bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of murine hepatocyte death by membrane-bound CD95 (Fas/APO-1)-ligand: Characterization of an in Vitro system
Authors: Schlosser Stephan F. , Azzaroli Francesco , Dao Tao , Hingorani Ravi , Crispe Nicholas I. , Boyer James L.
Published: December 2003

Hepatocytes constitutively express CD95 (also called Fas/APO-1) and are therefore potential targets for CD95-ligand (CD95L)-mediated injury. To study this mechanism of cell death in hepatocytes we developed an in vitro model of liver cell apoptosis using membrane-bound CD95L as the inducing agent. Primary mouse hepatocytes were cocultured with NIH 3T3 fibroblasts, stably transfected with mouse CD95L (FCD95L+). Fibroblasts stably transfected with vector only (FCD95L−) served as controls. Hepatocytes from mice expressing low levels of CD95 (Faslprmice) served as controls for effects unrelated to CD95. Morphologic and biochemical studies indicate that CD95 is expressed in cultured mouse hepatocytes. Membrane-bound CD95 from transfected fibroblasts destroyed all cocultured hepatocytes within 24 hours in the absence of protein synthesis inhibitors. Characteristic features of apoptosis were observed in dying hepatocytes and occurred in the following sequence: formation of cytoplasmic blebs and nuclear condensation after 3 hours; nuclear fragmentation and DNA strand breaks after 4 hours. These changes were observed only when normal hepatocytes were cocultured with FCD95L+ and were not observed with FCD95L− or in hepatocytes from Faslpr mice. Anti-CD95 antibody (Jo2) evoked similar changes in hepatocytes, although to a much lesser extent. We conclude that coculture of mouse hepatocytes with FCD95L+ is a useful in vitro model for CD95-mediated apoptosis induced by CD95L. The high incidence of apoptosis caused by membrane-bound CD95L differs from the much smaller effects induced by the Jo2 antibody. In view of the high sensitivity of hepatocytes towards CD95L we speculate that CD95L-induced liver damage in vivo may be minimized by restricting exposure of hepatocytes to CD95L.
A loss of memory in stratified momentum wakes
Authors: Meunier Patrice , Spedding Geoffrey R.
Published: December 2003

In this paper we compare the wakes of various bluff bodies in a stratified fluid at moderately high Froude numbers (F≡2UB/ND>8) and Reynolds numbers (Re≈5000). The size and amplitude of the long-lasting wakes clearly depend on the shape of the bluff body, the wake width being small for a streamlined object and large for an object with sharp edges. However, the wake width can be collapsed when it is normalized by an effective diameter based on the drag force, often called the momentum thickness. General laws for the wake width, the velocity defect, and the Strouhal number are thus deduced and fit the data well. Finally, the cross-fluctuations of the velocity and the turbulent kinetic energy are analyzed. Their amplitudes and widths are proportional to those of the mean profile. Thus, the wake remembers only the momentum flux given by the bluff body to the fluid and not any other aspects of its geometry.
The effects of thin and ultrathin liquid films on dynamic wetting
Authors: Chen X. , Ramé E. , Garoff S.
Published: December 2003

We examine the effects of thick (micron scale) fluid films and thin molecular scale (10–100 Å) films on the hydrodynamics near advancing contact lines by measuring the liquid–vapor interface shape of a meniscus and comparing the measurements to three models. Using flow visualization, we directly observe the fluid flow field near the moving contact line and give a qualitative description of the stagnation point and dividing streamline emanating from the contact line region. For thick films, when the capillary number satisfies Ca(a/d)3/2⩽O(1) (where d is the film thickness and a is the macroscopic length scale of the system), the liquid–vapor interface is bent only slightly by the viscous flow and the effective dynamic contact angle is close to zero. As Ca approaches O(1/ln(a/d)), a modulated wedge-like region appears at some distance from the film and expands both away from and toward the film as Ca increases. The dynamic contact angle approaches the classic power law behavior as this region expands. For molecularly thin films, the liquid–vapor interface shape within microns of the moving contact line is correctly described by theoretical models based purely on hydrodynamics and without disjoining pressure effects.
Free-surface entrainment into a rimming flow containing surfactants
Authors: Thoroddsen S. T. , Tan Y.-K.
Published: December 2003

We study experimentally the free-surface entrainment of tubes into a steady rimming flow formed inside a partially filled horizontally rotating cylinder. The liquid consists of a glycerin–water mixture containing surfactants (fatty acids). The phenomenon does not occur without the surfactants and the details are sensitive to their concentration. The entrainment of numerous closely spaced air tubes and/or surfactant columns can start intermittently along a two-dimensional stagnation line, but is usually associated with the appearance of an axially periodic vortex structure, the so-called shark teeth, which fixes the spanwise location of these tubes. The number of tubes is governed by the three-dimensional shape of the free surface, reducing from more than 10 to only two in each trough, as the rotation rate is increased. The tubes vary in diameter from 10–30 μm and can extend hundreds of diameters into the liquid layer before breaking up into a continuous stream of bubbles and/or drops. The tubes are driven through the stagnation line by the strong viscous shear and are stretched in the downstream direction. The entrainment starts when the Capillary number Ca=μωR/σ≃0.4.
Conformations and dynamics of Ets-1 ETS domain–DNA complexes
Authors: Reddy Swarnalatha Y. , Obika Satoshi , Bruice Thomas C.
Published: December 2003

Molecular dynamics studies have been performed for 3.5 ns on the ETS domain of Ets-1 transcription factor bound to the 14-bp DNA, d(AGTGCC
GGAA
ATGT), comprising the core sequence of high-affinity (GGAA), ETS–GGAA. In like manner, molecular dynamics simulations have been carried out for 3.9 ns on the mutant low-affinity core sequence, GGAG (ETS–GGAG). Analyses of the DNA backbone of ETS–GGAG show conformational interconversions from B
I
to B
II
substates. Also, crank shaft motions are noticed at the mutated nucleotide base pair step after 1,500 ps of dynamics. The corresponding nucleotide of ETS–GGAA is characteristic of a B
I
conformation and no crank shaft motions are observed. The single mutation of ETS–GGAA to ETS–GGAG also results in variations of helical parameters and solvent-accessible surface area around the major and minor grooves of the DNA. The presence of water contacts during the entire simulation proximal to the fourth base pair step of core DNA sequence is a characteristic feature of ETS–GGAA. Such waters are more mobile in ETS–GGAG at 100 ps and distant after 1,500 ps. Anticorrelated motions between certain amino acids of Ets-1 protein are predominant in ETS–GGAA but less so or absent in the mutant. These motions are reflected in the flexibility of amino acid residues of the protein backbone. We consider that these conformational features and water contacts are involved in stabilizing the hydrogen bond interactions between helix-3 residues of Ets-1 and DNA during the transcription process.
A pointwise estimate for fractionary derivatives with applications to partial differential equations
Authors: Córdoba Antonio , Córdoba Diego
Published: December 2003

This article emphasizes the role played by a remarkable pointwise inequality satisfied by fractionary derivatives in order to obtain maximum principles and

L
p

-decay of solutions of several interesting partial differential equations. In particular, there are applications to quasigeostrophic flows, in two space variables with critical viscosity, that model the Eckman pumping [see Baroud, Ch. N., Plapp, B. B., She, Z. S. & Swinney, H. L. (2002)
Phys. Rev. Lett.
88, 114501 and Constantin, P. (2002)
Phys. Rev. Lett.
89, 184501].
Altered starch structure is associated with endosperm modification in Quality Protein Maize
Authors: Gibbon Bryan C. , Wang Xuelu , Larkins Brian A.
Published: December 2003

The biochemical basis of modified kernel texture in Quality Protein Maize (QPM) is poorly understood. Proteomic analysis of several QPM lines indicated increased levels of granule-bound starch synthase I in the soluble nonzein protein fraction of these genotypes. Increased extraction of this enzyme reflected a change in starch structure, which was manifested as shorter amylopectin branches and increased starch-granule swelling. In mature kernels, these alterations in starch structure were associated with interconnections between starch granules that resulted in a vitreous kernel phenotype. Understanding the molecular basis for this previously uncharacterized starch structure will accelerate the development of QPM.
Direct and distinguishable inhibitory roles for SUMO isoforms in the control of transcriptional synergy
Authors: Holmstrom Sam , Van Antwerp Mary E. , Iñiguez-Lluhí Jorge A.
Published: December 2003

Functional interactions between factors bound at multiple sites on DNA often lead to a synergistic or more-than-additive transcriptional response. We previously defined a class of peptide sequences termed synergy control motifs (SC motifs) that function in multiple regulators by selectively inhibiting synergistic activity driven from multiple but not single response elements. By studying the prototypic SC motifs of the glucocorticoid receptor, we show that SC motifs inhibit transcription
per se
both in cis and in trans, and that a requirement for multiple contacts with DNA renders them selective for compound response elements. Notably, SC motifs are sites for SUMOylation, and the degree of modification correlates strongly with the extent of synergy control. Recruiting SUMO to the promoter either independently or as a fusion to the glucocorticoid receptor is sufficient to recapitulate the in trans and in cis inhibition by SC motifs without apparent changes in subcellular localization. Moreover, we find that the core ubiquitin fold domain of SUMO is sufficient for inhibition and that, independently of their potential for polySUMO chain formation, SUMO-2 and SUMO-3 are more effective inhibitors than SUMO-1.
Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation
Authors: Ye Jin , Wang Chunfu , Sumpter Rhea , Brown Michael S. , Goldstein Joseph L. , Gale Michael
Published: December 2003

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.
Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1
Authors: Margittai M. , Widengren J. , Schweinberger E. , Schröder G. F. , Felekyan S. , Haustein E. , König M. , Fasshauer D. , Grubmüller H. , Jahn R. , Seidel C. A. M.
Published: December 2003

Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble
N
-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.
Juvenile emotional experience alters synaptic composition in the rodent cortex, hippocampus, and lateral amygdala
Authors: Poeggel Gerd , Helmeke Carina , Abraham Andreas , Schwabe Tina , Friedrich Patricia , Braun Katharina
Published: December 2003

A quantitative anatomical study in the rodent anterior cingulate and somatosensory cortex, hippocampus, and lateral amygdala revealed region-, cell-, and dendrite-specific changes of spine densities in 3-week-old
Octodon degus
after repeated parental separation. In parentally separated animals significantly higher spine densities were found on the apical and basal dendrites of the cingulate cortex (up to 143% on apical and 138% on basal dendrite). Branching order analysis revealed that this effect is seen on all segments of the apical dendrite, whereas on the basal dendrites significantly higher spine densities were seen only on the outer branches (third to fifth dendritic segments). Increased spine densities were also observed on the hippocampal CA1 pyramidal neurons (up to 109% on the distal apical segments and up to 106% on the basal segment) compared with the control group. In contrast, significantly reduced spine densities were observed on the granule cell dendrites in the dentate gyrus (down to 92%) and on the apical dendrites in the medial nucleus of the amygdala (down to 95%). No significant changes of spine densities were seen in the somatosensory cortex (except for an increase in the proximal apical segments) and in the lateral nucleus of the dorsal amygdala (except for an increase in the proximal basal dendritic segments). These results demonstrate that repeated stressful emotional experience alters the balance of presumably excitatory synaptic inputs of pyramidal neurons in the limbic system. Such experience-induced modulations of limbic circuits may determine psychosocial and cognitive capacities during later life.
Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants
Authors: Costantino Nina , Court Donald L.
Published: December 2003

Homologous recombination can be used to generate recombinants on episomes or directly on the
Escherichia coli
chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage λ-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20–70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In
E. coli
, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.
High-resolution yeast phenomics resolves different physiological features in the saline response
Authors: Warringer Jonas , Ericson Elke , Fernandez Luciano , Nerman Olle , Blomberg Anders
Published: December 2003

We present a methodology for gene functional prediction based on extraction of physiologically relevant growth variables from all viable haploid yeast knockout mutants. This quantitative phenomics approach, here applied to saline cultivation, identified marginal but functionally important phenotypes and allowed the precise determination of time to adapt to an environmental challenge, rate of growth, and efficiency of growth. We identified ≈500 salt-sensitive gene deletions, the majority of which were previously uncharacterized and displayed salt sensitivity for only one of the three physiological features. We also report a high correlation to protein–protein interaction data; in particular, several salt-sensitive subcellular networks indicating functional modules were revealed. In contrast, no correlation was found between gene dispensability and gene expression. It is proposed that high-resolution phenomics will be instrumental in systemwide descriptions of intragenomic functional networks.
Membrane tube formation from giant vesicles by dynamic association of motor proteins
Authors: Koster Gerbrand , VanDuijn Martijn , Hofs Bas , Dogterom Marileen
Published: December 2003

The tubular morphology of intracellular membranous compartments is actively maintained through interactions with motor proteins and the cytoskeleton. Moving along cytoskeletal elements, motor proteins exert forces on the membranes to which they are attached, resulting in the formation of membrane tubes and tubular networks. To study the formation of membrane tubes by motor proteins, we developed an
in vitro
assay consisting of purified kinesin proteins directly linked to the lipids of giant unilamellar vesicles. When the vesicles are brought into contact with a network of immobilized microtubules, membrane tubes and tubular networks are formed. Through systematic variation of the kinesin concentration and membrane composition we study the mechanism involved. We show that a threshold concentration of motor proteins is needed and that a low membrane tension facilitates tube formation. Forces involved in tube formation were measured directly with optical tweezers and are shown to depend only on the tension and bending rigidity of the membrane. The forces were found to be higher than can be generated by individual motor proteins, indicating that multiple motors were working together to pull tubes. We propose a simple mechanism by which individual motor proteins can dynamically associate into clusters that provide the force needed for the formation of tubes, explaining why, in contrast to earlier findings [Roux, A., Cappello, G., Cartaud, J., Prost, J., Goud, B. & Bassereau, P. (2002)
Proc. Natl. Acad. Sci. USA
99, 5394–5399], motor proteins do not need to be physically linked to each other to be able to pull tubes.
Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy
Authors: Chen Yan , Wei Li-Na , Müller Joachim D.
Published: December 2003

Fluorescence fluctuation spectroscopy provides information about protein interactions in the intercellular environment from naturally occurring equilibrium fluctuations. We determine the molecular brightness of fluorescent proteins from the fluctuations by analyzing the photon counting histogram (PCH) or its moments and demonstrate the use of molecular brightness in probing the oligomerization state of proteins. We report fluorescence fluctuation measurements of enhanced GFP (EGFP) in cells up to concentrations of 10 μM by using an improved PCH theory. The molecular brightness of EGFP is constant in the concentration range studied. The brightness of a tandem EGFP construct, which carries two fluorophores, increases by a factor of two compared with EGFP alone, demonstrating the sensitivity of molecular brightness as a probe for protein complex formation. Oligomerization of nuclear receptors plays a crucial role in the regulation of gene expression. We probe the oligomerization state of the testicular receptor 4 and the ligand-binding domains of retinoid X receptor and retinoic acid receptor by observing molecular brightness changes as a function of protein concentration. The large concentration range accessible by experiment allows us to perform titration experiments on EGFP fusion proteins. An increase in the molecular brightness with protein concentration indicates the formation of homocomplexes. We observe the formation of homodimers of retinoid X receptor ligand binding domain upon addition of ligand. Resolving protein interactions in a cell is an important step in understanding cellular function on a molecular level. Brightness analysis promises to develop into an important tool for determining protein complex formation in cells.
Structural basis for PAS domain heterodimerization in the basic helix–loop–helix-PAS transcription factor hypoxia-inducible factor
Authors: Erbel Paul J. A. , Card Paul B. , Karakuzu Ozgur , Bruick Richard K. , Gardner Kevin H.
Published: December 2003

Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix–loop–helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFα and ARNT. Although this complex involves protein–protein interactions mediated by basic helix–loop–helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2α by NMR spectroscopy. We demonstrate that HIF-2α PAS-B binds the analogous ARNT domain
in vitro
, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2α central β-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains
in vitro
but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this β-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.
Domain movements of HAP2 in the cap–filament complex formation and growth process of the bacterial flagellum
Authors: Maki-Yonekura Saori , Yonekura Koji , Namba Keiichi
Published: December 2003

The cap at the growing end of the bacterial flagellum is essential for its growth, remaining stably attached while permitting the insertion of flagellin transported from the cytoplasm through the narrow central channel. We analyzed the structure of the isolated cap in its frozen hydrated state by electron cryomicroscopy. The 3D density map now shows detailed features of domains and their connections, giving reliable volumes and masses, making assignment of the domains to the amino acid sequence possible. A model of the cap–filament complex built with an atomic model of the filament allows a quantitative analysis of the cap domain movements on cap binding and rotation that promotes the efficient self assembly of flagellin during the filament growth process.
Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different
Authors: Lindahl Marika , Florencio Francisco J.
Published: December 2003

Light-dependent regulation of a growing number of chloroplast enzymatic activities has been found to occur through the reversible reduction of intra- or intermolecular disulphides by thioredoxins. In cyanobacteria, despite their similarity to chloroplasts, no proteins have hitherto been shown to interact with thioredoxins, and the role of the cyanobacterial ferredoxin/thioredoxin system has remained obscure. By using an immobilized cysteine 35-to-serine site-directed mutant of the
Synechocystis
sp. PCC 6803 thioredoxin TrxA as bait, we screened the
Synechocystis
cytosolic and peripheral membrane protein complements for proteins interacting with TrxA. The covalent bond between the isolated target proteins and mutated TrxA was confirmed by nonreducing/reducing two-dimensional SDS/PAGE. Thus, we have identified 18 cytosolic proteins and 8 membrane-associated proteins as candidate thioredoxin substrates. Twenty of these proteins have not previously been associated with thioredoxin-mediated regulation. Phosphoglucomutase, one of the previously uncharacterized thioredoxin-linked enzymes, has not earlier been considered a target for metabolic control through disulphide reduction. In this article, we show that phosphoglucomutase is inhibited under oxidizing conditions and activated by DTT and reduced wild-type TrxA
in vitro
. The results imply that thioredoxin-mediated redox regulation is as extensive in cyanobacteria as in chloroplasts but that the subjects of regulation are largely different.
Imaging linear birefringence and dichroism in cerebral amyloid pathologies
Authors: Jin Lee-Way , Claborn Kacey A. , Kurimoto Miki , Geday Morten A. , Maezawa Izumi , Sohraby Faranak , Estrada Marcus , Kaminksy Werner , Kahr Bart
Published: December 2003

New advances in polarized light microscopy were used to image Congo red-stained cerebral amyloidosis in sharp relief. The rotating-polarizer method was used to separate the optical effects of transmission, linear birefringence, extinction, linear dichroism, and orientation of the electric dipole transition moments and to display them as false-color maps. These effects are typically convolved in an ordinary polarized light microscope. In this way, we show that the amyloid deposits in Alzheimer's disease plaques contain structurally disordered centers, providing clues to mechanisms of crystallization of amyloid
in vivo
. Comparisons are made with plaques from tissues of subjects having Down's syndrome and a prion disease. In plaques characteristic of each disease, the Congo red molecules are oriented radially. The optical orientation in amyloid deposited in blood vessels from subjects having cerebral amyloid angiopathy was 90° out of phase from that in the plaques, suggesting that the fibrils run tangentially with respect to the circumference of the blood vessels. Our result supports an early model in which Congo red molecules are aligned along the long fiber axis and is in contrast to the most recent binding models that are based on computation. This investigation illustrates that the latest methods for the optical analysis of heterogeneous substances are useful for
in situ
study of amyloid.
Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases
Authors: Wolfman Neil M. , McPherron Alexandra C. , Pappano William N. , Davies Monique V. , Song Kening , Tomkinson Kathleen N. , Wright Jill F. , Zhao Liz , Sebald Suzanne M. , Greenspan Daniel S. , Lee Se-Jin
Published: December 2003

Myostatin is a transforming growth factor β family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated
in vitro
by treatment with acid; however, the mechanisms by which latent myostatin is activated
in vivo
are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin
in vivo
and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.
The early stages of duplicate gene evolution
Authors: Moore Richard C. , Purugganan Michael D.
Published: December 2003

Gene duplications are one of the primary driving forces in the evolution of genomes and genetic systems. Gene duplicates account for 8–20% of the genes in eukaryotic genomes, and the rates of gene duplication are estimated at between 0.2% and 2% per gene per million years. Duplicate genes are believed to be a major mechanism for the establishment of new gene functions and the generation of evolutionary novelty, yet very little is known about the early stages of the evolution of duplicated gene pairs. It is unclear, for example, to what extent selection, rather than neutral genetic drift, drives the fixation and early evolution of duplicate loci. Analysis of recently duplicated genes in the
Arabidopsis thaliana
genome reveals significantly reduced species-wide levels of nucleotide polymorphisms in the progenitor and/or duplicate gene copies, suggesting that selective sweeps accompany the initial stages of the evolution of these duplicated gene pairs. Our results support recent theoretical work that indicates that fates of duplicate gene pairs may be determined in the initial phases of duplicate gene evolution and that positive selection plays a prominent role in the evolutionary dynamics of the very early histories of duplicate nuclear genes.
Transgenic mouse model for echovirus myocarditis and paralysis
Authors: Hughes Scott A. , Thaker Harshwardhan M. , Racaniello Vincent R.
Published: December 2003

Echoviruses have been implicated in multiple human disease syndromes, including aseptic meningitis, paralysis, and heart disease, but no animal model is available for studying the pathogenesis of infection. Production of human integrin very late antigen 2, a receptor for echovirus type 1, in transgenic mice conferred susceptibility to viral infection. Intracerebral inoculation of newborn transgenic mice with echovirus leads to paralysis and wasting. No disease was observed in infected nontransgenic mice. In paralyzed mice significant damage was observed in the outer layers of the cerebrum, and numerous condensed neuronal nuclei were present. In contrast, intracerebral inoculation of adolescent (3- to 4-week-old) transgenic mice with echovirus type 1 did not lead to paralysis but an acute wasting phenotype and myocarditis. These findings establish human very late antigen 2 transgenic mice as a model for echovirus pathogenesis.
Phylogenetic and biochemical evidence for sterol synthesis in the bacterium
Gemmata obscuriglobus
Authors: Pearson Ann , Budin Meytal , Brocks Jochen J.
Published: December 2003

Sterol biosynthesis is viewed primarily as a eukaryotic process, and the frequency of its occurrence in bacteria has long been a subject of controversy. Two enzymes, squalene monooxygenase and oxidosqualene cyclase, are the minimum necessary for initial biosynthesis of sterols from squalene. In this work, 19 protein gene sequences for eukaryotic squalene monooxygenase and 12 protein gene sequences for eukaryotic oxidosqualene cyclase were compared with all available complete and partial prokaryotic genomes. The only unequivocal matches for a sterol biosynthetic pathway were in the proteobacterium,
Methylococcus capsulatus
, in which sterol biosynthesis is known, and in the planctomycete,
Gemmata obscuriglobus
. The latter species contains the most abbreviated sterol pathway yet identified in any organism. Analysis shows that the major sterols in
Gemmata
are lanosterol and its uncommon isomer, parkeol. There are no subsequent modifications of these products. In bacteria, the sterol biosynthesis genes occupy a contiguous coding region and possibly comprise a single operon. Phylogenetic trees constructed for both enzymes show that the sterol pathway in bacteria and eukaryotes has a common ancestry. It is likely that this contiguous reading frame was exchanged between bacteria and early eukaryotes via lateral gene transfer or endosymbiotic events. The primitive sterols produced by
Gemmata
suggest that this genus could retain the most ancient remnants of the sterol biosynthetic pathway.
Organismal complexity, protein complexity, and gene duplicability
Authors: Yang Jing , Lusk Richard , Li Wen-Hsiung
Published: December 2003

Although the evolutionary significance of gene duplication has long been recognized, it remains unclear what determines gene duplicability. We find protein complexity to be an important determinant because the proportion of unduplicated genes (
P
) increases with the number of subunits in a protein. However,
P
is high (≥65%) for both monomers and multimers in yeast, but ≤30% in human except for subunits of large multimers, implying that organismal complexity is a stronger determinant of gene duplicability than protein complexity. The same conclusion is reached from a comparison of family sizes in yeast and human.
Donation of catalytic residues to RNA polymerase active center by transcription factor Gre
Authors: Sosunova Ekaterina , Sosunov Vasily , Kozlov Maxim , Nikiforov Vadim , Goldfarb Alex , Mustaev Arkady
Published: December 2003

During transcription elongation, RNA polymerase (RNAP) occasionally loses its grip on the growing RNA end and backtracks on the DNA template. Prokaryotic Gre factors rescue the backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes the disengaged 3′ RNA segment. By using RNA-protein crosslinking in defined ternary elongating complexes, site-directed mutagenesis, discriminative biochemical assays, and docking of the two protein structures, we show that Gre acts by providing two carboxylate residues for coordination of catalytic Mg
2+
ion in the RNAP active center. A similar mechanism is suggested for the functionally analogous eukaryotic SII factor. The results expand the general two-metal model of RNAP catalytic mechanism whereby one of the Mg
2+
ions is permanently retained, whereas the other is recruited ad hoc by an auxiliary factor.
The tetraspanin CD63 enhances the internalization of the H,K-ATPase β-subunit
Authors: Duffield Amy , Kamsteeg Erik-Jan , Brown Andrea N. , Pagel Philipp , Caplan Michael J.
Published: December 2003

The tetraspanin CD63 resides in late endosomes, lysosomes, secretory vesicles, and at the plasma membrane, and it moves among these compartments. We find that CD63 is present also in tubulovesicular elements, the intracellular compartments that contain the H,K-ATPase in unstimulated gastric parietal cells. The H,K-ATPase β-subunit and CD63 colocalize in parietal cells and form a complex that can be coprecipitated. The β-subunit and CD63 also interact when they are coexpressed in COS-7 cells. Furthermore, expression with CD63 induces the redistribution of the β-subunit from the cell surface to CD63+intracellular compartments. Immunofluorescence and biochemical experiments reveal that this redistribution occurs by enhanced endocytosis of H,K-ATPase β-subunit complexed with CD63. Coexpression of the β-subunit with mutant CD63 polypeptides demonstrates that the enhanced internalization of the β-subunit depends on the capacity of CD63 to interact with adaptor protein complexes 2 and 3. These data indicate that CD63 serves as an adaptor protein that links its interaction partners to the endocytic machinery of the cell and suggest a previously uncharacterized protein-trafficking role for the tetraspanins.
Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium
in vitro
and
in vivo
Authors: Sakhalkar Harshad S. , Dalal Milind K. , Salem Aliasger K. , Ansari Ramin , Fu Jie , Kiani Mohammad F. , Kurjiaka David T. , Hanes Justin , Shakesheff Kevin M. , Goetz Douglas J.
Published: December 2003

We exploited leukocyte–endothelial cell adhesion chemistry to generate biodegradable particles that exhibit highly selective accumulation on inflamed endothelium
in vitro
and
in vivo
. Leukocyte–endothelial cell adhesive particles exhibit up to 15-fold higher adhesion to inflamed endothelium, relative to noninflamed endothelium, under
in vitro
flow conditions similar to that present in blood vessels, a 6-fold higher adhesion to cytokine inflamed endothelium relative to non-cytokine-treated endothelium
in vivo
, and a 10-fold enhancement in adhesion to trauma-induced inflamed endothelium
in vivo
due to the addition of a targeting ligand. The leukocyte–inspired particles have adhesion efficiencies similar to that of leukocytes and were shown to target each of the major inducible endothelial cell adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) that are up-regulated at sites of pathological inflammation. The potential for targeted drug delivery to inflamed endothelium has significant implications for the improved treatment of an array of pathologies, including cardiovascular disease, arthritis, inflammatory bowel disease, and cancer.
Gene expression profiles of primary breast tumors maintained in distant metastases
Authors: Weigelt Britta , Glas Annuska M. , Wessels Lodewyk F. A. , Witteveen Anke T. , Peterse Johannes L. , van't Veer Laura J.
Published: December 2003

It has been debated for decades how cancer cells acquire metastatic capability. It is unclear whether metastases are derived from distinct subpopulations of tumor cells within the primary site with higher metastatic potential, or whether they originate from a random fraction of tumor cells. Here we show, by gene expression profiling, that human primary breast tumors are strikingly similar to the distant metastases of the same patient. Unsupervised hierarchical clustering, multidimensional scaling, and permutation testing, as well as the comparison of significantly expressed genes within a pair, reveal their genetic similarity. Our findings suggest that metastatic capability in breast cancer is an inherent feature and is not based on clonal selection.
Limited boundaries for extensive horizontal gene transfer among
Salmonella
pathogens
Authors: Brown Eric W. , Mammel Mark K. , LeClerc J. Eugene , Cebula Thomas A.
Published: December 2003

Recombination is thought to be rare within
Salmonella
, as evidenced by absence of gene transfer among SARC strains that represent the broad genetic diversity of the eight primary subspecies of this common facultative intracellular pathogen. We adopted a phylogenetic approach to assess recombination within the
mutS
gene of 70 SARB strains, a genetically homogeneous population of
Salmonella enterica
subspecies I strains, which have in common the ability to infect warm-blooded animals. We report here that SARB strains show evidence for widespread recombinational exchange in contrast to results obtained with strains exhibiting species-level genetic variation. Besides extensive allele shuffling, SARB strains showed notably larger recombinagenic patch sizes for
mutS
(at least ≈1.1 kb) than previously reported for
S. enterica
SARC strains. Explaining these experimental dichotomies provides important insight for understanding microbial evolution, because they suggest likely ecologic and genetic barriers that limit extensive gene transfer in the feral setting.
Autocrine laminin-5 ligates α6β4 integrin and activates RAC and NFκB to mediate anchorage-independent survival of mammary tumors
Authors: Zahir Nastaran , Lakins Johnathon N. , Russell Alan , Ming WenYu , Chatterjee Chandrima , Rozenberg Gabriela I. , Marinkovich M. Peter , Weaver Valerie M.
Published: December 2003

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express α6β4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail–truncated α6β4 integrin. α6β4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of β4 integrin is necessary for basal and epidermal growth factor–induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional β1 and β4 integrin through activation of NFκB, and overexpression of NFκB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5–α6β4 integrin–RAC–NFκB signaling.
Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz
Authors: Wilhelm James E. , Hilton Meredith , Amos Quinlan , Henzel William J.
Published: December 2003

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end–directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation.
Cup-ling oskar RNA localization and translational control
Authors: Lasko Paul
Published: December 2003

RNA localization and spatially restricted translational control can serve to deploy specific proteins to particular places within a cell. oskar (osk) RNA is a key initiatior of posterior patterning and germ cell specification in Drosophila, and its localization and translation are under elaborate control. In this issue, Wilhelm et al. (2003) show that the protein Cup both promotes osk localization and participates in repressing translation of unlocalized osk.
Kuhn, Nominalism, and Empiricism
Authors: Bird Alexander
Published: December 2003

In this paper I draw a connection between Kuhn and the empiricist legacy, specifically between his thesis of incommensurability, in particular in its later taxonomic form, and van Fraassen's constructive empiricism. I show that if it is the case the empirically equivalent but genuinely distinct theories do exist, then we can expect such theories to be taxonomically incommensurable. I link this to Hacking's claim that Kuhn was a nominalist. I also argue that Kuhn and van Fraassen do not differ as much as might be thought as regards the claim that observation is theory laden.
The Dark Matter Double Bind: Astrophysical Aspects of the Evidential Warrant for General Relativity
Authors: Vanderburgh William L.
Published: December 2003

The dark matter problem in astrophysics exposes an underappreciated weakness in the evidential warrant for General Relativity (GR). The “dark matter double bind” entails that GR gets no differential evidential support from dynamical phenomena occurring at scales larger than our solar system, as compared to members of a significant class of rival gravitation theories. These rivals are each empirically indistinguishable from GR for phenomena taking place at solar system scales, but make predictions that may differ radically from GR's at larger scales. Thus the typical confidence in the universal applicability of GR is insufficiently warranted in the present evidential context.
Identifying periodically expressed transcripts in microarray time series data
Authors: Wichert Sofia , Fokianos Konstantinos , Strimmer Korbinian
Published: December 2003

Motivation: Microarray experiments are now routinely used to collect large-scale time series data, for example to monitor gene expression during the cell cycle. Statistical analysis of this data poses many challenges, one being that it is hard to identify correctly the subset of genes with a clear periodic signature. This has lead to a controversial argument with regard to the suitability of both available methods and current microarray data.

Methods: We introduce two simple but efficient statistical methods for signal detection and gene selection in gene expression time series data. First, we suggest the average periodogram as an exploratory device for graphical assessment of the presence of periodic transcripts in the data. Second, we describe an exact statistical test to identify periodically expressed genes that allows one to distinguish periodic from purely random processes. This identification method is based on the so-called g-statistic and uses the false discovery rate approach to multiple testing.

Results: Using simulated data it is shown that the suggested method is capable of identifying cell-cycle-activated genes in a gene expression data set even if the number of the cyclic genes is very small and regardless the presence of a dominant non-periodic component in the data. Subsequently, we re-examine 12 large microarray time series data sets (in part controversially discussed) from yeast, human fibroblast, human HeLa and bacterial cells. Based on the statistical analysis it is found that a majority of these data sets contained little or no statistical significant evidence for genes with periodic variation linked to cell cycle regulation. On the other hand, for the remaining data the method extends the catalog of previously known cell-cycle-specific transcripts by identifying additional periodic genes not found by other methods. The problem of distinguishing periodicity due to generic cell cycle activity and to artifacts from synchronization is also discussed.

Availability: The approach has been implemented in the R package GeneTS available from http://www.stat.uni-muenchen.de/~strimmer/software.html under the terms of the GNU General Public License.
Prediction of protein subcellular locations using fuzzy k-NN method
Authors: Huang Ying , Li Yanda
Published: December 2003

Motivation: Protein localization data are a valuable information resource helpful in elucidating protein functions. It is highly desirable to predict a protein's subcellular locations automatically from its sequence.

Results: In this paper, fuzzy k-nearest neighbors (k-NN) algorithm has been introduced to predict proteins' subcellular locations from their dipeptide composition. The prediction is performed with a new data set derived from version 41.0 SWISS-PROT databank, the overall predictive accuracy about 80% has been achieved in a jackknife test. The result demonstrates the applicability of this relative simple method and possible improvement of prediction accuracy for the protein subcellular locations. We also applied this method to annotate six entirely sequenced proteomes, namely Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Oryza sativa, Arabidopsis thaliana and a subset of all human proteins.

Availability: Supplementary information and subcellular location annotations for eukaryotes are available at http://166.111.30.65/hying/fuzzy_loc.htm