bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophoresis of a thin charged disk
Authors: Sherwood J. D. , Stone H. A.
Published: July 2002

The electrophoretic velocity of a charged disk of zero thickness is computed in the limit of small surface potentials, but with arbitrary double layer thickness. The disk represents an idealized clay particle, and has uniform surface charge over its flat surface, together with a uniform line charge around its edge. The contributions of these two charges to the electrophoretic velocity are considered separately. Asymptotic results are obtained for thin and thick double layers, and intermediate results are obtained by numerical integration. The singularities in both the electrical and hydrodynamic fields at the edge of the particle enhance the importance of the edge charge when the double layer thickness κ−1 is small compared to the disk radius a.
Nonlinear dynamics of modulated flow between a porous injector and an impermeable substrate
Authors: Medina J. Antonio , Schwartz Daniel T.
Published: July 2002

The stable, time-periodic flow between a porous injector disk and an impermeable substrate disk is explored for the case where fluid is injected into the gap region with a spatially uniform time-periodic velocity V(τ)=V0(1+α cos(στ)), where V0 is the mean injection velocity, α is the flow modulation amplitude, and σ is the flow modulation frequency. Fourier series expansions in time (τ) are combined with regular perturbation expansions of the Fourier coefficients in powers of α to describe the linear and nonlinear frequency dispersion in the system when 0<α<1. Generalized analytical expressions are obtained for the quasisteady response, and the method of matched asymptotic expansions is used to analyze the high frequency linear response of the system. Finite difference methods are used to calculate the frequency dispersion of the system for a wide range of modulation frequencies (σ) and Reynolds numbers. Oscillating harmonics are shown to interact destructively (via nonlinear inertial terms), resulting in the nullification of certain Fourier modes in the flow field. The Reynolds number and frequency dependence of harmonic nullification events are explored and their implications for creating multilayered alloys are briefly discussed.
A low-order model for vortex shedding patterns behind vibrating flexible cables
Authors: Olinger D. J.
Published: July 2002

A recent focus in studies of vortex shedding behind circular cylinders has been on the use of low-order dynamical systems such as circle maps to predict wake dynamics. These purely temporal models have been limited by their inability to describe three-dimensional spatial flow variations along the cylinder span, a hallmark of transitional flows such as the cylinder wake. In the present work this limitation is overcome through development of a spatial-temporal map lattice which utilizes a series of coupled circle map oscillators along the cylinder span. This model allows for the study of vortex shedding patterns and wake dynamics behind vibrating flexible cables. Required input for the model includes the forcing frequency, amplitude, mode shape, aspect ratio and wavelength of the cable, Reynolds number, vortex convection velocity, and various phase angles. Model output parameters studied in this work include vortex shedding patterns and wake response frequency. Standing wave mode shapes and traveling waves along the cable span are modeled. Lacelike vortex patterns are observed for the standing wave case. A physical mechanism for the lacelike patterns is postulated. For traveling waves oblique shedding patterns are confirmed. Nonharmonic forcing outside the classical lock-on region yields vortex dislocation patterns in the wake. Honeycomb patterns are also observed for higher-order mode shapes at large forcing amplitudes. The current work establishes a new class of models based on circle maps for modeling spatially varying cylinder wakes.
Maximum entropy versus minimum enstrophy vortices
Authors: Brands H. , Chavanis P. H. , Pasmanter R. , Sommeria J.
Published: July 2002

Motivated by the experimental study of Huang and Driscoll [Phys. Rev. Lett. 72, 2187 (1994)], we carry a systematic comparison between maximum entropy and restricted minimum enstrophy vortices. We find that, when the angular momentum constraint is reconsidered, the restricted minimum enstrophy principle provides a good and useful approximation of the statistical theory, except for large energies. In the case investigated by Huang and Driscoll, this approximation even yields a better agreement with experiments than the full statistical theory because the relaxation of the system toward the maximum entropy state is not complete. However, we show that this agreement cannot hold in more general cases, due to physical inconsistencies of minimum enstrophy principles. We also discuss the relevance of generalized thermodynamics (Tsallis entropy) to predict the equilibrium state.
Anomalous losses of deuterium–deuterium fusion products in the Tokamak Fusion Test Reactor*
Authors: Zweben S. J. , Bush C. E. , Chang C. S. , Chang Z. , Darrow D. S. , Fredrickson E. D. , Herrmann H. W. , Mynick H. E. , Schivell J. , Bell M. , Boivin R. , Budny R. V. , Cheng C. Z. , Ernst D. , Hammett G. , Johnson L. C. , McCune D. , Murakami M. , Owens D. K. , Park J. , Phillips C.-K. , Redi M. H. , Scott S. , Strachan J. D. , Taylor G. , Tuszewski M. , White R. B. , Wilson J. R. , Zarnstorff M.
Published: July 2002

In the Tokamak Fusion Test Reactor (TFTR) [International Conference on Plasma Physics and Controlled Nuclear Fusion Research, Wurzburg, Paper No. A-2-2 (International Atomic Energy Agency, Vienna, 1993)] there have been at least three types of anomalous loss of alpha-like deuterium–deuterium (D–D) fusion products: (1) a magnetohydrodynamic (MHD)-induced loss of D–D fusion products correlated with Mirnov and fishbone-type oscillations and sawtooth crashes, (2) a slow ‘‘delayed’’ loss of partially thermalized D–D fusion products occurring without large MHD activity, and (3) ion cyclotron resonance heating (ICRH)-induced loss of D–D fusion products ions observed during direct electron heating experiments, and possibly also during 3He minority heating. In this paper each of these will be reviewed, concentrating on those due to MHD activity, which are the largest of these anomalous losses. The experimental results are compared with numerical models of various fusion product transport mechanisms.
Excitation of lower hybrid waves by a density-modulated electron beam in a plasma cylinder
Authors: Sharma Suresh C. , Srivastava M. P. , Sugawa M. , Tripathi V. K.
Published: July 2002

A density-modulated electron beam propagating through a plasma cylinder drives electrostatic lower hybrid waves to instability via Cerenkov interaction. The lower hybrid wave instability has the largest growth rate γ when the frequency and wave number of the modulation are comparable to that of the unstable wave. The growth rate of the instability increases with the modulation index Δ and is maximized for Δ=1. For Δ=0, γ turns out to be ∼0.43×107 sec−1. The growth rate scales as the one-third power of the beam density. The real frequency of the unstable wave increases as almost the square root of the beam voltage. The results of the theory are applied to explain some of the experimental observations of Chang [Phys. Rev. Lett. 35, 285 (1975)].
Experimental evidence of long-range correlations and self-similarity in plasma fluctuations
Authors: Carreras B. A. , van Milligen B. Ph. , Pedrosa M. A. , Balbı́n R. , Hidalgo C. , Newman D. E. , Sánchez E. , Bravenec R. , McKee G. , Garcı́a-Cortés I. , Bleuel J. , Endler M. , Riccardi C. , Davies S. , Matthews G. F. , Martines E. , Antoni V.
Published: July 2002

To better understand long time transport dynamics, techniques to investigate long-range dependences in plasma fluctuations have been applied to data from several confinement devices including tokamaks, stellarators, and reversed field pinch. The results reveal the self-similar character of the edge plasma fluctuations. This implies that the tail of the autocorrelation function decays as a power law and suggests that there is a superdiffusive component of the anomalous transport. Rescaled fluctuation and turbulent flux spectra from different devices also show a strong similarity. For a range of parameters corresponding to the tokamak ohmic regime and equivalent power for other devices, the spectral decay index may show a universal character.
Numerical investigations on strong pattern selecting Eckhaus instabilities in neon glow discharges
Authors: Jonas P. , Bruhn B. , Koch B.-P. , Dinklage A.
Published: July 2002

Starting from the hydrodynamic description of the positive column in a neon glow discharge, a numerical approach is used in order to study the nonlinear properties of ionization waves. Within the instability region of the homogeneous equilibrium state, a secondary instability of the Eckhaus type is found. Compared to the classical results, the plasma system shows some peculiarities, e.g., an asymmetric stability band and strong selection of periodic patterns. The dependency of the shape and the width of this band on the discharge parameters is investigated. The spatiotemporal dynamics connected with the transitions from the stability band to the instability region have been studied showing different behavior on the upper and lower border of the stability region. Normally a subcritical Eckhaus instability has been revealed. Moreover, at selected sets of plasma parameters the phenomenon of spatiotemporal intermittency is found.
Charging of silica particles in an argon dusty plasma
Authors: Thomas Edward , Watson Michael
Published: July 2002

The Fisk Plasma Source (FPS) is a plasma device operating at Fisk University. Dusty plasmas in the FPS device are produced by suspending 40 μm diameter silica (SiO2) particles in an argon dc (direct current) glow discharge plasma. Experiments are performed at pressures ranging from 90 to 150 mTorr. A 20 mW HeNe laser is used to illuminate the dust particles. Langmuir probes and video images are used to diagnose the charging and confinement of the dust particles in the plasma. A modified version of the dust grain charging model is developed and is used to estimate the charge. The silica particles are shown to become negatively charged in the plasma with up to 105 electrons. The presence of the negatively charged dust grains is correlated to a reduction in the electron saturation current in the plasma.
Electron-hole recombination at the Si-SiO2 interface
Authors: Yablonovitch E. , Swanson R. M. , Eades W. D. , Weinberger B. R.
Published: July 2002

We have measured the surface recombination current J(ns,ps) at high quality thermally grown Si-SiO2 interfaces as a function of the surface density of electrons and holes, ns and ps. We find that the recombination is dominated by centers whose electron capture cross section is about 100 times greater than their hole capture cross section. Therefore, the maximum recombination occurs when ps≊100ns. Recombination is minimized under extreme electron or hole accumulation and is coincidentally the same in both cases: exp(qV/kT)×10−14 A/cm2.
Nanometer molecular lithography
Authors: Douglas Kenneth , Clark Noel A. , Rothschild Kenneth J.
Published: July 2002

We demonstrate a three-step parallel process for the fabrication of a 1-nm-thick metal (Ta/W) film with 15-nm-diam holes periodically arranged on a triangular lattice of parameter 22 nm. In this process, a two-dimensional crystalline protein monolayer is deposited on a smooth substrate, metal coated by evaporation, and then ion milled. Under ion milling this protein-metal heterostructure exhibits differential metal removal and rearrangement which varies on a protein molecular length scale. The spatial ordering of the resulting holes has the same nanometer periodicity as the protein lattice.
Second harmonic generation in poled polymer films
Authors: Singer K. D. , Sohn J. E. , Lalama S. J.
Published: July 2002

We report the observation of second harmonic generation in a new class of organic polymeric materials, namely, electric field poled polymer glasses. Both the observed second harmonic coefficient (d33=6.0±1.3×10−9 esu, at 1.58 μm wavelength) and the poling process are described by a thermodynamic model that we have developed. The ability to form thin films from these materials may qualify them for integrated optics applications.
Design of optimized high-speed depletion-edge-translation optical waveguide modulators in III-V semiconductors
Authors: Coldren L. A. , Mendoza-Alvarez J. G. , Yan R. H.
Published: July 2002

A new first-principle theory that includes the higher order field and carrier effects in semiconductor waveguide refractive index modulators has been developed. For the first time, the relative importance of the electrorefractive, band filling, linear electro-optic, and plasma effects is shown. The theory agrees well with measurements from experimental AlGaAs/GaAs depletion-edge-translation (DET) phase modulators that have given record levels of phase shift per volt per unit length, and it suggests an improved DET device design with much larger phase shifting efficiency. The required drive voltage/bandwidth figure of merit, Vπ/ Δf, for this configuration (without including quantum wells) is lower than for any other existing optical index modulator.
Effect of circuit oscillations on the dc current-voltage characteristics of double barrier resonant tunneling structures
Authors: Young Jeff F. , Wood B. M. , Liu H. C. , Buchanan M. , Landheer D. , SpringThorpe A. J. , Mandeville P.
Published: July 2002

The influence of external circuit parameters on the measured current-voltage behavior of double barrier resonant tunneling structures is studied both experimentally and through modeling. It is demonstrated that characteristic ‘‘plateaulike’’ structures, which often appear in the region of negative differential resistance, can be accounted for in terms of the oscillating nature of the circuit, given that only average currents and voltages are monitored.
Planetary exploration in the time of astrobiology: Protecting against biological contamination
Authors: Rummel John D.
Published: July 2002

These are intriguing times in the exploration of other
solar-system bodies. Continuing discoveries about life on Earth and the
return of data suggesting the presence of liquid water environments on
or under the surfaces of other planets and moons have combined to
suggest the significant possibility that extraterrestrial life may
exist in this solar system. Similarly, not since the Viking missions of
the mid-1970s has there been as great an appreciation for the potential
for Earth life to contaminate other worlds. Current plans for the
exploration of the solar system include constraints intended to prevent
biological contamination from being spread by solar-system exploration
missions.
Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues
Authors: Rousseau F. , Schymkowitz J. W. H. , Wilkinson H. R. , Itzhaki L. S.
Published: July 2002

p13suc1 has two native states, a monomer and a domain-swapped
dimer. We show that their folding pathways are connected by the
denatured state, which introduces a kinetic barrier between monomer and
dimer under native conditions. The barrier is lowered under conditions
that speed up unfolding, thereby allowing, to our knowledge for the
first time, a quantitative dissection of the energetics of domain
swapping. The monomer–dimer equilibrium is controlled by two conserved
prolines in the hinge loop that connects the exchanging domains. These
two residues exploit backbone strain to specifically direct dimer
formation while preventing higher-order oligomerization. Thus, the loop
acts as a loaded molecular spring that releases tension in the monomer
by adopting its alternative conformation in the dimer. There is an
excellent correlation between domain swapping and aggregation,
suggesting they share a common mechanism. These insights have allowed
us to redesign the domain-swapping propensity of suc1 from a fully
monomeric to a fully dimeric protein.
Initiating oncogenic event determines gene-expression patterns of human breast cancer models
Authors: Desai Kartiki V. , Xiao Nianqing , Wang Weili , Gangi Lisa , Greene John , Powell John I. , Dickson Robert , Furth Priscilla , Hunter Kent , Kucherlapati Raju , Simon Richard , Liu Edison T. , Green Jeffrey E.
Published: July 2002

Molecular expression profiling of tumors initiated by transgenic overexpression of
c-myc
,
c-neu
,
c-ha-ras
, polyoma middle T antigen (
PyMT
) or simian virus 40 T/t antigen (T-ag) targeted to the mouse mammary gland have identified both common and oncogene-specific events associated with tumor formation and progression. The tumors shared great similarities in their gene-expression profiles as compared with the normal mammary gland with an induction of cell-cycle regulators, metabolic regulators, zinc finger proteins, and protein tyrosine phosphatases, along with the suppression of some protein tyrosine kinases. Selection and hierarchical clustering of the most variant genes, however, resulted in separating the mouse models into three groups with distinct oncogene-specific patterns of gene expression. Such an identification of targets specified by particular oncogenes may facilitate development of lesion-specific therapeutics and preclinical testing. Moreover, similarities in gene expression between human breast cancers and the mouse models have been identified, thus providing an important component for the validation of transgenic mammary cancer models.
A theoretical examination of the factors controlling the catalytic efficiency of the DNA-(adenine-
N
6
)-methyltransferase from
Thermus aquaticus
Authors: Newby Zachary E. R. , Lau Edmond Y. , Bruice Thomas C.
Published: July 2002

Ab initio
and density functional calculations have been carried out to more fully understand the factors controlling the catalytic activity of the
Thermus aquaticus
DNA methyltransferase (M
Taq
I) in the
N
-methylation at the N
6
of an adenine nucleobase. The noncatalyzed reaction was modeled as a methyl transfer from trimethylsulfonium to the N
6
of adenine. Activation barriers of 32.0 kcal/mol and 24.0 kcal/mol were predicted for the noncatalyzed reaction in the gas phase by MP2/6–31+G(d,p)//HF/6–31+G(d,p) and B3LYP/6–31+G(d,p) calculations, respectively. Calculations performed to evaluate the effect of substrate positioning in the active site of M
Taq
I on the reaction determine the barrier to be 23.4 kcal/mol and 17.3 kcal/mol for the MP2/6–31+G(d,p)//HF/6–31+G(d,p) and B3LYP/6–31+G(d,p) gas phase calculations, respectively. The effect of hydrogen bonding between the N
6
of adenine and the terminal oxygen of Asn-105 on the activation barrier was also studied. A formamide molecule was modeled into the system to mimic the function of active site residue Asn-105. The activation barrier for this reaction was found to be 21.8 kcal/mol and 15.8 kcal/mol as determined from the MP2/6–31+G(d,p)//HF/6–31+G(d,p) and B3LYP/6–31+G(d,p) calculations, respectively. This result predicts a contribution of less than 2 kcal/mol to the lowering of the activation barrier from amide hydrogen bonding between formamide and N
6
of adenine. Comparison of the reaction coordinates suggest that it is not the hydrogen bonding of the Asn-105 that lends to the catalytic prowess of the enzyme since the organization of the substrates in the active site of the enzyme has a far greater effect on reducing the activation barrier. The results also suggest a stepwise mechanism for the removal of the hydrogen from the N
6
of adenine as opposed to a concerted reaction in which a proton is abstracted simultaneously with the transfer of the methyl group. The hydrogen on the N
6
of the intermediate methyl adenine product is far more acidic than in the reactant complex and may be subsequently abstracted by basic groups in the active site that are too weak to abstract the proton before the full sp
3
hybridization of the attacking nitrogen.
Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-α
Authors: Carter Percy H. , Scherle Peggy A. , Muckelbauer Jodi A. , Voss Matthew E. , Liu Rui-Qin , Thompson Lorin A. , Tebben Andrew J. , Solomon Kimberly A. , Lo Yvonne C. , Li Zhong , Strzemienski Paul , Yang Gengjie , Falahatpisheh Nikoo , Xu Meizhong , Wu Zhongren , Farrow Neil A. , Ramnarayan Kal , Wang Jing , Rideout Darryl , Yalamoori Venkatachalapathi , Domaille Peter , Underwood Dennis J. , Trzaskos James M. , Friedman Steven M. , Newton Robert C. , Decicco Carl P.
Published: July 2002

The binding of tumor necrosis factor alpha (TNF-α) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that
N
-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein–protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-α to TNFRc1 (IC
50
= 50 nM) and also blocked TNF-stimulated phosphorylation of Iκ-B in Ramos cells (IC
50
= 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 μM. Detailed evaluation of this and related molecules revealed that compounds in this class are “photochemically enhanced” inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (
ca.
40–100 μM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-α to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-α–TNFRc1 interaction.
Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation
Authors: Davis Benjamin B. , Thompson David A. , Howard Laura L. , Morisseau Christophe , Hammock Bruce D. , Weiss Robert H.
Published: July 2002

Atherosclerosis, in its myriad incarnations the foremost killer disease in the industrialized world, is characterized by aberrant proliferation of vascular smooth muscle (VSM) cells in part as a result of the recruitment of inflammatory cells to the blood vessel wall. The epoxyeicosatrienoic acids are synthesized from arachidonic acid in a reaction catalyzed by the cytochrome P450 system and are vasoactive substances. Metabolism of these compounds by epoxide hydrolases results in the formation of compounds that affect the vasculature in a pleiotropic manner. As an outgrowth of our observations that urea inhibitors of the soluble epoxide hydrolase (sEH) reduce blood pressure in spontaneously hypertensive rats as well as the findings of other investigators that these compounds possess antiinflammatory actions, we have examined the effect of sEH inhibitors on VSM cell proliferation. We now show that the sEH inhibitor 1-cyclohexyl-3-dodecyl urea (CDU) inhibits human VSM cell proliferation in a dose-dependent manner and is associated with a decrease in the level of cyclin D1. In addition,
cis
-epoxyeicosatrienoic acid mimics the growth-suppressive activity of CDU; there is no evidence of cellular toxicity or apoptosis in CDU-treated cells when incubated with 20 μM CDU for up to 48 h. These results, in light of the antiinflammatory and antihypertensive properties of these compounds that have been demonstrated already, suggest that the urea class of sEH inhibitors may be useful for therapy for diseases such as hypertension and atherosclerosis characterized by exuberant VSM cell proliferation and vascular inflammation.
Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.
Authors: Frey W , Schief W R , Pack D W , Chen C T , Chilkoti A , Stayton P , Vogel V , Arnold F H
Published: July 2002

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.
The replication initiator protein pi of the plasmid R6K specifically interacts with the host-encoded helicase DnaB.
Authors: Ratnakar P V , Mohanty B K , Lobert M , Bastia D
Published: July 2002

The replication initiator protein pi of plasmid R6K is known to interact with the seven iterons of the gamma origin/enhancer and activate distant replication origins alpha and beta (ori alpha and ori beta) by pi-mediated DNA looping. Here we show that pi protein specifically interacts in vitro with the host-encoded helicase DnaB. The site of interaction of pi on DnaB has been localized to a 37-aa-long region located between amino acids 151 and 189 of DnaB. The surface of pi that interacts with DnaB has been mapped to the N-terminal region of the initiator protein between residues 1 and 116. The results suggest that during initiation of replication, the replicative helicase DnaB is first recruited to the gamma enhancer by the pi protein. In a subsequent step, the helicase probably gets delivered from ori gamma to ori alpha and ori beta by pi-mediated DNA looping.
Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context.
Authors: Rasmussen P B , Holst B , Valentin-Hansen P
Published: July 2002

Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context. A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon. In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR. Thus, CRP functions either as an activator or as a corepressor. Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription. When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation. Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.
ORK1, a potassium-selective leak channel with two pore domains cloned from
Drosophila melanogaster
by expression in
Saccharomyces cerevisiae
Authors: Goldstein Steve A. N. , Price Laura A. , Rosenthal David N. , Pausch Mark H.
Published: July 2002

A K
+
channel gene has been cloned from
Drosophila melanogaster
by complementation in
Saccharomyces cerevisiae
cells defective for
K
+
uptake. Naturally expressed in the neuromuscular tissues
of adult flies, this gene confers K
+
transport capacity on
yeast cells when heterologously expressed. In
Xenopus
laevis
oocytes, expression yields an ungated
K
+
-selective current whose attributes resemble the
“leak” conductance thought to mediate the resting potential of
vertebrate myelinated neurons but whose molecular nature has long
remained elusive. The predicted protein has two pore (P) domains and
four membrane-spanning helices and is a member of a newly recognized
K
+
channel family. Expression of the channel in flies and
yeast cells makes feasible studies of structure and
in
vivo
function using genetic approaches that are not possible in
higher animals.
Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.
Authors: Lackmann M , Bucci T , Mann R J , Kravets L A , Viney E , Smith F , Moritz R L , Carter W , Simpson R J , Nicola N A , Mackwell K , Nice E C , Wilks A F , Boyd A W
Published: July 2002

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.
Binding of SecB to ribosome-bound polypeptides has the same characteristics as binding to full-length, denatured proteins
Authors: Randall Linda L. , Topping Traci B. , Hardy Simon J. S. , Pavlov Michael Y. , Freistroffer David V. , Ehrenberg Måns
Published: July 2002

The interaction of the chaperone SecB with ribosome-bound polypeptides that are in the process of elongation has been studied using an
in vitro
protein synthesis system. The binding is characterized by the same properties as those demonstrated for the binding of SecB to full-length proteins that are in nonnative conformation: it is readily reversible and has no specificity for the leader peptide. In addition, it is shown that the growing polypeptide chains must achieve a critical length to bind tightly enough to allow their isolation in complex with SecB. This explains the longstanding observation that, even when export is cotranslational, it begins late in synthesis. Furthermore, the required length is approximately the same as the length that defines the binding frame within denatured, full-length proteins bound to SecB.
Escherichia coli
DNA polymerase II catalyzes chromosomal and episomal DNA synthesis
in vivo
Authors: Rangarajan Savithri , Gudmundsson Gudmundur , Qiu Zhihao , Foster Patricia L. , Goodman Myron F.
Published: July 2002

We have investigated a role for
Escherichia coli
DNA polymerase II (Pol II) in copying chromosomal and episomal DNA in dividing cells
in vivo
. Forward mutation frequencies and rates were measured at two chromosomal loci,
rpoB
and
gyrA
, and base substitution and frameshift mutation frequencies were measured on an F′(
lacZ
) episome. To amplify any differences in polymerase error rates, methyl-directed mismatch repair was inactivated. When wild-type Pol II (
polB
+
) was replaced on the chromosome by a proofreading-defective Pol II exo
−
(
polBex1
), there was a significant increase in mutation frequencies to rifampicin resistance (Rif
R
) (
rpoB
) and nalidixic acid resistance (Nal
R
) (
gyrA
). This increased mutagenesis occurred in the presence of an antimutator allele of
E. coli
DNA polymerase III (Pol III) (
dnaE915
), but not in the presence of wild-type Pol III (
dnaE
+
), suggesting that Pol II can compete effectively with DnaE915 but not with DnaE
+
. Sequencing the Rif
R
mutants revealed a G → A hot spot highly specific to Pol II exo
−
. Pol II exo
−
caused a significant increase in the frequency of base substitution and frameshift mutations on F′ episomes, even in
dnaE
+
cells, suggesting that Pol II is able to compete with Pol III for DNA synthesis on F episomes.
Stepped versus continuous rotatory motors at the molecular scale
Authors: Sabbert Dirk , Junge Wolfgang
Published: July 2002

Nature invented molecular rotatory devices such as the flagellar motor and ATP synthase. Photoselection techniques have been frequently used to detect the rotational random walk of proteins but only rarely for the rotational drift of subunits in proteins. Pertinent theories predict an oscillatory behavior of the polarization anisotropy,
r
, for unidirectional rotational drift, as opposed to a monotonic relaxation of
r
for bidirectional random walk. The underlying assumption of an angular continuum is questionable for intersubunit rotation in proteins. We developed a theory for stepped rotatory devices. It predicts the damped oscillation of
r
under unidirectional drift. Damping increases with decreasing number of steps. For only three steps a quasi-monotonic relaxation of
r
is predicted for both random walk and drift. In photoselection experiments with active F-ATPase we observed the relaxation of
r
when a spectroscopic probe was attached to the central γ-subunit. This behavior is compatible with the expectation for a three-stepped rotatory device.
Alleviation of x-irradiation-based deficit in memory-based learning by
d
-amphetamine: Suggestions for attention deficit–hyperactivity disorder
Authors: Highfield D. A. , Hu D. , Amsel A.
Published: July 2002

Selective exposure to x-irradiation during infancy, from postnatal days (PND) 2–11 in the rat, results in severe hippocampal granule cell hypoplasia. Preweanling (PND 17–18) rats, which suffer such hippocampal granule-cell agenesis, show deficits in patterned single alternation (PSA), a form of memory-based learning. Deficits in short-term memory along with increased arousal have been suggested as characteristic of children diagnosed with attention deficit–hyperactivity disorder (ADHD). We report here on the ameliorating effects of
d
-amphetamine, a drug commonly used in the treatment of ADHD, before Ritalin, on PSA, after infantile (PND 2–15) exposure to x-irradiation. After i.p. injections of 0.3 mg/kg
d
-amphetamine, the onset and magnitude of the PSA memory-based discrimination in the x-irradiated preweanling rats was restored to about the level of controls. These results, showing alleviation of x-irradiation-related deficits in short-term memory by
d
-amphetamine injections, along with our earlier and present results, showing substantial deficits after x-irradiation alone, encourage the hypothesis that hippocampal granule-cell hypoplasia, which would occur in humans prenatally and is Altman’s model of “minimal brain dysfunction” [Altman, J. (1986) in
Learning Disabilities and Prenatal Risk
, ed. Lewis, M. (Univ. of Illinois Press, Urbana), pp. 241–304], may be a factor in at least some forms of ADHD and may provide a basis for an animal model of the disease.
A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis
Authors: Wentworth Paul , Liu Yunqi , Wentworth Anita D. , Fan Ping , Foley Matthew J. , Janda Kim D.
Published: July 2002

General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate
1
was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2′-hydroxyl group of the anhydroribitol phosphodiester substrate
2
. After murine immunization with hapten
1
, mAb production yielded a library of 35 antibodies that bound to a BSA-
1
conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester
2
. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25°C showed that the most active antibody, MATT.F-1, obeyed classical Michaelis–Menten kinetics with a
K
m
= 104 μM, a
k
cat
= 0.44 min
−
1
, and a
k
cat
/
k
uncat
= 1.7 × 10
3
. Hapten
1
stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody–catalyst (1.6 × 10
7
M
−
1
) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.
The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the
Escherichia coli
periplasm
Authors: Debarbieux Laurent , Beckwith Jon
Published: July 2002

Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of
Escherichia coli
. One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase. Thioredoxin 1 is kept in a reduced state by thioredoxin reductase. In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation
in vivo
. In the
Escherichia coli
periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD. In a
dsbD
null mutant, DsbC accumulates in the oxidized form. This oxidized form is then able to promote disulfide bond formation. In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves. Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the
E. coli
periplasm. In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a
dsbA
strain defective for disulfide bond formation. Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant. We conclude that the
in vivo
redox function of thioredoxin 1 depends on the redox environment in which it is localized.
Age-associated neuronal atrophy occurs in the primate brain and is reversible by growth factor gene therapy
Authors: Smith D. E. , Roberts J. , Gage F. H. , Tuszynski M. H.
Published: July 2002

The effects of normal aging on the primate brain are incompletely understood. Although both human and nonhuman primates demonstrate clear functional declines in selective attention, “executive” functions, and some components of declarative memory with aging, most studies have failed to demonstrate extensive neuronal
atrophy
or
loss
as a substrate for these degenerative changes in primates. In particular, extensive age-related neuronal loss in memory-related brain regions such as the hippocampus and entorhinal cortex has not been found. However, it is possible that neuronal loss or atrophy might occur in subcortical nuclei that modulate the activity of neocortical regions, thereby accounting for altered cognitive function with aging. In the present study, we describe, to our knowledge for the first time, a significant and extensive decline in the
number
and
size
of immunolabeled neurons in subcortical cholinergic basal forebrain regions of aged rhesus monkeys, the best animal model of human aging, by using stereological methods. Notably, the loss of subcortical cholinergic neuronal markers in aged monkeys was nearly completely reversed by human nerve growth factor gene delivery. These findings (
i
) identify
reversible
cellular atrophy as a potential mechanism contributing to age-related cognitive decline in primates, (
ii
) suggest, when considered with other studies, that subcortical brain regions exhibit greater vulnerability to the effects of aging than cortical regions, and (
iii
) indicate that neurotrophin gene transfer may be an effective means of preventing neuronal atrophy or degeneration in age-related neurodegenerative disorders.
Emergent mechanical properties of self-assembled polymeric capsules
Authors: Castellano Ronald K. , Clark Ross , Craig Stephen L. , Nuckolls Colin , Rebek Julius
Published: July 2002

Synthetic self-assembled systems combine responsiveness and
reversibility with the ability to perform chemical tasks such as
molecular recognition and catalysis. An unmet challenge is the
construction of polymeric materials that, like nature's tubulin, are
simultaneously reversible and capable of useful
physical
tasks. We report here a class of reversibly formed polymers that show
covalent-polymer mechanical integrity in solution and in the solid
state. Non-Newtonian, polymeric behavior is observed despite the low
molecular weight of the individual subunits and the seemingly weak
forces holding the assemblies together. These polymers assemble through
self-complementary hydrogen bonding and by physical encapsulation of
small molecules; accordingly, the emergent macroscopic structure and
function can be controlled by appropriate chemical signals.
Rearrangement and Expression of Immunoglobulin Light Chain Genes Can Precede Heavy Chain Expression during Normal B Cell Development in Mice
Authors: Novobrantseva Tatiana I. , Martin Verena M. , Pelanda Roberta , Müller Werner , Rajewsky Klaus , Ehlich Andreas
Published: July 2002

In mouse mutants incapable of expressing μ chains, VκJκ joints are detected in the CD43+ B cell progenitors. In agreement with these earlier results, we show by a molecular single cell analysis that 4–7% of CD43+ B cell progenitors in wild-type mice rearrange immunoglobulin (Ig)κ genes before the assembly of a productive VHDHJH joint. Thus, μ chain expression is not a prerequisite to Igκ light chain gene rearrangements in normal development. Overall, ∼15% of the total CD43+ B cell progenitor population carry Igκ gene rearrangements in wild-type mice. Together with the results obtained in the mouse mutants, these data fit a model in which CD43+ progenitors rearrange IgH and Igκ loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors VκJκ joining rapidly initiates κ chain expression, irrespective of the presence of a μ chain.
Differential Requirement for LAT and SLP-76 in GPVI versus T Cell Receptor Signaling
Authors: Judd Barbi A. , Myung Peggy S. , Obergfell Achim , Myers Erin E. , Cheng Alec M. , Watson Stephen P. , Pear Warren S. , Allman David , Shattil Sanford J. , Koretzky Gary A.
Published: July 2002

Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells. Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage. Both SLP-76 and LAT are important for optimal platelet activation downstream of the collagen receptor, GPVI. In addition, SLP-76 is involved in signaling mediated by integrin αIIbβ3. Because SLP-76 and LAT function coordinately in T cell signal transduction, yet their roles appear to differ in hemostasis, we investigated in detail the functional consequences of SLP-76 and LAT deficiencies in platelets. Previously we have shown that LAT−/− platelets exhibit defective responses to the GPVI-specific agonist, collagen-related peptide (CRP). Consistent with this, we find that surface expression of P-selectin in response to high concentrations of GPVI ligands is reduced in both LAT- and SLP-76–deficient platelets. However, platelets from LAT−/− mice, but not SLP-76−/− mice, aggregate normally in response to high concentrations of collagen and convulxin. Additionally, unlike SLP-76, LAT is not tyrosine phosphorylated after fibrinogen binding to integrin αIIbβ3, and collagen-stimulated platelets deficient in LAT spread normally on fibrinogen-coated surfaces. Together, these findings indicate that while LAT and SLP-76 are equally required for signaling via the T cell antigen receptor (TCR) and pre-TCR, platelet activation downstream of GPVI and αIIbβ3 shows a much greater dependency on SLP-76 than LAT.
Technical note: Check software for use with trans‐rectal ultrasound guided I‐125 seed prostate implants
Authors: Kermode R. H. , Locks S. M.
Published: July 2002

In‐house software has been developed to enable the dose distribution of a commercial treatment planning system to be verified prior to permanent trans‐rectal ultrasound guided I‐125 seed implantation of the prostate. Printouts enabling the required loading pattern to be communicated to other staff groups are also generated. This software can be obtained via the authors and the AAPM software exchange (URL: http://aapm.org/medphys/resources/software/index.htm).
Quantitative cardiac SPECT
Authors: Peace Richard A.
Published: July 2002

This thesis studied automated statistical mapping in myocardial perfusion SPECT to detect coronary artery disease (CAD). Registering myocardial studies to a 3D template allows an analysis on a voxel by voxel basis. Normal mean and standard deviation templates were created for each sex by registering 25 male and 25 female studies to a standard shape and position. A test group of 104 patients undergoing dipyridamole technetium‐99m tetrofosmin SPECT and angiography were used to assess the automated method. Patients were divided into those with angiographic evidence of CAD (n=56) and those without (n=48). The test studies were registered to the templates and count normalized by minimizing the sum of absolute differences. A Z‐score map of the statistical differences between registered study and template were calculated for all voxels within the myocardium. The contrast (Z‐score) and extent (number of voxels in a cluster exceeding the contrast threshold) thresholds for detection of CAD were optimized using receiver operating characteristic (ROC) analysis. The optimal thresholds resulted in a sensitivity of 73% and a specificity of 92% for automatic detection of CAD. The area under the fitted ROC curve (±1 SE) was 0.86±0.08 for a Z‐score contrast threshold of 5. The performance of this method and that of three experienced observers was compared by continuous ROC analysis. There was no statistically significant difference between the performances of the three observers and that of automatic detection in terms of the area under the ROC curves (p⩾0.25). The use of this automated statistical mapping approach shows a performance comparable with experienced observers, but avoids observer variability.