ability to correctly treat chains in which the bonds have symmetric torsional potential energy functions, as shown by application to polyethylene, where each bead on the high coordination lattice represents C2H4.
Charge and fission of droplets in electrostatic sprays
Authors: Gomez Alessandro , Tang Keqi
Published: July 2002

Measurements of the charge and size of heptane droplets generated by electrostatic sprays showed that the droplet charge-to-volume ratio is a monotonically decreasing function of size. In the useful range of electrospray operation, characterized by droplets smaller than the size of the orifice from which the liquid is issued, it was found that the larger were the droplets the closer they were to the Rayleigh limit. In particular, when droplets had charging levels between 70% and 80% of such limit, they were observed to rupture because the repulsive force due to surface charge evidently overcame surface tension. The rupture phenomenon, here termed Coulomb fission, was also captured in microphotographs that typically showed a droplet with one or two, diametrically opposed, conical protrusions terminating in a fine jet ejecting a stream of much smaller, apparently equisized offsprings. The process appeared swift and, yet, well ordered, quite different from the common view of a violent, convulsive explosion. Corroborating evidence on the disruption pattern was also gathered by quantitative measurements of the evolution of the droplet size distribution in evaporating sprays using phase Doppler anemometry (PDA). Implications of these findings are finally discussed in the context of a particular application of electrostatic sprays, electrospray ionization, a technique that is revolutionizing the mass-spectrometric analysis of large biomolecules.
Details of the drag curve near the onset of vortex shedding
Authors: Henderson Ronald D.
Published: July 2002

A closer look at the drag curve for a circular cylinder near the onset of vortex shedding reveals that there is a sharp transition in the forces acting on a body moving through a fluid when it produces an unsteady wake. In this Letter results from high-resolution computer simulations are presented to quantify the change in viscous drag, pressure drag, and base pressure coefficients.
Capillary breakup of a viscous thread surrounded by another viscous fluid
Authors: Lister John R. , Stone Howard A.
Published: July 2002

Previous long-wavelength analyses of capillary breakup of a viscous fluid thread in a perfectly inviscid environment show that the asymptotic self-similar regime immediately prior to breakup is given by a balance between surface tension, inertia, and extensional viscous stresses in the thread. In contrast, it is shown here that if viscosity in the external fluid, however small, is included then the asymptotic balance is between surface tension and viscous stresses in the two fluids while inertia is negligible. Scaling estimates for this new balance suggest that both axial and radial scales decrease linearly with time to breakup, so that the aspect ratio remains O(1) with time but scales with viscosity ratio like (μint/μext)1/2 for μint≫μext, where μint and μext are the internal and external viscosities. Numerical solutions to the full Stokes equations for μint=μext confirm the scalings with time and give self-similar behavior near pinching. However, the self-similar pinching region is embedded in a logarithmically large axial advection driven by the increasing range of scales intermediate between that of the pinching region and that of the macroscopic drop. The interfacial shape in the intermediate region is conical with angles of about 6° on one side and 78° on the other.
Hydrodynamics and stability of a deformable body moving in the proximity of interfaces
Authors: Galper A. R. , Miloh T.
Published: July 2002

The motion of a deformable body embedded in an inviscid irrotational nonuniform ambient flow field in the proximity of interfaces is treated here using a newly developed Hamiltonian formalism. The corresponding dynamic equations governing the motion of the body are derived and their integrability is investigated. We find that the presence of boundaries results in an additional chaotization of a body’s motion. Based on the derived Hamiltonian formalism the Liapunov stability of the motion of a body translating parallel or towards a remote flat wall is also considered using the Energy-Casimir approach. The appropriate stability criteria are derived. Finally some applications for bubble dynamics concerning an influence of a periodical deformation of a bubble on its motion is presented.
Large eddy simulation of a plane jet
Authors: Le Ribault C. , Sarkar S. , Stanley S. A.
Published: July 2002

Large eddy simulations of spatially evolving planar jets have been performed using the standard Smagorinsky, the dynamic Smagorinsky, and the dynamic mixed models and model performance evaluated. Computations have been performed both at a low Reynolds number, Red=3000, in order to make comparisons with a previous DNS at the same Reynolds number, and at a higher value, Red=30 000, to compare with high Reynolds number experiments. Model predictions with respect to the evolution of jet half-width, centerline velocity decay, mean velocity profiles, and profiles of turbulence intensity are evaluated. Some key properties of the SGS models such as the eddy-viscosity constant and the subgrid dissipation are also compared. It is found that the standard Smagorsinsky model is much too dissipative and severely underpredicts the evolution of the jet half-width and centerline velocity decay. The dynamic versions of the Smagorinsky model and the mixed model allow for streamwise and transverse variation of the constant in the eddy-viscosity expression which results in much better performance and good agreement with experimental and DNS data. The mixed model has an additional scale-similarity part which, in a priori tests against filtered jet DNS data, is found to predict the subgrid shear stress profile. Although the subgrid shear stress obtained by the dynamic Smagorinsky model is substantially smaller than that obtained in the a priori tests using the jet DNS data, surprisingly, in the a posteriori computations, the dynamic Smagorinsky model performs as well as the dynamic mixed model. Analysis of the mean momentum equation gives the reason for such behavior: the resolved stress in computations with the dynamic Smagorinsky model is larger than it should be and compensates for the underprediction of the subgrid shear stress by the Smagorinsky model. The numerical discretization errors have been quantified. The error due to noncommutativity of spatial differentiation and physical space filtering on nonuniform grids is found to be small because of the relatively mild stretching used in the present LES. The modeling error is found to be generally smaller than the discretization error with the standard Smagorinsky model having the largest modeling error.
Effect of acoustic streaming on the mass transfer from a sublimating sphere
Authors: Kawahara N. , Yarin A. L. , Brenn G. , Kastner O. , Durst F.
Published: July 2002

The effect of the acoustic streaming on the mass transfer from the surface of a sphere positioned in an ultrasonic acoustic levitator is studied both experimentally and theoretically. Acoustic levitation using standing ultrasonic waves is an experimental tool for studying the heat and mass transfer from small solid or liquid samples, because it allows an almost steady positioning of a sample at a fixed location in space. However, the levitator introduces some difficulties. One of the main problems with acoustic levitation is that an acoustic streaming is induced near the sample surface, which affects the heat and mass transfer rates, as characterized by increased Nusselt and Sherwood numbers. The transfer rates are not uniform along the sample surface, and the aim of the present study is to quantify the spatial Sherwood number distribution over the surface of a sphere. The experiments are based on the measurement of the surface shape of a sphere layered with a solid substance as a function of time using a charge-coupled device (CCD) camera with backlighting. The sphere used in this research is a glass sphere layered with a volatile solid substance (naphthalene or camphor). The local mass transfer from the surface both with and without an ultrasonic acoustic field is investigated in order to evaluate the effect of the acoustic streaming. The experimental results are compared with predictions following from the theory outlined [A. L. Yarin, M. Pfaffenlehner, and C. Tropea, J. Fluid Mech. 356, 65 (1998); A. L. Yarin, G. Brenn, O. Kastner, D. Rensink, and C. Tropea, ibid. 399, 151 (1999)] which describes the acoustic field and the resulting acoustic streaming, and the mass transfer at the surface of particles and droplets located in an acoustic levitator. The results are also compared with the experimental data and with the theoretical predictions of Burdukov and Nakoryakov [J. Appl. Mech. Tech. Phys. 6, 51 (1965)], which are valid only in the case of spherical particles much smaller than the sound wavelength. Good agreement between experiment and the theory of Yarin et al. is demonstrated. The time-averaged heat and mass transfer rates over a sphere surface are greatest at the sphere’s equator and least at its poles in the experiment as predicted by the theory (the ultrasonic standing wave spans the vertical axis passing through the poles). The measured distribution of the mass transfer rate over the sphere surface also agrees with the theoretical predictions, which shows that in strong acoustic fields sublimation (or evaporation) results from the acoustic streaming.
The AF-1 activation-function of ERα may be dispensable to mediate the effect of estradiol on endothelial NO production in mice
Authors: Pendaries C. , Darblade B. , Rochaix P. , Krust A. , Chambon P. , Korach K. S. , Bayard F. , Arnal J.-F.
Published: July 2002

Two isoforms of estrogen receptor (ER) have been described: ERα and ERβ. The initial gene targeting of ERα, consisting in the introduction of a Neo cassette in exon 1 [αERKO, hereafter called ERα-Neo KO (knockout)], was reported in 1993. More recently, another mouse deficient in ERα because of the deletion of exon 2 (ERαKO, hereafter called ERα-Δ2 KO) was generated. In ovariectomized ERα-wild-type mice, estradiol (E
2
) increases uterine weight and basal production of endothelial nitric oxide (NO). Both of these effects are abolished in ERα-Δ2 KO mice. In contrast, we show here that both of these effects of E
2
are partially (uterine weight) or totally (endothelial NO production) preserved in ERα-Neo KO. We also confirm the presence of two ERα mRNA splice variants in uterus and aorta from ERα-Neo KO mice. One of them encodes a chimeric ERα protein (ERα55), partially deleted in the A/B domain, that was detected in both uterus and aorta by Western blot analysis. The other ERα mRNA splice variant codes for an isoform deleted for the A/B domain (ERα46), which was detected in uterus of ERα-Neo KO, and wild-type mice. This protein isoform was not detected in aorta. The identification of these two N-terminal modified isoforms in uterus, and at least one of them in aorta, probably explains the persistence of the E
2
effects in ERα-Neo KO mice. Furthermore, ERα-Neo KO mice may help in the elucidation of the specific functions of full-length ERα (ERα66) and ERα46, both shown to be physiologically generated
in vivo
.
Foreign-investment-induced Exo-urbanisation in the Pearl River Delta, China
Authors: Sit Victor F. S. , Yang Chun
Published: July 2002

Urbanisation in the People's Republic of China during 1949-78 was mainly driven by internal forces under a self-reliant, centrally planned economic system. Since the Opening and Reform initiated in 1978, external forces, especially foreign investment inflow, have emerged as a new driving force of urbanisation in some parts of China, particularly in the Pearl River Delta (the Delta) of South China. This study investigates the nature and pattern of this new type of urbanisation, which we label as exo(genous)-urbanisation. Foreign direct investment (FDI) into the Delta is mainly characterised by small and medium-scale, labour-intensive, processing-types of manufacturing and trade-creative investment coming from Hong Kong and Macao. It shows a spatial tendency within the host region's urban hierarchy in favour of small cities and counties and a distinct border orientation. This contrasts with the concentration in large metropolises or economic core regions found in most less developing countries. Such FDI has interacted with internal reforms to effect significant changes in the Delta during 1978-93, such as economic restructuring, rural industrialisation, the emergence of an export-oriented economy and its rapid integration with the world economy. It has also generated massive population in-migration and dramatic landscape transformation there. Put simply, exo-urbanisation in the Delta shows a predominant growth of small urban centres particularly those along the border with Hong Kong and Macao and hence a more equal level of urbanisation in the Delta and a declining primacy of Guangzhou, the regional primate city. However, exo-urbanisation in the Delta also raises doubts on social and environmental fronts as well as on its durability. The FDI inflow and the newly created export-oriented industrialisation and urban growth in the Delta are obviously subject to vicissitudes of the world market. In spite of this, contemporary urbanisation theories should give attention to the role of external forces. Further studies on exo-urbanisation are not only practical for development and planning in areas under intensive inflow of foreign investment, but could also enrich the literature on the globalisation process and its impacts on the developing countries.
Parental communication of Holocaust experiences and interpersonal patterns in offspring of Holocaust survivors
Authors: Wiseman Hadas , Barber Jacques P. , Raz Alon , Yam Idit , Foltz Carol , Livne-Snir Sharon
Published: July 2002

This study examined the interpersonal problems and central relationship patterns of Holocaust Survivors’ Offspring (HSO) who were characterised by different patterns of parental communication of their parents’ Holocaust trauma. Fifty-six adults born to mothers who were survivors of Nazi concentration camps and 54 adults born to parents who immigrated to Israel before 1939 with their own parents (non-HSO) were recruited randomly from an Israeli sample. While the groups did not differ in their current mental health, HSO who reported nonverbal communication with little information about their mother’s trauma endorsed more interpersonal distress than HSO who experienced informative verbal communication and less affiliation than either HSO who experienced informative verbal communication or non-HSO. They also differed in their central relationship patterns with their parents and spouses. The findings are discussed in the context of the unique dynamics of growing up with the silent presence of the mother’s trauma.
Two New Ypt GTPases Are Required for Exit From the Yeast trans-Golgi Compartment
Authors: Jedd Gregory , Mulholland Jon , Segev Nava
Published: July 2002

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the transGolgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/ 32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.
A Glycine-rich RNA-binding Protein Mediating Cold-inducible Suppression of Mammalian Cell Growth
Authors: Nishiyama Hiroyuki , Itoh Katsuhiko , Kaneko Yoshiyuki , Kishishita Masamichi , Yoshida Osamu , Fujita Jun
Published: July 2002

In response to low ambient temperature, mammalian cells as well as microorganisms change various physiological functions, but the molecular mechanisms underlying these adaptations are just beginning to be understood. We report here the isolation of a mouse cold-inducible RNA-binding protein (cirp) cDNA and investigation of its role in cold-stress response of mammalian cells. The cirp cDNA encoded an 18-kD protein consisting of an amino-terminal RNAbinding domain and a carboxyl-terminal glycine-rich domain and exhibited structural similarity to a class of stress-induced RNA-binding proteins found in plants. Immunofluorescence microscopy showed that CIRP was localized in the nucleoplasm of BALB/3T3 mouse fibroblasts. When the culture temperature was lowered from 37 to 32°C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37°C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37°C with prolongation of G1 phase of the cell cycle. These results indicate that CIRP plays an essential role in cold-induced growth suppression of mouse fibroblasts. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in mammalian cells.
Linking Integrin α6β4-based Cell Adhesion to the Intermediate Filament Cytoskeleton: Direct Interaction between the β4 Subunit and Plectin at Multiple Molecular Sites
Authors: Rezniczek Günther A. , de Pereda José M. , Reipert Siegfried , Wiche Gerhard
Published: July 2002

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the β4 subunit of the basement membrane laminin receptor integrin α6β4 that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin β4 cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin β4 subunits and cytokeratin filaments.
Targeting of Protein Kinase Cα to Caveolae
Authors: Mineo Chieko , Ying Yun-Shu , Chapline Christine , Jaken Susan , Anderson Richard G.W.
Published: July 2002

Previously, we showed caveolae contain a population of protein kinase Cα (PKCα) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCα and measured the interaction of recombinant PKCα with these membranes. PKCα bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require the addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A 68-kD PKCα-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCα binding. A 100–amino acid sequence from the middle of sdr competitively blocked PKCα binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.
Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases
Authors: Jou Tzuu-Shuh , Schneeberger Eveline E. , James Nelson W.
Published: July 2002

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.
KDEL Receptor (Erd2p)-mediated Retrograde Transport of the Cholera Toxin A Subunit from the Golgi Involves COPI, p23, and the COOH Terminus of Erd2p
Authors: Majoul Irina , Sohn Kai , Wieland Felix Theodor , Pepperkok Rainer , Pizza Mariagrazia , Hillemann Jörg , Söling Hans-Dieter
Published: July 2002

A cholera toxin mutant (CTX–K63) unable to raise cAMP levels was used to study in Vero cells the retrograde transport of the toxin A subunit (CTX-A–K63), which possesses a COOH-terminal KDEL retrieval signal. Microinjected GTP-γ-S inhibits the internalization as well as Golgi–ER transport of CTX-A–K63. The appearance of CTX-A–K63 in the Golgi induces a marked dispersion of Erd2p and p53 but not of the Golgi marker giantin. Erd2p is translocated under these conditions most likely to the intermediate compartment as indicated by an increased colocalization of Erd2p with mSEC13, a member of the mammalian coat protein II complex. IgGs as well as Fab fragments directed against Erd2p, β-COP, or p23, a new member of the p24 protein family, inhibit or block retrograde transport of CTX-A–K63 from the Golgi without affecting its internalization or its transport to the Golgi. Anti-Erd2p antibodies do not affect the binding of CTX-A to Erd2p, but inhibit the CTX-K63–induced translocation of Erd2p and p53.
Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin Function
Authors: Longtine Mark S. , Fares Hanna , Pringle John R.
Published: July 2002

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother–bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Δ vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Δ cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605–616).
Tropomyosin-containing Actin Cables Direct the Myo2p-dependent Polarized Delivery of Secretory Vesicles in Budding Yeast
Authors: Pruyne David W. , Schott Daniel H. , Bretscher Anthony
Published: July 2002

The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth. Tropomyosin localizes specifically to actin cables and not cortical patches. Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle–associated Rab GTPase Sec4p depolarize rapidly. Bud growth ceases and the mother cell grows isotropically. When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays. Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p-dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence. With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables. Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth.
Dual Function of Cyk2, a cdc15/PSTPIP Family Protein, in Regulating Actomyosin Ring Dynamics and Septin Distribution
Authors: Lippincott John , Li Rong
Published: July 2002

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.
The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization
Authors: Miller Rita K. , Matheos Dina , Rose Mark D.
Published: July 2002

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)– Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Δ, pea2Δ, bud6Δ, and bni1Δ, required for normal polarization and actin cytoskeleton functions and, of these, bni1Δ affected Kar9p localization most severely. Like kar9Δ, bni1Δ mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Δ, the bni1Δ mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Δ bni1Δ double mutants suggested that Kar9p retained some function in bni1Δ mitotic cells. Unlike the polarization mutants, kar9Δ shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Δ. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.
A Cdc24p-Far1p-Gβγ Protein Complex Required for Yeast Orientation during Mating
Authors: Nern Aljoscha , Arkowitz Robert A.
Published: July 2002

Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During mating, haploid Saccharomyces cerevisiae cells orient their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the bud site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here we show that a two- hybrid interaction between Cdc24p and Gβ requires Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and Gβ. Binding experiments demonstrate that Cdc24p, Far1p, and Gβ form a complex in which pairwise interactions can occur in the absence of the third protein. Cdc24p localizes to sites of polarized growth suggesting that this complex is localized. In the absence of CDC24-FAR1-mediated chemotropism, a bud site selection protein, Bud1p/Rsr1p, is essential for morphological changes in response to pheromone. These results suggest that formation of a Cdc24p-Far1p-Gβγ complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal.
p21-Activated Kinase 1 (Pak1) Regulates Cell Motility in Mammalian Fibroblasts
Authors: Sells Mary Ann , Boyd Jonathan T. , Chernoff Jonathan
Published: July 2002

The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all, eukaryotic cells. We and others have established that Pak's effects on these parameters are mediated by changes in the organization of cortical actin. Because cell motility requires polarized rearrangements of the actin/myosin cytoskeleton, we examined the role of Pak1 in regulating cell movement. We established clonal tetracycline-regulated NIH-3T3 cell lines that inducibly express either wild-type Pak1, a kinase-dead, or constitutively-active forms of this enzyme, and examined the morphology, F-actin organization, and motility of these cells. Expression of any of these forms of Pak1 induced dramatic changes in actin organization which were not inhibited by coexpression of a dominant-negative form of Rac1. Cells inducibly expressing wild-type or constitutively-active Pak1 had large, polarized lamellipodia at the leading edge, were more motile than their normal counterparts when plated on a fibronectin-coated surface, and displayed enhanced directional movement in response to an immobilized collagen gradient. In contrast, cells expressing a kinase-dead form of Pak1 projected multiple lamellipodia emerging from different parts of the cell simultaneously. These cells, though highly motile, displayed reduced persistence of movement when plated on a fibronectin-coated surface and had defects in directed motility toward immobilized collagen. Expression of constitutively activated Pak1 was accompanied by increased myosin light chain (MLC) phosphorylation, whereas expression of kinase-dead Pak1 had no effect on MLC. These results suggest that Pak1 affects the phosphorylation state of MLC, thus linking this kinase to a molecule that directly affects cell movement.
Aip1p Interacts with Cofilin to Disassemble Actin Filaments
Authors: Rodal Avital A. , Tetreault Jonathan W. , Lappalainen Pekka , Drubin David G. , Amberg David C.
Published: July 2002

Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid–based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches.
An Exclusively Nuclear RNA-Binding Protein Affects Asymmetric Localization of ASH1 mRNA and Ash1p in Yeast
Authors: Long Roy M. , Gu Wei , Meng Xiuhua , Gonsalvez Graydon , Singer Robert H. , Chartrand Pascal
Published: July 2002

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3′-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3′-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.
Synergism of Xist Rna, DNA Methylation, and Histone Hypoacetylation in Maintaining X Chromosome Inactivation
Authors: Csankovszki Györgyi , Nagy András , Jaenisch Rudolf
Published: July 2002

Xist RNA expression, methylation of CpG islands, and hypoacetylation of histone H4 are distinguishing features of inactive X chromatin. Here, we show that these silencing mechanisms act synergistically to maintain the inactive state. Xist RNA has been shown to be essential for initiation of X inactivation, but not required for maintenance. We have developed a system in which the reactivation frequency of individual X-linked genes can be assessed quantitatively. Using a conditional mutant Xist allele, we provide direct evidence for that loss of Xist RNA destabilizes the inactive state in somatic cells, leading to an increased reactivation frequency of an X-linked GFP transgene and of the endogenous hypoxanthine phosphoribosyl transferase (Hprt) gene in mouse embryonic fibroblasts. Demethylation of DNA, using 5-azadC or by introducing a mutation in Dnmt1, and inhibition of histone hypoacetylation using trichostatin A further increases reactivation in Xist mutant fibroblasts, indicating a synergistic interaction of X chromosome silencing mechanisms.
FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal
Authors: Dijkers Pascale F. , Birkenkamp Kim U. , Lam Eric W.-F. , Thomas N. Shaun B. , Lammers Jan-Willem J. , Koenderman Leo , Coffer Paul J.
Published: July 2002

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim−/− mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling.

These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB–mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.