bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple scale modeling of turbulent nonequilibrium boundary layer flows
Authors: Gleize V. , Schiestel R. , Couaillier V.
Published: July 2002

Multiple scale models are investigated in order to improve modeling of turbulent nonequilibrium boundary layers. The model is first calibrated on the standard equilibrium boundary layer and then applied to several nonequilibrium flows. Applications include the flow around an airfoil, the shock–boundary layer interaction, the transonic bump flow, and a three-dimensional bump. The multiple scale model predictions are compared with experimental results and with standard one-scale models. Several improvements are obtained that make the multiple scale model an interesting tool for tackling the prediction of nonequilibrium turbulent flows.
Bifurcation phenomena in incompressible sudden expansion flows
Authors: Drikakis D.
Published: July 2002

A numerical study of laminar incompressible flows in symmetric plane sudden expansions was carried out. Computations were performed for various Reynolds number and expansion ratios. The results revealed that the flow remains symmetric up to a certain Reynolds number depending on the expansion ratio, while asymmetries appear at higher Reynolds numbers. The computations indicated that the critical Reynolds number of the symmetry-breaking bifurcation reduces when increasing the expansion ratio while the flow regains symmetry downstream of an initial channel length. The flow asymmetries were verified by comparing several discretization schemes up to fourth order of accuracy as well as various iterative solvers.
Stabilization mechanisms of short waves in stratified gas–liquid flow
Authors: Sangalli Massimo , McCready Mark J. , Chang Hsueh-Chia
Published: July 2002

Interfacial waves grow in a cocurrent, stratified gas–liquid flow by extracting energy from the main flow. The most unstable mode typically has a wavelength comparable to or less than the liquid depth. Experiments show that these short waves can saturate at small amplitude with no generation of long-wave or transverse modes. By decomposing the typical Stuart–Landau analysis into three components, it is found that saturation usually occurs by cubic self-interaction of the fundamental mode but quadratic resonant interaction with the first overtone is also possible. Interaction with mean flow modes is usually much less important. Experiments confirm the predictions of weakly nonlinear theory. The measured overtone is found to be O(|A1|2) and is phase-locked with the fundamental except near a 1–2 resonance point where the fundamental and the overtone have comparable speeds. Near this resonance, the amplitudes are of the same order and the phase angle between them is observed to jump irregularly as predicted by modern dynamical systems theory for intermittent chaos near a heteroclinic cycle. The phase and magnitude of the overtone interaction specify the shape, chaotic dynamics and symmetry of the waves across resonance which are analyzed and confirmed experimentally.
Are flows electromagnetically forced in thin stratified layers two dimensional?
Authors: Paret J. , Marteau D. , Paireau O. , Tabeling P.
Published: July 2002

The relaxation of three-dimensional perturbations in flows generated in thin density stratified layers is discussed. It is found that the relaxation time for such perturbations is small compared to the other characteristic times of the system. These results offer a basis to assess the two dimensionality of freely decaying turbulence prepared in such configurations.
Shear sheltering and the continuous spectrum of the Orr–Sommerfeld equation
Authors: Jacobs Robert G. , Durbin Paul A.
Published: July 2002

The expansion into eigenfunctions of a general disturbance in a viscous flow is possible only when both the discrete and continuous modes of the Orr–Sommerfeld equation are employed. Proper implementation of the boundary conditions and a method for computation of the continuous modes are developed. The unique phenomenon known as shear sheltering is discussed and illustrated. It is shown that the penetration depth of disturbances into the boundary layer has a dependence on frequency and Reynolds number similar to that of a Stokes layer. A simple model that captures this dependence is developed.
Direct numerical simulation of vortex structures and turbulent scalar transfer across a free surface in a fully developed turbulence
Authors: Nagaosa Ryuichi
Published: July 2002

Dynamics of well-organized tube-like coherent structures under a free surface and turbulent scalar transfer across the free surface in fully developed turbulent flow in an open channel is investigated. A direct numerical simulation of the three-dimensional Navier–Stokes equations is used to obtain the structure of the free-surface turbulence. First, the effect of the free surface on fully developed turbulence statistics is described. Anisotropy of velocity and vorticity under the free surface are given. Next, the dynamics of the intermittent vortex tubes beneath the free surface are stated. The genesis and development of these coherent structures and their interactions with the free surface are demonstrated. The role of the vortex/surface interactions on the dynamics of turbulence under the free surface, particularly intercomponent energy transfer due to the pressure–strain effect, is discussed. In addition, passive scalar transfer across the free surface is studied. Finally, the promotion of turbulent scalar transfer at the free surface by the vortex/interface interactions is discussed.
Turbulence inside a vortex
Authors: Cotel Aline J. , Breidenthal Robert E.
Published: July 2002

Following Bradshaw’s analogy between rotating and stratified flows, the turbulence within a vortex is analyzed using a new model for stratified entrainment. At the vortex radius where the tangential velocity is a maximum, the model predicts that the flow is so strongly “stratified” that even the smallest turbulent eddies are incapable of transporting fluid there. The growth of the vortex is thus limited by molecular viscosity, even though the vortex Reynolds number is large. The model prediction is compared to experiments in the literature of wingtip vortices. The result is consistent with the remarkable observations of laminar-like growth of this turbulent flow.
Re-examining the thermal mixing layer with numerical simulations
Authors: de Bruyn Kops S. M. , Riley J. J.
Published: July 2002

The question of whether a temperature mixing layer evolves in a self-similar manner is of importance in developing and validating theories about scalar mixing. The simplicity of the flow encourages the thought that it is self-similar, but several laboratory experiments at moderate Peclet numbers have found inconsistencies with self-similar behavior. The experimentalists are limited, however, by the length of the wind tunnels and by difficulties in aligning the virtual origins of the scalar and velocity fields. Direct numerical simulations virtually eliminate both these problems, and large-eddy simulations add the ability to study an approximation to the case of an infinite Peclet number. These two simulation techniques are used in this paper to show that the mixing layer at a moderate Peclet number very nearly evolves with a single length and time scale, and that behavior consistent with self-similarity is observed in the case of an infinite Peclet number. In addition, the results show that direct numerical simulations can accurately reproduce the data from wind tunnel experiments downstream of a turbulence grid, and that large-eddy simulations are a valuable research tool for studying the large-scale characteristics of mixing.
Alpha particle effects on the internal kink and fishbone modes
Authors: Wu Yanlin , Cheng C. Z. , White R. B.
Published: July 2002

The effects of alpha particles on the internal kink and fishbone modes are studied analytically. The nonadiabatic contribution from untrapped alpha particles is negligible. Finite inverse aspect ratio, plasma β, and plasma shaping effects can significantly enhance the trapped particle drift reversal domain in the pitch angle space and reduce the bounce-averaged magnetic drift frequency. The decrease of the drift magnitude and drift reversal effects on the ideal kink mode is small, but the βα threshold for the fishbone mode can be much lower than previously predicted [B. Coppi, S. Migliuolo, F. Pegoraro, and F. Porcelli, Phys. Fluids B 2, 927 (1990)]. Moreover, the fishbone mode could be excited by alpha particles, even when the plasma is stable in the ideal magnetohydrodynamic (MHD) limit. In addition, the ion diamagnetic drift frequency (finite ion Larmor radius effect) has a strong destabilizing effect on the fishbone mode when it is comparable with the trapped alpha-averaged precessional drift frequency, even though it stabilizes the plasma in the ideal MHD limit.
The influence of collisions on the plasma sheath transition
Authors: Riemann K.-U.
Published: July 2002

In the asymptotic limit λD/λ→0 (where λD is the electron Debye length and λ represents the ion mean free path) the boundary layer of a collision dominated plasma is split up in a collision free planar sheath (scale λD) and a quasi-neutral presheath (scale λ). The “sheath edge” separating the presheath and the sheath is defined by the Bohm criterion. To clear up controversial statements on the validity of the plasma sheath concept and on the role of the Bohm criterion for finite values of λD/λ, a simple fluid model of the ions is used to account for collisions and for space charges in the boundary layer. It is shown that the asymptotic solution on an “intermediate scale” (scale length λ1/5λD4/5) is suitable to match the presheath and sheath solutions smoothly and to construct a convenient approximation for small but finite λD/λ. The intermediate scale, again, is closely related to the Bohm criterion. Various attempts to derive a “generalized” Bohm criterion accounting for collisions are inconsistent.
Influence of particle trapping on the propagation of ion cyclotron waves
Authors: Abbasi H. , Tsintsadze N. L. , Tskhakaya D. D.
Published: July 2002

The influence of particle trapping on the nonlinear dynamics of ion cyclotron waves is investigated. It is shown that in the case when the frequency of externally applied electromagnetic waves is close to that of the ion cyclotron waves, the nonlinear coupling between the ion cyclotron waves and the resulting longitudinal perturbations is mainly caused by the pondermotive potential of ions. Due to the trapping of particles, the characteristics of the propagation of the stationary ion cyclotron waves, as well as the condition for their existence, change. Under some circumstances there is no possibility for their propagation. Depending on the amplitude, oscillatory and localized shapes are possible. Trapping of particles makes the characteristic length of modulation larger than the case when it is neglected.
Highly efficient Raman amplification in a polarization-preserving optical fiber
Authors: Nakazawa Masataka
Published: July 2002

In a forward pump configuration, a Raman gain coefficient of 4.8×10−11 cm/W and a gain as high as 40 dB/W have been successfully attained with a combination of 1.34-μm pump and 1.42-μm signal pulses. A 20-dB gain at a wavelength of 1.52 μm was also obtained at a 3.7-W input. The test fiber is a 1-km-long polarization-preserving and absorption-reducing type polarization-preserving fiber which has a GeO2 highly doped core with a small core diameter of 4.5 μm. It is also shown that the 3-dB gain bandwidth is around 130–135 cm−1 and that optical power ratio of the amplified signal to the spontaneous emission is about 24 dB for the 1.42-μm 1st Stokes line.
Time-dependent compositional variation in SiO2 films nitrided in ammonia
Authors: Han C. J. , Moslehi M. M. , Helms C. R. , Saraswat K. C.
Published: July 2002

We report on selected results of an extensive study of SiO2 nitridation in an NH3 ambient obtained primarily using Auger sputter profiling. By investigating a large matrix of samples, we have sorted out the discrepancies present in the literature concerning the kinetics of the nitridation process. Two major new results obtained are the quantitative observation of oxygen depletion in the nitroxide ‘‘bulk’’ region and, consistent with a recent report by R. Vasquez, M. Hecht, F. Grunthaner, and M. Naiman [Appl. Phys. Lett. 44, 969 (1984)], the reoxidation of the interface by oxygen presumably liberated by the bulk exchange reaction.
Observation of plasmons coupled with optical phonons in n-AlxGa1−xAs by Raman scattering
Authors: Yuasa T. , Naritsuka S. , Mannoh M. , Shinozaki K. , Yamanaka K. , Nomura Y. , Mihara M. , Ishii M.
Published: July 2002

Raman scattering from Si-doped n-AlxGa1−xAs (x=0.25) grown by molecular beam epitaxy has been studied at room temperature. In addition to the optical phonon lines, three new Raman lines have been clearly observed in samples with high carrier concentrations (n≳1×1018 cm−3). These lines are identified as the coupled plasmon-longitudinal optical (LO) phonon modes because their frequencies and relative intensities to the LO phonon lines depend on the excitation wavelength.
Analysis of switching transients in KNO3 ferroelectric memories
Authors: Araujo C. , Scott J. F. , Godfrey R. Bruce , McMillan L.
Published: July 2002

We have measured the switching current transient i(t) versus time for 75-nm-thick KNO3 nonvolatile memories having address voltages of 6.3 V and switching times 20–30 ns. The currents are minimum (nearly zero) at t=0 and exhibit a symmetric shape about their maxima at 23±2 ns. This is compatible with Fatuzzo’s theory of sideways domain growth [Phys. Rev. 127, 1999 (1962)] as well as with our earlier results on thickness dependence of coercive field Ec∼d−1.3 and field dependence of switching times ts∼E−1.5. All of the results show that domain growth speed transverse to the applied field is the rate-limiting parameter.
Electrical measurement of the formation of the platinum-rich metal silicides by metal-silicon reaction
Authors: Gas P. , Tardy J. , LeGoues F. , d’Heurle F. M.
Published: July 2002

I n situ measurements of resistance have been used to study the interaction of Pt with a limited amount of Si deposited in adjacent layers on oxidized Si wafers. One observes in sequence the formation of Pt2Si, Pt12Si5, and Pt3Si, as seen previously by others using time-sequence annealing. The present method has the advantage of being quite rapid and simple. Changing the heating rate provides one with a measure of the activation energies for several reactions, including, for the first time, values of the apparent activation energy for phases whose formation is dominated by nucleation and growth, rather than by diffusion. A phase transformation was observed during the cooling of Pt3Si. The final Pt3Si phase examined by transmission electron microscopy reveals an interesting Widmanstätten structure. The resistivities of the different phases have been measured: 29, 32, and 53±10 μΩ cm, respectively, in order of increasing Pt content.
Acoustic investigation of the elastic properties of ZnO films
Authors: Carlotti G. , Socino G. , Petri A. , Verona E.
Published: July 2002

The elastic properties of ZnO films deposited by rf magnetron sputtering on Al2O3 substrates have been analyzed by means of an acoustic investigation technique. The phase velocities of a spectrum of acoustic modes propagating along the layered structure have been measured and the results exploited for determining the complete set of elastic constants of the film. The effective constants of the film are lower than those of the bulk material by amounts which depend on the elastic constant considered and range from −1.2% for c33 to −24.8% for c11. The values obtained were used for determining the dispersion curves of acoustic modes propagating along ZnO layers deposited on fused quartz and silicon and showed good agreement with experimental results.
A direct role of SRY and SOX proteins in pre-mRNA splicing
Authors: Ohe Kenji , Lalli Enzo , Sassone-Corsi Paolo
Published: July 2002

The mammalian testis determining factor SRY and its related Sox factors are critical developmental regulators. They share significant similarity in their high mobility group (HMG) domain and display discrete patterns of tissue-specific expression. Here we show that SRY and the Sox protein SOX6 colocalize with splicing factors in the nucleus and are dynamically redistributed following the blockage of splicing in living cells. Anti-SOX6 antibodies supershift the spliceosomal complex from assembled splicing reactions and inhibit splicing
in vitro
of multiple pre-mRNA substrates. Most importantly, SOX6-depleted nuclear extracts have impaired splicing activity, which is efficiently restored by addition of the recombinant SOX6 HMG domain and also by recombinant SRY and the SOX9 HMG domain. These results reveal an unexpected biological function of the
SRY
,
SOX6
, and
SOX9
gene products and provide a functional link to the biochemical mechanisms operating in mammalian sex determination and in other developmental processes regulated by
Sox
genes.
Chemical amplification with encapsulated reagents
Authors: Chen Jian , Körner Steffi , Craig Stephen L. , Lin Shirley , Rudkevich Dmitry M. , Rebek Julius
Published: July 2002

Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.
“Size-selectivity” in the template-directed assembly of dinuclear triple-stranded helicates
Authors: Albrecht Markus , Blau Oliver , Fröhlich Roland
Published: July 2002

The self-assembly of supramolecular structures depends on a subtle interplay of a series of different control mechanisms. The geometric as well as electronic complementarity of the molecular building blocks is crucial for the specific formation of defined supramolecular species. In addition, secondary effects, like templating, also have an important function. The templating ability of different cations in the formation of triple-stranded helicate-type complexes from alkyl-bridged di(8-hydroxyquinoline) ligands is investigated by introduction of alkyl chains of different length as ligand spacers. Hereby a “size-selectivity” between the cations and the dinuclear helicate-type complexes {(ligand)
3
M
2
} is observed. Large cations support the formation of big dinuclear complexes, whereas small cations are able to template the formation of small complexes.
Evaluating the arrayed primer extension resequencing assay of TP53 tumor suppressor gene
Authors: Tõnisson Neeme , Zernant Jana , Kurg Ants , Pavel Hendrik , Slavin Georg , Roomere Hanno , Meiel Aune , Hainaut Pierre , Metspalu Andres
Published: July 2002

Identification of mutations in the tumor suppressor gene TP53 has implications for the molecular epidemiology and for the molecular pathology of human cancer. We have developed and evaluated an arrayed primer extension assay for covering both strands of a region of the coding sequence containing more than 95% of the mutations described so far in TP53. On average, 97.5% of the arrayed TP53 gene sequence can be analyzed from either sense or antisense strands, and 81% from both strands. A patient DNA sample is amplified and annealed to arrayed primers, which then promote DNA polymerase extension reactions with four fluorescently labeled dideoxynucleotides. The TP53 gene chip spans exons 2–9 plus two introns from both strands. The performance of the assay was evaluated by using freshly extracted genomic DNA, as well as DNA extracted from archival (paraffin-embedded) DNA samples. The arrayed primer extension-based TP53 gene test provides an accurate and efficient tool for DNA sequence analysis of this frequently mutated gene for both research and clinical applications.
Using multimodal ligands to influence network topology in silver(I) coordination polymers
Authors: Oxtoby Neil S. , Blake Alexander J. , Champness Neil R. , Wilson Claire
Published: July 2002

A range of Ag(I) one- and two-dimensional coordination frameworks has been prepared and structurally characterized by using the multimodal ligand 3,6-di-pyrazin-2-yl-(1,2,4,5)-tetrazine, which offers both monodentate and chelating binding sites. It is demonstrated that multimodal ligands can be used to prepare coordination frameworks with novel and unusual topologies and to influence the precise geometrical arrangement of both ligands and metal centers within such supramolecular arrays.
Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA
Authors: Yu Xiong , Jacobs Steven A. , West Stephen C. , Ogawa Tomoko , Egelman Edward H.
Published: July 2002

Both the bacterial RecA protein and the eukaryotic Rad51 protein form helical nucleoprotein filaments on DNA that catalyze strand transfer between two homologous DNA molecules. However, only the ATP-binding cores of these proteins have been conserved, and this same core is also found within helicases and the F1-ATPase. The C-terminal domain of the RecA protein forms lobes within the helical RecA filament. However, the Rad51 proteins do not have the C-terminal domain found in RecA, but have an N-terminal extension that is absent in the RecA protein. Both the RecA C-terminal domain and the Rad51 N-terminal domain bind DNA. We have used electron microscopy to show that the lobes of the yeast and human Rad51 filaments appear to be formed by N-terminal domains. These lobes are conformationally flexible in both RecA and Rad51. Within RecA filaments, the change between the “active” and “inactive” states appears to mainly involve a large movement of the C-terminal lobe. The N-terminal domain of Rad51 and the C-terminal domain of RecA may have arisen from convergent evolution to play similar roles in the filaments.
Spatial and temporal control of RNA stability
Authors: Bashirullah Arash , Cooperstock Ramona L. , Lipshitz Howard D.
Published: July 2002

Maternally encoded RNAs and proteins program the early development of all animals. A subset of the maternal transcripts is eliminated from the embryo before the midblastula transition. In certain cases, transcripts are protected from degradation in a subregion of the embryonic cytoplasm, thus resulting in transcript localization. Maternal factors are sufficient for both the degradation and protection components of transcript localization. Cis-acting elements in the RNAs convert transcripts progressively (
i
) from inherently stable to unstable and (
ii
) from uniformly degraded to locally protected. Similar mechanisms are likely to act later in development to restrict certain classes of transcripts to particular cell types within somatic cell lineages. Functions of transcript degradation and protection are discussed.
Biologic consequences of Stat1-independent IFN signaling
Authors: Gil M. Pilar , Bohn Erwin , O'Guin Andrew K. , Ramana Chilakamarti V. , Levine Beth , Stark George R. , Virgin Herbert W. , Schreiber Robert D.
Published: July 2002

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.
Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor
Authors: Lin Hui-Kuan , Yeh Shuyuan , Kang Hong-Yo , Chang Chawnshang
Published: July 2002

Whereas several apoptosis-related proteins have been linked to the antiapoptotic effects of Akt serine–threonine kinase, the search continues to explain the Akt signaling role in promoting cell survival via antiapoptotic effects. Here, we demonstrate that Akt phosphorylates the androgen receptor (AR) at Ser-210 and Ser-790. A mutation at AR Ser-210 results in the reversal of Akt-mediated suppression of AR transactivation. Activation of the phosphatidylinositol-3-OH kinase/Akt pathway results in the suppression of AR target genes, such as p21, and the decrease of androgen/AR-mediated apoptosis, which may involve the inhibition of interaction between AR and AR coregulators. Together, these findings provide a molecular basis for cross-talk between two signaling pathways at the level of Akt and AR–AR coregulators that may help us to better understand the roles of Akt in the androgen/AR-mediated apoptosis.
Reovirus reverse genetics: Incorporation of theCATgene into the reovirus genome
Authors: Roner Michael R. , Joklik Wolfgang K.
Published: July 2002

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which theCATgene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein σ2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.
GTP-binding protein βγ subunits mediate presynaptic calcium current inhibition by GABA
B
receptor
Authors: Kajikawa Yoshinao , Saitoh Naoto , Takahashi Tomoyuki
Published: July 2002

A variety of GTP-binding protein (G protein)-coupled receptors are expressed at the nerve terminals of central synapses and play modulatory roles in transmitter release. At the calyx of Held, a rat auditory brainstem synapse, activation of presynaptic γ-aminobutyric acid type B receptors (GABA
B
receptors) or metabotropic glutamate receptors inhibits presynaptic P/Q-type Ca
2+
channel currents via activation of G proteins, thereby attenuating transmitter release. To identify the heterotrimeric G protein subunits involved in this presynaptic inhibition, we loaded G protein βγ subunits (Gβγ) directly into the calyceal nerve terminal through whole-cell patch pipettes. Gβγ slowed the activation of presynaptic Ca
2+
currents (
I
pCa
) and attenuated its amplitude in a manner similar to the externally applied baclofen, a GABA
B
receptor agonist. The effects of both Gβγ and baclofen were relieved after strong depolarization of the nerve terminal. In addition, Gβγ partially occluded the inhibitory effect of baclofen on
I
pCa
. In contrast, guanosine 5′-
O
-(3-thiotriphosphate)-bound G
o
α loaded into the calyx had no effect. Immunocytochemical examination revealed that the subtype of G proteins G
o
, but not the G
i
, subtype, is expressed in the calyceal nerve terminal. These results suggest that presynaptic inhibition mediated by G protein-coupled receptors occurs primarily by means of the direct interaction of G
o
βγ subunits with presynaptic Ca
2+
channels.
Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skininvitro
Authors: Bernerd Françoise , Asselineau Daniel , Vioux Corinne , Chevallier-Lagente Odile , Bouadjar Bakar , Sarasin Alain , Magnaldo Thierry
Published: July 2002

Sun exposure has been clearly implicated in premature skin aging and neoplastic development. These features are exacerbated in patients with xeroderma pigmentosum (XP), a hereditary disease, the biochemical hallmark of which is a severe deficiency in the nucleotide excision repair of UV-induced DNA lesions. To develop an organotypic model of DNA repair deficiency, we have cultured several strains of primary XP keratinocytes and XP fibroblasts from skin biopsies of XP patients. XP skin comprising both a full-thickness epidermis and a dermal equivalent was succesfully reconstructedin vitro. Satisfactory features of stratification were obtained, but the expression of epidermal differentiation products, such as keratin K10 and loricrin, was delayed and reduced. In addition, the proliferation of XP keratinocytes was more rapid than that of normal keratinocytes. Moreover, increased deposition of cell attachment proteins, α-6 and β-1 integrins, was observed in the basement membrane zone, and β-1 integrin subunit, the expression of which is normally confined to basal keratinocytes, extended into several suprabasal cell layers. Most strikingly, thein vitroreconstructed XP skin displayed numerous proliferative epidermal invasions within dermal equivalents. Epidermal invasion and higher proliferation rate are reminiscent of early steps of neoplasia. Compared with normal skin, the DNA repair deficiency ofin vitroreconstructed XP skin was documented by long-lasting persistence of UVB-induced DNA damage in all epidermal layers, including the basal layer from which carcinoma develops. The availability ofin vitroreconstructed XP skin provides opportunities for research in the fields of photoaging, photocarcinogenesis, and tissue therapy.
Protection and storage of chlorophyll in overwintering evergreens
Authors: Gilmore Adam M. , Ball Marilyn C.
Published: July 2002

How evergreen species store and protect chlorophyll during exposure
to high light in winter remains unexplained. This study reveals that
the evergreen snow gum (
Eucalyptus pauciflora
Sieb. ex
Spreng.) stores and protects its chlorophylls by forming special
complexes that are unique to the winter-acclimated state. Our
in
vivo
spectral and kinetic characterizations reveal a prominent
component of the chlorophyll fluorescence spectrum around 715 nm at 77
K. This band coincides structurally with a loss of chlorophyll and an
increase in energy-dissipating carotenoids. Functionally, the band
coincides with an increased capacity to dissipate excess light energy,
absorbed by the chlorophylls, as heat without intrathylakoid
acidification. The increased heat dissipation helps protect the
chlorophylls from photo-oxidative bleaching and thereby facilitates
rapid recovery of photosynthesis in spring.
Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart
Authors: Leri Annarosa , Barlucchi Laura , Limana Federica , Deptala Andrzej , Darzynkiewicz Zbigniew , Hintze Thomas H. , Kajstura Jan , Nadal-Ginard Bernardo , Anversa Piero
Published: July 2002

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity
in vivo
is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20–30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.
Glutamatergic modulation of hyperactivity in mice lacking the dopamine transporter
Authors: Gainetdinov Raul R. , Mohn Amy R. , Bohn Laura M. , Caron Marc G.
Published: July 2002

In the brain, dopamine exerts an important modulatory influence
over behaviors such as emotion, cognition, and affect as well as
mechanisms of reward and the control of locomotion. The dopamine
transporter (DAT), which reuptakes the released neurotransmitter into
presynaptic terminals, is a major determinant of the intensity and
duration of the dopaminergic signal. Knockout mice lacking the dopamine
transporter (DAT-KO mice) display marked changes in dopamine
homeostasis that result in elevated dopaminergic tone and pronounced
locomotor hyperactivity. A feature of DAT-KO mice is that their
hyperactivity can be inhibited by psychostimulants and serotonergic
drugs. The pharmacological effect of these drugs occurs without any
observable changes in dopaminergic parameters, suggesting that other
neurotransmitter systems in addition to dopamine might contribute to
the control of locomotion in these mice. We report here that the
hyperactivity of DAT-KO mice can be markedly further enhanced when
N-
methyl-
d
-aspartate receptor-mediated
glutamatergic transmission is blocked. Conversely, drugs that enhance
glutamatergic transmission, such as positive modulators of
l
-α-amino-3-hydroxy-5-methylisoxazole-4-propionate
glutamate receptors, suppress the hyperactivity of DAT-KO mice.
Interestingly, blockade of
N-
methyl-
d
-aspartate receptors prevented
the inhibitory effects of both psychostimulant and serotonergic drugs
on hyperactivity. These findings support the concept of a reciprocal
functional interaction between dopamine and glutamate in the basal
ganglia and suggest that agents modulating glutamatergic transmission
may represent an approach to manage conditions associated with
dopaminergic dysfunction.
Supermolecular structure of the enteropathogenic
Escherichia coli
type III secretion system and its direct interaction with the EspA-sheath-like structure
Authors: Sekiya Kachiko , Ohishi Minako , Ogino Tomoaki , Tamano Koichi , Sasakawa Chihiro , Abe Akio
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) secretes
several Esp proteins via the type III secretion system (secreton).
EspA, EspB, and EspD are required for translocation of the effector
proteins into host cells, in which EspB and EspD are thought to form a
pore in the host membrane. Recent study has shown that EspA forms a
filamentous structure that assembles as a physical bridge between
bacteria and host cell surfaces, which then functions as a conduit for
the translocation of bacterial effectors into host cells. To
investigate the supermolecular structure of the type III secreton in
EPEC, we partially purified it from the bacteria membrane and observed
it via transmission electron microscopy. The EPEC type III secreton was
composed of a basal body and a needle part and was similar to those of
Salmonella
and
Shigella
, except for a
sheath-like structure at the tip of the needle. The length of
sheath-like structures varied; it extended more than 600 nm and was 10
times longer than the
Shigella
needle part. The putative
major needle component, EscF, was required for both secretion of Esp
proteins and needle complex formation. Interestingly, elongation of the
sheath-like structure was observed under constitutive expression of
EspA but not of EscF. Furthermore, the transmission electron microscopy
view with immunogold labeled anti-EspA antibodies clearly showed that
EspA is a component of the sheath-like structure. This study revealed,
to our knowledge for the first time, the supermolecular structure of
the EPEC type III secreton and its direct association with the
EspA-sheath-like structure.
Identification of molecular interactions between P-site tRNA and the ribosome essential for translocation
Authors: Feinberg Jason S. , Joseph Simpson
Published: July 2002

Translocation of the tRNA–mRNA complex is a fundamental step in
the elongation cycle of protein synthesis. Our studies show that the
ribosome can translocate a P-site-bound tRNA
Met
with a
break in the phosphodiester backbone between positions 56 and 57 in the
TΨC-loop. We have used this fragmented P-site-bound
tRNA
Met
to identify two 2′-hydroxyl groups at positions 71
and 76 in the 3′-acceptor arm that are essential for translocation.
Crystallographic data show that the 2′-hydroxyl group at positions 71
and 76 contacts the backbone of 23S rRNA residues 1892 and 2433–2434,
respectively, in the ribosomal E site. These results establish a set of
functional interactions between P-site tRNA and 23S rRNA that are
essential for translocation.
Genome-wide analysis of the
Drosophila
immune response by using oligonucleotide microarrays
Authors: De Gregorio Ennio , Spellman Paul T. , Rubin Gerald M. , Lemaitre Bruno
Published: July 2002

To identify new
Drosophila
genes involved in the immune response, we monitored the gene expression profile of adult flies in response to microbial infection by using high-density oligonucleotide microarrays encompassing nearly the full
Drosophila
genome. Of 13,197 genes tested, we have characterized 230 induced and 170 repressed by microbial infection, most of which had not previously been associated with the immune response. Many of these genes can be assigned to specific aspects of the immune response, including recognition, phagocytosis, coagulation, melanization, activation of NF-κB transcription factors, synthesis of antimicrobial peptides, production of reactive oxygen species, and regulation of iron metabolism. Additionally, we found a large number of genes with unknown function that may be involved in control and execution of the immune response. Determining the function of these genes represents an important challenge for improving our knowledge of innate immunity. Complete results may be found at
http://www.fruitfly.org/expression/immunity/
.
The anti-HIV pentameric pseudopeptide HB-19 is preferentially taken up
in vivo
by lymphoid organs where it forms a complex with nucleolin
Authors: Krust B. , Vienet R. , Cardona A. , Rougeot C. , Jacotot E. , Callebaut C. , Guichard G. , Briand J. P. , Grognet J. M. , Hovanessian A. G. , Edelman L.
Published: July 2002

The HB-19 pseudopeptide 5[Kψ(CH
2
N)PR]-TASP, ψ(CH
2
N) for reduced peptide bond, is a specific inhibitor of HIV infection in different CD4
+
cell lines and in primary T-lymphocytes and macrophages. It blocks virus-particle attachment to permissive cells by binding and forming a stable complex with nucleolin expressed on the cell surface. Here, we have investigated the tissue distribution of the tritiated HB-19 by using β-radio imager whole-body mapping in rats. A rapid, selective, and stable distribution and accumulation of the systematically administered HB-19 was demonstrated within the spleen, liver, bone, and kidney as soon as 5 min following its administration. No apparent uptake of HB-19 occurred in the brain and the muscle tissue. Interestingly and despite its rapid clearance from the blood, at 24 h postexposure a significant proportion of HB-19 was still recovered from target organs, of which 16–37% could be acounted for intact pseudopeptide. The elimination of HB-19 mainly occurred by renal glomerular filtration and most of the excreted radioactivity appeared to be HB-19 metabolites. Finally, injection of the biotin-labeled HB-19 pseudopeptide but not its control counterpart allowed the recovery of the HB-19–nucleolin complex from the liver, spleen, thymus, and bone marrow, thus indicating that the
in vivo
molecular target of HB-19 is surface nucleolin. Our results demonstrate the preferential uptake and stability of HB-19 in lymphoid organs that are the site of HIV propagation.
Mutants of
Arabidopsis
defective in a sequence-specific mRNA degradation pathway
Authors: Johnson Mark A. , Pérez-Amador Miguel A. , Lidder Preetmoninder , Green Pamela J.
Published: July 2002

One of the ways a cell can rapidly and tightly regulate gene
expression is to target specific mRNAs for rapid decay. A number of
mRNA instability sequences that mediate rapid mRNA decay have been
identified, particularly from multicellular eukaryotes, but pinpointing
the cellular components that play critical roles in sequence-specific
decay
in vivo
has been more difficult. In contrast,
general pathways of mRNA degradation in yeast have been well
established through the analysis of mutants affecting the general mRNA
decay machinery. Strategies to isolate mutants in sequence-specific
mRNA decay pathways, although extremely limited so far, have the
potential to be just as powerful. In the study reported here, a
selection in transgenic plants allowed the isolation of rare mutants of
Arabidopsis thaliana
that elevate the abundance of mRNAs
that contain the plant mRNA instability sequence called DST (downstream
element). This instability sequence is highly conserved in unstable
small auxin up RNA (
SAUR
) transcripts. Genetic analysis
of two
dst
mutants isolated via this selection showed
that they are incompletely dominant and represent two independent loci.
In addition to affecting DST-containing transgene mRNAs, mutations at
both loci increased the abundance of the endogenous
DST-containing
SAUR-AC1
mRNA, but not controls lacking
DST sequences. That these phenotypes are caused by deficiencies in
DST-mediated mRNA decay was supported by mRNA stability measurements in
transgenic plants. Isolation of the
dst
mutants provides
a means to study sequence-specific mRNA degradation
in
vivo
and establishes a method to isolate similar mutants from
other organisms.
Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy
Authors: Cotlet Mircea , Hofkens Johan , Habuchi Satoshi , Dirix Gunter , Van Guyse Maarten , Michiels Jan , Vanderleyden Jos , De Schryver Frans C.
Published: July 2002

The photophysics and photochemistry taking place in
the DsRed protein, a recently cloned red fluorescent protein from a
coral of the
Discosoma
genus, are investigated here by
means of ensemble and single-molecule time-resolved detection and
spectroscopic measurements. Ensemble time-resolved data reveal that
25% of the immature green chromophores are present in tetramers
containing only this immature form. They are responsible for the weak
fluorescence emitted at 500 nm. The remaining 75% of the immature
green chromophores are involved in a fluorescence resonance energy
transfer process to the red species. The combination of time-resolved
detection with spectroscopy at the single-molecule level reveals, on
543-nm excitation of individual DsRed tetramers, the existence of a
photoconversion of the red chromophore emitting at 583 nm and decaying
with a 3.2-ns time constant into a super red one emitting at 595 nm and
for which the decay time constant ranges between 2.7 and 1.5 ns. The
phenomenon is further corroborated at the ensemble level by the
observation of the creation of a super red form and a blue absorbing
species on irradiation with 532-nm pulsed light at high excitation
power. Furthermore, single-molecule experiments suggest that a similar
photoconversion process might occur in the immature green species on
488-nm excitation.
Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors.
Authors: Andjelković M , Jakubowicz T , Cron P , Ming X F , Han J W , Hemmings B A
Published: July 2002

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both RAC-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKalpha activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.
Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.
Authors: Stahl F , Wende W , Jeltsch A , Pingoud A
Published: July 2002

Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the ecorV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.