bluuebunny/crossref_metadata_2025 huggingface dataset:
An ab initio derived torsional potential energy surface for (H2O)3. II. Benchmark studies and interaction energies
Authors: Klopper Wim , Schütz Martin , Lüthi Hans P. , Leutwyler Samuel
Published: July 2002

A torsional potential energy surface for the cyclic water trimer was calculated at the level of second-order Mo/ller–Plesset perturbation theory. For the construction of this ab initio surface, the first-order wave function was expanded in a many-electron basis which linearly depends on the interelectronic coordinates r12. The one-electron basis of Gaussian orbitals was calibrated on the water monomer and dimer to ensure that the ab initio surface computed represents the (near- ) basis set limit for the level of theory applied. The positions of the free O—H bonds are described by three torsional angles. The respective three-dimensional torsional space was investigated by 70 counterpoise corrected single-point calculations for various values of these angles, providing a grid to fit an analytical representation of the potential energy surface. The four symmetry unique stationary points previously found at the Hartree–Fock and conventional Mo/ller–Plesset levels [Schütz et al., J. Chem. Phys. 99, 5228 (1993)] were studied in detail: Relative energies of the structures were calculated by applying second-order Mo/ller–Plesset and coupled cluster methods; harmonic vibrational frequencies were calculated at the second-order Mo/ller–Plesset level with a 6-311++G(d,p) basis set at these stationary points. It is expected that the present torsional potential energy surface for the water trimer will play an important role in the understanding of the vibrational transitions observed by far-infrared vibration–rotation–tunneling spectroscopy in terms of a nearly free pseudorotational interconversion on a cyclic vibrational–tunneling path.
Concerning the applicability of density functional methods to atomic and molecular negative ions
Authors: Galbraith John Morrison , Schaefer Henry F.
Published: July 2002

There is concern within theoretical chemistry that density functional methods are fundamentally in error for negative ions. We have tested this hypothesis by evaluating electron affinities for F and F2 with a variety of density functionals and extremely large, diffuse basis sets. In addition, we have observed the behavior of a known unbound system Ne−. We have found no convincing evidence to support the claims of negative ion instability with density functional methods.
Two-photon dissociation and ionization of liquid water studied by femtosecond transient absorption spectroscopy
Authors: Thomsen C. L. , Madsen D. , Keiding S. R. , Tho/gersen J. , Christiansen O.
Published: July 2002

The photodissociation and photoionization of liquid water following two-photon absorption at 266 nm is studied in the spectral range from 213 to 1108 nm with subpicosecond time resolution. Probing in the UV enables the first direct simultaneous observation of the photoproducts eaq−, Haq, and OHaq. This makes it possible to follow the geminate recombination kinetics between the photoproducts and to determine the relative yields of the dissociation and ionization channels. The concentration of hydrated electrons deduced from the visible and near-infrared transient absorption measurements decays by 40%±2% within the first 90 ps due to recombination with OHaq and H3O+. Analyzing our measurements of the hydrated electron concentration using the independent reaction time approximation results in the relative yields of 82%±3% and 18%±3% for recombination with OHaq and with H3O+, respectively. This is in excellent agreement with the relative yield of 82%±10% for recombination with OHaq determined directly from our ultraviolet transient absorption measurements. The contribution of hydrated electrons from direct ionization is insignificant when liquid water is excited below 9.32 eV and the ionization is likely to occur via dissociation and proton transfer. The transient ultraviolet absorption data shows that if ionization exclusively occurs via dissociation, 65% of the produced H(hot) atoms react with the surrounding solvent molecules to produce hydrated electrons. If proton transfer, on the other hand, is the only process responsible for the ionization, our measurements show that the ratio between dissociation and ionization is 55%. Geminate recombination of OHaq and Haq fragments following the photodissociation at 9.32 eV is not observed indicating that the translation energy of at least one of the fragments is sufficient to penetrate the water solvent cage. Finally, we have measured the two-photon absorption spectrum of liquid water from 110 to 160 nm, and the spectrum is in good agreement with our ab initio gas-phase calculations of the two-photon absorption cross sections for the transitions involved.
Theoretical study of polymeric mixtures with different sequence statistics. I. Ising class: Linear random copolymers with different statistical sequences and ternary blends of linear random copolymers with homopolymers
Authors: Qi Shuyan , Chakraborty Arup K.
Published: July 2002

We derive a Landau free energy functional for polymeric mixtures containing components with different sequence statistics. We then apply this general field theory to two mixtures that belong to the Ising universality class: mixtures of two different linear random copolymers, and ternary systems of linear random copolymers and two homopolymers. We discuss the instability conditions for the homogeneous state of these mixtures, and calculate the structure factors for different components in the homogeneous state. The structure factors show interesting features which can directly be compared with scattering experiments carried out with selectively deuterated samples. We also work out the eigenmodes representing the least stable concentration fluctuations for these mixtures. The nature of these concentration fluctuations provides information regarding the ordered phases and the kinetic pathways that lead to them. We find various demixing modes for different characteristics of the two mixtures (e.g., average compositions, statistical correlation lengths, and volume fractions).
The high-frequency limit in general relativity
Authors: Burnett Gregory A.
Published: July 2002

A precise characterization of the high-frequency limit in general relativity (vacuum space-times) is presented. The averaging schemes used in earlier works are avoided by the use of weak limits, giving the characterization the advantage that the high-frequency limit can be rigorously analyzed. Using this characterization, a theorem to the effect that a certain ‘‘effective stress-energy’’ tensor acts as a source of curvature of the smooth background space-time is proved. It is shown in the special case corresponding to the presence of a single wave that this tensor has the same form as the stress-energy tensor of a null fluid. Also, the extension of this characterization of the high-frequency limit to other fields, such as electromagnetic fields, and to nonvacuum space-times is briefly discussed.
Relativistic Gamow vectors
Authors: Antoniou I. , Gadella M. , Prigogine I. , Pronko G. P.
Published: July 2002

The Friedrichs model has often been used in order to obtain explicit formulas for eigenvectors associated to complex eigenvalues corresponding to lifetimes. Such eigenvectors are called Gamow vectors and they acquire meaning in extensions of the conventional Hilbert space of quantum theory to the so-called rigged Hilbert space. In this paper, Gamow vectors are constructed for a solvable model of an unstable relativistic field. As a result, we obtain a time asymmetric relativistic extension of the Fock space. This extension leads to two distinct Poincaré semigroups. The time reversal transformation maps one semigroup to the other. As a result, the usual PCT invariance should be extended. We show that irreversibility as expressed by dynamical semigroups is compatible with the requirements of relativity.
The century of the incomplete revolution: Searching for general relativistic quantum field theory
Authors: Rovelli Carlo
Published: July 2002

In fundamental physics, this has been the century of quantum mechanics and general relativity. It has also been the century of the long search for a conceptual framework capable of embracing the astonishing features of the world that have been revealed by these two “first pieces of a conceptual revolution.” The general requirements on the mathematics and some specific developments toward the construction of such a framework are discussed. Examples of covariant constructions of (simple) generally relativistic quantum field theories have been obtained as topological quantum field theories, in nonperturbative zero-dimensional string theory and its higher-dimensional generalizations, and as spin foam models. A canonical construction of a general relativistic quantum field theory is provided by loop quantum gravity. Remarkably, all these diverse approaches have turned out to be related, suggesting an intriguing general picture of general relativistic quantum physics.
Classification of states and macroscopic degeneracy in an open XY-chain in transverse field
Authors: Loginov A. V. , Pereverzev Yu. V.
Published: July 2002

The spectral structure for an open X–Y chain is determined rigorously, and one-fermion states are classified for all values of anisotropy and transverse field. Quasidegeneracy is interpreted as well as the general nature of ordering in a certain class of systems with a locally nondegenerate ground state.
Transitions toward turbulence in a curved channel
Authors: Bland S. B. , Finlay W. H.
Published: July 2002

A numerical study of the transitions that occur with increasing Reynolds number in a curved channel with radius ratio η=0.875 is performed using spectral simulations of the three-dimensional, incompressible, time-dependent Navier–Stokes equations. Periodic boundary conditions are used in the spanwise and streamwise directions. At Reynolds number Re=6.31 Rec temporally periodic wavy (twisting) Dean vortices occur (Rec is the Reynolds number for the transition from laminar curved channel Poiseuille flow to steady, streamwise-oriented Dean vortices). At Re=8.84 Rec, a three-frequency flow is discovered in which two new incommensurate frequencies modulate the wavy vortices. At Re=10.10 Rec the two modulation frequencies are phase locked producing a two-frequency modulated wavy vortex flow that is similar in some ways to that seen in Taylor–Couette flow. The spatial and temporal characteristics of the modulation frequencies are discussed.
Tracer dispersion in porous media with a double porosity
Authors: Magnico P. , Leroy C. , Bouchaud J. P. , Gauthier C. , Hulin J. P.
Published: July 2002

Tracer dispersion in double porosity packings of porous grains obtained by grinding sintered glass samples of various porosities and permeabilities is studied experimentally. The dispersivity lD increases faster with velocity than for single porosity packings: this variation is predicted by modeling the medium as a one-dimensional (1-D) sequence of identical cells with one slow and one fast path in parallel. The grain size and the permeability contrast between the inside and the outside of the grains can also be determined. At low grain porosities, the transit time τi inside individual grains becomes of the order of the mean transit time across the whole sample and non-Gaussian dispersion curves are observed.
Excitation of lower-hybrid instability by longitudinal currents
Authors: Satyanarayana P. , Chaturvedi P. K.
Published: July 2002

The stability of a homogeneous plasma with currents along the magnetic field is investigated. It is found that electrons streaming along the magnetic field lines in a neutralizing positive ion background could excite lower-hybrid instability. The critical drift velocity should exceed the parallel phase velocity for instability: Vd>ωr/k∥, where ωr is the lower-hybrid frequency (ΩeΩi)1/2. This condition is found to be easily met in the laboratory and space plasmas and is applicable both in the collisionless and collisional palsmas.
Reply to comments of Hill
Authors: Paxton A. H. , Baker Louis , Gardner R. L.
Published: July 2002

Hill notes that the calculated energy loss rate for a lightning channel found in the original paper [Phys. Fluids 29, 2736 (1986)] is significantly smaller than the rate he obtained. This is explained primarily by his much smaller electrical conductivity. The value obtained in the original paper is justified and other features of the results are clarified.
Particle and fluid simulations of resistive current-driven electrostatic ion cyclotron waves
Authors: Seyler Charles E. , Providakes Jason
Published: July 2002

The results from 1-D numerical simulations of electrostatic ion cyclotron waves (EIC) are presented for a model in which the electrons are a resistive (collisional) fluid. Simulations of both the kinetic and fluid descriptions are performed and compared in order to assess the fundamental limitations of fluid theory for EIC waves. The effect of ion–neutral collisions is also included using a simple Monte Carlo technique. It is found that a small ion–neutral collision frequency destroys the frequency harmonic coupling of kinetic EIC waves and tends to validate the fluid description. The saturation amplitude of the current driven EIC instability is in agreement with recent laboratory experiments. The coherent nature (extremely narrow spectral width) and phase velocity agree with ground based (coherent backscatter radars) and in situ observations of current-driven EIC waves in the high latitude ionosphere.
Alkaline-mediated differential interaction (AMDI): A simple automatable single-nucleotide polymorphism assay
Authors: Bartlett Sylvia , Straub Josef , Tonks Susan , Wells Rush Spencer , Bodmer Julia G. , Bodmer Walter F.
Published: July 2002

The key requirements for high-throughput single-nucleotide
polymorphism (SNP) typing of DNA samples in large-scale disease
case-control studies are automatability, simplicity, and robustness,
coupled with minimal cost. In this paper we describe a fluorescence
technique for the detection of SNPs that have been amplified by using
the amplification refractory mutation system (ARMS)-PCR procedure. Its
performance was evaluated using 32 sequence-specific primer mixes to
assign the HLA-DRB alleles to 80 lymphoblastoid cell line DNAs chosen
from our database for their diversity. All had been typed previously by
alternative methods, either direct sequencing or gel electrophoresis.
We believe the detection system that we call AMDI (alkaline-mediated
differential interaction) satisfies the above criteria and is suitable
for general high-throughput SNP typing.
Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography
Authors: Marsh Brad J. , Mastronarde David N. , Buttle Karolyn F. , Howell Kathryn E. , McIntosh J. Richard
Published: July 2002

The positional relationships among all of the visible organelles in
a densely packed region of cytoplasm from an insulin secreting,
cultured mammalian cell have been analyzed in three dimensions (3-D) at
≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15
cell that included a large portion of the Golgi ribbon was
reconstructed in 3-D by electron tomography. The reconstructed volume
(3.1 × 3.2 × 1.2 μm
3
) allowed sites of
interaction between organelles, and between microtubules and organellar
membranes, to be accurately defined in 3-D and quantitatively analyzed
by spatial density analyses. Our data confirm that the Golgi in an
interphase mammalian cell is a single, ribbon-like organelle composed
of stacks of flattened cisternae punctuated by openings of various
sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979)
Am.
J. Anat.
154, 455–476]. The data also show that the
endoplasmic reticulum (ER) is a single continuous compartment that
forms close contacts with mitochondria, multiple trans Golgi cisternae,
and compartments of the endo-lysosomal system. This ER traverses the
Golgi ribbon from one side to the other via cisternal openings.
Microtubules form close, non-random associations with the cis Golgi,
the ER, and endo-lysosomal compartments. Despite the dense packing of
organelles in this Golgi region, ≈66% of the reconstructed volume is
calculated to represent cytoplasmic matrix. We relate the intimacy of
structural associations between organelles in the Golgi region, as
quantified by spatial density analyses, to biochemical mechanisms for
membrane trafficking and organellar communication in mammalian cells.
The interaction between the coactivator dCBP and Modulo, a chromatin-associated factor, affects segmentation and melanotic tumor formation in
Drosophila
Authors: Bantignies Frédéric , Goodman Richard H. , Smolik Sarah M.
Published: July 2002

The development of
Drosophila
requires the function of the CREB-binding protein, dCBP. In flies, dCBP serves as a coactivator for the transcription factors Cubitus interruptus, Dorsal, and Mad, and as a cosuppressor of
Drosophila
T cell factor. Current models propose that CBP, through its intrinsic and associated histone acetyltransferase activities, affects transient chromatin changes that allow the preinitiation complex to access the promoter. In this report, we provide evidence that dCBP may regulate the formation of chromatin states through interactions with the
modulo
(
mod
) gene product, a protein that is thought to be involved in chromatin packaging. We demonstrate that dCBP and Modulo bind
in vitro
and
in vivo
, that mutations in
mod
enhance the embryonic phenotype of a dCBP mutation, and that dCBP mutations enhance the melanotic tumor phenotype characteristic of
mod
homozygous mutants. These results imply that, in addition to its histone acetyltransferase activity, dCBP may affect higher-order chromatin structure.
Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms
Authors: Sun Hong , Taneja Reshma
Published: July 2002

Stra13 is a transcriptional repressor related within its basic helix–loop–helix domain with the
Drosophila
Hairy, Enhancer of Split, and the mouse Hes1 proteins that interact with the corepressor Groucho. Because Stra13 lacks the conserved WRPW motif for interaction with Groucho, we examined the function and mechanism of transcriptional repression mediated by Stra13 that exhibits several distinctive features. Here, we report that Stra13 expression is closely associated with cell growth arrest induced by several triggers such as retinoic acid and trichostatin A (TSA; a specific histone deacetylase inhibitor) as well as by serum starvation. Stra13 expression is transcriptionally repressed and maintained at a low level in cells through a negative autoregulatory mechanism that is brought about by its interaction with the corepressor histone deacetylase (HDAC1). This interaction requires the Stra13 C-terminal domain containing three α-helices, which are also functionally critical to its repressive activity. Thus, inhibition of HDAC activity by TSA abrogates Stra13-mediated repression of its promoter, resulting in induction of Stra13 expression that is coincident with TSA-induced growth arrest. Further, once induced, Stra13 strongly represses the expression of the cell proliferation-associated gene c-Myc through an HDAC1-independent pathway that involves its interaction with the basal transcription factor TFIIB. Our studies indicate that Stra13 may play a key role in signaling pathways that lead to growth arrest and terminal differentiation by repression of target genes via HDAC-dependent and HDAC-independent mechanisms.
Nebular and auroral emission lines of [Cl
iii
] in the optical spectra of planetary nebulae
Authors: Keenan Francis P. , Aller Lawrence H. , Ramsbottom Catherine A. , Bell Kenneth L. , Crawford Fergal L. , Hyung Siek
Published: July 2002

Electron impact excitation rates in Cl III, recently determined
with the R-matrix code, are used to calculate electron temperature
(
T
e
) and density
(
N
e
) emission line ratios involving both the
nebular (5517.7, 5537.9 Å) and auroral (8433.9, 8480.9, 8500.0 Å)
transitions. A comparison of these results with observational data for
a sample of planetary nebulae, obtained with the Hamilton Echelle
Spectrograph on the 3-m Shane Telescope, reveals that the
R
1
=
I
(5518
Å)/
I
(5538 Å) intensity ratio provides estimates of
N
e
in excellent agreement with the values
derived from other line ratios in the echelle spectra. This agreement
indicates that
R
1
is a reliable density
diagnostic for planetary nebulae, and it also provides observational
support for the accuracy of the atomic data adopted in the line ratio
calculations. However the [Cl
iii
] 8433.9 Å line is
found to be frequently blended with a weak telluric emission feature,
although in those instances when the [Cl
iii
] intensity
may be reliably measured, it provides accurate determinations of
T
e
when ratioed against the sum of the 5518
and 5538 Å line fluxes. Similarly, the 8500.0 Å line, previously
believed to be free of contamination by the Earth's atmosphere, is
also shown to be generally blended with a weak telluric emission
feature. The [Cl
iii
] transition at 8480.9 Å is found to
be blended with the He
i
8480.7 Å line, except in
planetary nebulae that show a relatively weak He
i
spectrum, where it also provides reliable estimates of
T
e
when ratioed against the nebular lines.
Finally, the diagnostic potential of the near-UV [Cl
iii
]
lines at 3344 and 3354 Å is briefly discussed.
Induction of CD4+T cell-dependent CD8+type 1 responses in humans by a malaria DNA vaccine
Authors: Wang Ruobing , Epstein Judith , Baraceros Fe Maria , Gorak Edward J. , Charoenvit Yupin , Carucci Daniel J. , Hedstrom Richard C. , Rahardjo Nancy , Gay Tanya , Hobart Peter , Stout Rick , Jones Trevor R. , Richie Thomas L. , Parker Suezanne E. , Doolan Denise L. , Norman Jon , Hoffman Stephen L.
Published: July 2002

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-γ responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8+and CD4+T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8+-dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-γ responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.
Fully competent dendritic cells as inducers of T cell anergy in autoimmunity
Authors: Quaratino Sonia , Duddy Linda P. , Londei Marco
Published: July 2002

Mature immunologically competent dendritic cells are the most efficient antigen-presenting cells that powerfully activate T cells and initiate and sustain immune responses. Indeed, dendritic cells are able to efficiently capture antigens, express high levels of costimulatory molecules, and produce the combination of cytokines required to create a powerful immune response. They are also considered to be important in initiating autoimmune disease by efficiently presenting autoantigens to self-reactive T cells that, in this case, will mount a pathogenic autoimmune reaction. Triggering T cells is not a simple on–off procedure, as T cell receptor responds to minor changes in ligand with gradations of T cell activation and effector functions. These “misfit” peptides have been called Altered Peptide Ligands, and have been shown to have important biological significance. Here, we show that fully capable dendritic cells may present, upon natural antigen processing, a self-epitope with Altered Peptide Ligands features that can unexpectedly induce anergy in a human autoreactive T cell clone. These results indicate that presentation of a self-epitope by immunologically competent dendritic cells does not always mean “danger” and show a mechanism involved in the fine balance between activation and tolerance induction in humans.
Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of
Mertk
Authors: Vollrath Douglas , Feng Wei , Duncan Jacque L. , Yasumura Douglas , D'Cruz Patricia M. , Chappelow Aimee , Matthes Michael T. , Kay Mark A. , LaVail Matthew M.
Published: July 2002

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene
Mertk
likely corresponds to the retinal dystrophy (
rdy
) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of
Mertk
to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat
Mertk
to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of
Mertk
underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of
MERTK
mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.
Engineering a nicking endonuclease N.
Alw
I by domain swapping
Authors: Xu Yan , Lunnen Keith D. , Kong Huimin
Published: July 2002

Changing enzymatic function through genetic engineering still presents a challenge to molecular biologists. Here we present an example in which changing the oligomerization state of an enzyme changes its function. Type IIs restriction endonucleases such as
Alw
I usually fold into two separate domains: a DNA-binding domain and a catalytic/dimerization domain. We have swapped the putative dimerization domain of
Alw
I with a nonfunctional dimerization domain from a nicking enzyme, N.
Bst
NBI. The resulting chimeric enzyme, N.
Alw
I, no longer forms a dimer. Interestingly, the monomeric N.
Alw
I still recognizes the same sequence as
Alw
I but only cleaves the DNA strand containing the sequence 5′-GGATC-3′ (top strand). In contrast, the wild-type
Alw
I exists as a dimer in solution and cleaves two DNA strands; the top strand is cleaved by an enzyme binding to that sequence, and its complementary bottom strand is cleaved by the second enzyme dimerized with the first enzyme. N.
Alw
I is unable to form a dimer and therefore nicks DNA as a monomer. In addition, the engineered nicking enzyme is at least as active as the wild-type
Alw
I and is thus a useful enzyme. To our knowledge, this is the first report of creating a nicking enzyme by domain swapping.
Hydrated myoglobin's anharmonic fluctuations are not primarily due to dihedral transitions.
Authors: Steinbach P J , Brooks B R
Published: July 2002

To characterize the functionally important anharmonic motions of proteins, simulations of carboxymyoglobin (MbCO) dynamics have been performed during which dihedral transitions were prohibited. Comparison of torsionally restrained and unrestrained protein dynamics simulated at three levels of hydration and at temperatures ranging from 100 to 400 K suggests that hydration "catalyzes" protein mobility by facilitating collective anharmonic motions that do not require dihedral transitions. When dihedral transitions were prohibited, dehydrated MbCO, to a good approximation, exhibited only harmonic fluctuations, whereas hydrated MbCO exhibited both harmonic and anharmonic motions. The fluctuation of helix centers of mass also remained highly anharmonic in the torsionally restrained hydrated system. Atomic mean-square fluctuation at 300 K was reduced upon prohibition of dihedral transitions by only 28% and 10% for MbCO hydrated by 350 and 3830 water molecules, respectively.
Malignant conversion of chemically transformed normal human cells.
Authors: Milo G E , Li D , Casto B C , Theil K , Shuler C , Noyes I , Chen J
Published: July 2002

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.
Pattern of gp120 sequence divergence linked to a lack of clinical progression in human immunodeficiency virus type 1 infection.
Authors: Wang W K , Essex M , McLane M F , Mayer K H , Hsieh C C , Brumblay H G , Seage G , Lee T H
Published: July 2002

Differential rates of AIDS development and/or T4 lymphocyte depletion in HIV-1-infected individuals remain unexplained. The hypothesis that qualitative differences in selection pressure in vivo may account for different rates of disease progression was addressed in nine eligible study participants from a cohort of 315 homosexual men who have been followed since 1985. Disproportionately fewer changes in variable regions and more in C3 of gp12O were found to be significantly associated with slower disease progression. Our finding provides the first example to demonstrate that differential selection pressure related to the emergence of HIV-1 variants is associated with long term nonprogression. Candidate vaccines that elicit strong selection pressure on C3 of gp120 are likely to provide better protection than those targeting variable regions.
Evidence for a novel DNA damage binding protein in human cells.
Authors: Ghosh R , Peng C H , Mitchell D L
Published: July 2002

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.
Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.
Authors: Kuznetsov G , Bush K T , Zhang P L , Nigam S K
Published: July 2002

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.
Deoxycytidine kinase and deoxyguanosine kinase of
Lactobacillus acidophilus
R-26 are colinear products of a single gene
Authors: Ma Ning , Ikeda Seiichiro , Guo Shenyuan , Fieno Angela , Park Inshik , Grimme Stephen , Ikeda Takehisa , Ives David H.
Published: July 2002

Three of the four deoxynucleoside kinases required for growth of
Lactobacillus acidophilus
R-26 exist as heterodimeric
pairs specific for deoxyadenosine (dAK) and deoxycytidine (dCK) or dAK
and deoxyguanosine (dGK). However, only two tandem genes,
dak/dgk
, are found, and are expressed only as
dAK/dGK in transformed
Escherichia coli
. Sequencing
peptides spanning 63% of the native dCK subunit revealed a sequence
identical to that deduced from
dgk
(beginning
MTVIVL···), except that dCK lacks residues 2 and 3 (dCK
is M··IVL; dGK is ·TVIVL). Also, mass spectrometry
indicates that native dCK and dGK subunits are identical in mass
adjusted for the first three residues. Furthermore, the native enzymes
have identical isoelectric pH values, indicating an equal number of
charged residues. To enable
E. coli
to express peptide
having the native dCK sequence, codons 2 and 3 were deleted from the
dgk
portion of the tandem genes, resulting in expression
of protein having the specificities and regulatory properties of native
dAK/dCK, including heterotropic stimulation of dAK activity by
deoxycytidine or dCTP (not deoxyguanosine or dGTP) and end-product
inhibition of the respective activities by dATP and dCTP. Subcloning
normal and mutant
dgk
yielded homodimeric dGK and dCK,
respectively. The dCK homodimer strongly resembles human dCK, with a
low
K
m
for deoxycytidine, the ability to
phosphorylate deoxyadenosine and deoxyguanosine at much higher
K
m
values, and end-product inhibition by
dCTP. Thus two distinct and specific enzymes evidently are derived from
a single
Lactobacillus
gene. The mechanism by which this
occurs
in vivo
has yet to be elucidated.
Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution
Authors: Provance D. William , Wei Michael , Ipe Vinu , Mercer John A.
Published: July 2002

Mutant alleles at the dilute unconventional myosin heavy chain
locus cause diluted coat color, opisthotonic seizures, and death. The
dilute coat color phenotype is caused by irregular clumping of pigment
in the hair, but amounts of melanin are unchanged from wild-type
controls. The melanocyte phenotype has been described as adendritic,
since hair bulb and Harderian gland melanocytes appear to be rounded in
tissue sections. These observations do not exclude the possibility that
the processes lack pigment, since the melanocyte shape was judged by
the distribution of melanin. We have tested this hypothesis by
culturing primary melanocytes from dilute mutant and wild-type mice.
The mutant melanocytes do not lack processes; instead, they exhibit a
concentrated perinuclear distribution of melanosomes, while wild-type
melanocytes have a very uniform cytoplasmic distribution of
melanosomes. Electron micrographs show no detectable differences in
melanosome morphology or maturation between dilute and wild-type
melanocytes. Immunofluorescence experiments indicate that the dilute
protein is concentrated in regions of the cytoplasm that contain
melanosomes. These experiments show that the dilute myosin is necessary
for the localization of melanosomes, either by active transport or
tethering.
Preserved speech abilities and compensation following prefrontal damage.
Authors: Buckner R L , Corbetta M , Schatz J , Raichle M E , Petersen S E
Published: July 2002

Lesions to left frontal cortex in humans produce speech production impairments (nonfluent aphasia). These impairments vary from subject to subject and performance on certain speech production tasks can be relatively preserved in some patients. A possible explanation for preservation of function under these circumstances is that areas outside left prefrontal cortex are used to compensate for the injured brain area. We report here a direct demonstration of preserved language function in a stroke patient (LF1) apparently due to the activation of a compensatory brain pathway. We used functional brain imaging with positron emission tomography (PET) as a basis for this study.
Genetic evidence that the RAG1 protein directly participates in V(D)J recombination through substrate recognition.
Authors: Roman C A , Baltimore D
Published: July 2002

RAG1 protein is essential for the activation of V(D)J recombination in developing lymphocytes (V, variable; D, diversity; J, joining). However, it has not been determined whether its role involves substrate recognition and catalysis. A single amino acid substitution mutation in the RAG1 gene has now been identified that renders its activity sensitive to the sequence of the coding region abutting the heptamer site in the recombination signal sequence. These results strongly imply that RAG1 interacts directly with DNA.
Photocarcinogenesis and inhibition of intercellular adhesion molecule 1 expression in cells of DNA-repair-defective individuals
Authors: Ahrens Constanze , Grewe Markus , Berneburg Mark , Grether-Beck Susanne , Quilliet Xavier , Mezzina Mauro , Sarasin Alain , Lehmann Alan R. , Arlett Colin F. , Krutmann Jean
Published: July 2002

Cells from patients with xeroderma pigmentosum complementation group D (XP-D) and most patients with trichothiodystrophy (TTD) are deficient in excision repair of ultraviolet (UV) radiation-induced DNA damage. Although in both syndromes this defect is based on mutations in the same gene,
XPD
, only XP-D, not TTD, individuals have an increased risk of skin cancer. Since the reduction in DNA repair capacity is similar in XP-D and TTD patients, it cannot account for the difference in skin cancer risk. The features of XP-D and TTD might therefore be attributable to differences in the immune response following UV-irradiation, a factor which is presumed to be important for photocarcinogenesis. We have measured the capacity of UVB radiation to inhibit expression of the immunological key molecule intercellular adhesion molecule 1 (ICAM-1) in cells from three healthy individuals in comparison to cells from three XP-D and three TTD patients. Cells from XP-D patients, but not from TTD patients, exhibited an increased susceptibility to UVB radiation-induced inhibition of ICAM-1 expression. Transfection of XP-D cells with the wild-type
XPD
cDNA, but not with
XPC
cDNA, corrected this abnormal phenotype. Thus, the skin cancer risk in DNA repair-defective individuals correlated with the susceptibility of their cells to UVB radiation-induced inhibition of ICAM-1 expression, rather than with their defect in DNA repair. The XPD protein has dual roles: in DNA repair and transcription. The transcriptional role might be important for the control of expression of immunologically relevant genes and thereby contribute to the skin cancer risk of a DNA-repair-deficient individual.
X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase
Authors: Xu Yingwu , Sellam Olivier , Moréra Solange , Sarfati Simon , Biondi Ricardo , Véron Michel , Janin Joël
Published: July 2002

To be effective as antiviral agent, AZT (3′-azido-3′-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-Å x-ray structure of a complex with the N119A point mutant of
Dictyostelium
NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3′OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the β- and γ-phosphate, which plays an important part in phosphate transfer.
Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate
Authors: Chow Ming , Rubin Harry
Published: July 2002

A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20–50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9–12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD/D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD/D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of confluence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.
Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state
Authors: Sugiura Isamu , Furie Bruce , Walsh Christopher T. , Furie Barbara C.
Published: July 2002

The vitamin K-dependent γ-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to γ-carboxyglutamic acid in precursor proteins containing the γ-carboxylation recognition site (γ-CRS). During this reaction, glutamic acid is converted to γ-carboxyglutamic acid while vitamin KH
2
is converted to vitamin K 2,3-epoxide. Recombinant bovine carboxylase was purified free of γ-CRS-containing propeptide and endogenous substrate in a single-step immunoaffinity procedure. We show that in the absence of γ-CRS-containing propeptide and/or glutamate-containing substrate, carboxylase has little or no epoxidase activity. Epoxidase activity is induced by Phe-Leu-Glu-Glu-Leu (FLEEL) (9.2 pmol per min per pmol of enzyme), propeptide, residues −18 to −1 of proFactor IX (3.4 pmol per min per pmol of enzyme), FLEEL and propeptide (100 pmol per min per pmol of enzyme), and proPT28 (HVFLAPQQARSLLQRVRRANTFLEEVRK, residues −18 to +10 of human acarboxy-proprothrombin), (5.3 pmol per min per pmol of enzyme). These results indicate that in the absence of propeptide or glutamate-containing substrate, oxygenation of vitamin K by the carboxylase does not occur. Upon addition of propeptide or glutamate-containing substrate, the enzyme is converted to an active epoxidase. This regulatory mechanism prevents the generation of a highly reactive vitamin K intermediate in the absence of a substrate for carboxylation.