bluuebunny/crossref_metadata_2025 huggingface dataset:
Intense nanosecond duration source of 10–250 keV x rays suitable for imaging projectile-induced cavitation in human cadaver tissue
Authors: Boyer Craig N. , Holland Glenn E. , Seely John F.
Published: March 2005

The design, fabrication, and performance of a repetitive nanosecond x-ray source having a pumped field-emission x-ray tube are described. A compact Marx generator, 61 cm in length and storing 12 J energy, directly drives the field-emission tube with voltage pulses >380kV and with <4ns rise time from an equivalent generator impedance of 52Ω. The x-ray dose is 520 μSv at a distance of 30.5 cm. A numerical simulation model is used in which the x-ray tube’s cathode width and anode-cathode gap spacing are permitted to change with time, while electron flow between the cathode and anode is space charge limited and nonrelativistic. The x-ray tube model is coupled to an equivalent circuit representation of the Marx generator that includes the capacitance variation with charging voltage of the BaTiO3 capacitors. The capabilities of the x-ray source for flash radiography have been demonstrated by the study of the evolution of cavitation in human cadaver legs induced by high-velocity projectiles.
Production of photoemission-modulated beams in a thermionic electron gun
Authors: Neumann J. G. , Harris J. R. , Quinn B. , O’Shea P. G.
Published: March 2005

The generation and evolution of perturbations and modulations in intense charged particle beams are of key importance for many accelerator applications. Prior work focused on perturbations and modulations produced in gridded electron guns with thermionic cathodes. By using a drive laser, photoemission can produce perturbations within a longer beam generated by thermionic emission. These perturbations affect beam density only, while previous experiments with gridded guns produced perturbations in both beam density and velocity. We have extended these capabilities by developing a flexible system to produce multiple perturbations whose timing and amplitude can be easily adjusted for beam research applications. In this article we describe this apparatus and give preliminary results.
200 kHz electro-optic switch for ultrafast laser systems
Authors: Nickel Detlef , Stolzenburg Christian , Beyertt Angelika , Giesen Adolf , Häußermann Jürgen , Butze Frank , Leitner Martin
Published: March 2005

A method for obtaining very high electro-optic switching rates is demonstrated and employed for regenerative amplification of ultrashort pulses at high repetition rates. The intracavity polarization switch consists of a transverse-field BBO Pockels cell and a dielectric thin film polarizer. By multiple-shift usage of a single BBO crystal as Pockels cell, it is possible to keep thermal dissipation losses in the Pockels cell driver below 200 W. Using this intracavity polarization switch in an ultrafast thin-disk Yb:KYW regenerative amplifier, pulse energies of about 9 μJ with pulse durations of 280 fs at repetition rates of 200 kHz were generated.
Soft x-ray resonant Kerr rotation measurement and simulation of element-resolved and interface-sensitive magnetization reversals in a NiFe∕FeMn∕Co trilayer structure
Authors: Kim Sang-Koog , Lee Ki-Suk , Kortright J. B. , Shin Sung-Chul
Published: March 2005

We report experimental observations of element- and buried interface-resolved magnetization reversals in an oppositely exchange-biased NiFe∕FeMn∕Co trilayer structure by soft x-ray resonant Kerr rotation measurements. Not only Co-, Ni-, Fe-specific exchange-biased loops but also interfacial uncompensated (UC) Fe reversal loops coupled to the individual Co and NiFe layers are separately observed. From the experimental results interpreted with the help of the model simulations of soft x-ray resonant Kerr rotation, the effective thicknesses of interfacial UC regions at the buried interfaces of both FeMn∕Co and NiFe∕FeMn are found to be tUC=13±2Å and 6±4Å, respectively. The depth sensitivity as well as element specificity of the x-ray resonant Kerr effect offer an elegant way into the investigations of element- and depth-resolved magnetization reversals of ferromagnetic ultrathin regions at buried interfaces in multicomponent multilayer films.
Electron trap distribution in thin oxide after high-field stress
Authors: Cheung K. P. , Hits D. , Wang Y.
Published: March 2005

The centroid of neutral electron trap distribution in 80 Å SiO2 film after high-field electrical stress is determined using trap-filling measurements that can eliminate the contributions from trapped holes and interface trapped charges—complications that introduce ambiguity in previous studies. The centroid is found to be roughly half way between the midpoint of the oxide and the injecting electrode, implying an extremely nonuniform distribution. Such a highly nonuniform distribution is at odds with the assumption used in most oxide breakdown models. The impact of a highly nonuniform neutral trap distribution on thin oxide reliability projection could be important.
No Place like Home: Rooming Houses in Contemporary Urban Context
Authors: Mifflin Erin , Wilton Robert
Published: March 2005

In this paper we connect existing work on rooming houses to literature dealing with the meaningful nature of ‘home’ and its impact on individuals' health and social well-being. We then examine the extent to which rooming houses provide low-income tenants with ‘homes’, drawing from in-depth interviews with rooming-house tenants living in Hamilton, a city of 450 000 people in southern Ontario, Canada. Our analysis raises concerns about the capacity of rooming houses to provide affordable and stable accommodation. Poor living conditions and poor relations with rooming-house landlords worked directly against the capacity of rooms to offer private, controllable spaces and a degree of ontological security. Analysis also raised concerns about rooming houses as sites for social relations. Many respondents saw rooming houses as unpredictable and sometimes unhealthy social spaces, forcing them to seek other environments to cultivate and sustain relationships with friends and family. In general, respondents' experiences point to the shortcomings of rooming houses as ‘home’ environments, with implications for the health and social lives of the tenant population. Conceptual and policy implications are discussed in conclusion.
Willingness-to-Pay Estimation with Mixed Logit Models: Some New Evidence
Authors: Sillano Mauricio , de Dios Ortúzar Juan
Published: March 2005

Mixed-logit models are currently the state of the art in discrete-choice modelling, and their estimation in various forms (in particular, mixing revealed-preference and stated-preference data) is becoming increasingly popular. Although the theory behind these models is fairly simple, the practical problems associated with their estimation with empirical data are still relatively unknown and certainly not solved to everybody's satisfaction. In this paper we use a stated-preference dataset—previously used to derive willingness to pay for reduction in atmospheric pollution and subjective values of time—to estimate random parameter mixed logit models with different estimation methods. We use our results to discuss in some depth the problems associated with the derivation of willingness to pay with this class of models.
The Impact of Simplification in a Sequential Rule-Based Model of Activity-Scheduling Behavior
Authors: Moons Elke A L M G , Wets Geert P M , Aerts Marc , Arentze Theo A , Timmermans Harry J P
Published: March 2005

The aim of this paper is to gain a better understanding of the impact of simplification on a sequential model of activity-scheduling behavior which uses feature-selection methods. To that effect, the predictive performance of the Albatross model, which incorporates nine different facets of activity–travel behavior, based on the original full decision trees, is compared with the performance of the model based on trimmed decision trees. The results indicate that significantly smaller decision trees can be used for modeling the different choice facets of the sequential model system without losing much in predictive power. The performance of the models is compared at three levels: the choice-facet level, the activity-pattern level (comparing the observed and generated sequences of activities), and the trip-matrix level, comparing the correlation coefficients that determine the strength of the associations between the observed and the predicted origin–destination matrices. The results indicate that the model based on the trimmed decision trees predicts activity-diary schedules with a minimum loss of accuracy at the decision level. Moreover, the results indicate a slightly better performance at the activity-pattern and the trip-matrix level.
Exploring Ethos? Discourses of ‘Charity’ in the Provision of Emergency Services for Homeless People
Authors: Cloke Paul , Johnsen Sarah , May Jon
Published: March 2005

This paper examines the ethos of organisations providing emergency services for homeless people in Britain. Drawing on extensive surveys of nonstatutory organisations we present a discourse analysis of statements of ‘mission’, ‘values’, and ‘ethics’, arguing that, although care needs to be exercised in translating organisational ethos into likely practices of care, these overarching messages of ethos are significant waymarkers in the moral landscapes of caring for homeless people. Using Coles's rethinking of the politics of generosity, we interrogate ethos in terms of three ideal types—Christian caritas, secular humanism, and postsecular charity—concluding that the principal fault-line in current services divides organisations which expect particular behavioural outcomes from homeless people (including Christian ‘conversion’ and more secular assumptions of self-responsibility), and those which provide care regardless of individual response.
“Where We Stayed was very Bad …”: Migrant Children's Perspectives on Life in Informal Rented Accommodation in Two Southern African Cities
Authors: Ansell Nicola , van Blerk Lorraine
Published: March 2005

Most research and initiatives relating to children's experiences of urban space have focused on the physical environment. Housing policies in Third World countries have also emphasised the provision of physical infrastructure and buildings, and urban aesthetics. In this paper the authors draw on the voices of young informants from Maseru (Lesotho), and Blantyre (Malawi), who, in discussions concerning moving house, chose to talk about social and economic aspects of life in the informal sector rented accommodation that is increasingly characteristic of these and many other African cities. The children offer insight into the peopling of urban space, mapping unruly environments characterised by disorder, gossip, and social contestation, far removed from the hard technocratic spaces imagined by planners. Their observations are important not only because children represent a very large and relatively neglected proportion of African urban dwellers but also because they offer a unique insight into the dynamic character of urban environments. As close observers of adult decisionmaking processes, children are informed commentators on motivations for moving house as well as the impacts of urban environments on their own lives. Not only do the children highlight the inadequacies of the informal private rental sector but they also offer a window onto why it is inadequate.
Polycentric Urban Development and Spatial Clustering of Condominium Property Values: Singapore in the 1990s
Authors: Han Sun Sheng
Published: March 2005

Spatial decentralization is a common measure used by public planners to redistribute urban social and economic activities in order to resolve escalating problems such as crowdedness, pollution, and high cost of living in the central cities. Findings from studies assessing the changing rent gradient of cities, and probability of development around suburban centers, provide insights to the various impacts of spatial decentralization policies. The author makes use of global and local spatial auto-correlation statistics and seeks to explore the spatial clustering of property values in the context of polycentric urban development. Data were collected from the property sales transaction database in Singapore and analyzed employing geographic information systems and spatial statistics. Empirical results suggest that there was a remarkable redistribution among the planning regions as regards the number of condominium projects in the 1990s, which was in line with the polycentric urban development policy. A decline in the global Moran's I indicates a change from a strong to a relatively weak positive clustering. However, the local Moran statistics show that a spatial pattern of property-value clusters continued during the study period. Findings of this research capture a market signal indicating that the spatial distribution of property values may not readily follow planning guidelines, at least in a given short time frame.
The Lure of the Multiplex? The Interplay of Time, Distance, and Cinema Attendance
Authors: Collins Alan , Hand Chris , Ryder Andrew
Published: March 2005

Models of cinema demand have tended to employ aggregate data and focus on price and income as key variables. Surprisingly, the effects of travel time and location have not been formally investigated. The authors do so, following developments in the environmental economics literature, by presenting estimates of individual travel cost models for multiplex and nonmultiplex cinemas. A key finding is that travel time has a significant negative effect on nonmultiplex cinema trips, but that this does not hold for multiplex trips; reasons for this are advanced. In the case of multiplex cinema trips, a range of phenomena relating to minimising time-cost uncertainty are shown to be significant. The authors also contribute to an explanation for the ‘overscreening’ phenomenon observed in the United Kingdom and the USA, which has led to the closure of some relatively recently built multiplexes.
The Contradictions and Intersections of Class and Gender in a Global City: Placing Working Women's Lives on the Research Agenda
Authors: McDowell Linda , Perrons Diane , Fagan Colette , Ray Kath , Ward Kevin
Published: March 2005

In this paper we examine the relationships between class and gender in the context of current debates about economic change in Greater London. It is a common contention of the global city thesis that new patterns of inequality and class polarisation are apparent as the expansion of high-status employment brings in its wake rising employment in low-status, poorly paid ‘servicing’ occupations. Whereas urban theorists tend to ignore gender divisions, feminist scholars have argued that new class and income inequalities are opening up between women as growing numbers of highly credentialised women enter full-time, permanent employment and others are restricted to casualised, low-paid work. However, it is also argued that working women's interests coincide because of their continued responsibility for domestic obligations and still-evident gender discrimination in the labour market. In this paper we counterpose these debates, assessing the consequences for income inequality, for patterns of childcare and for work–life balance policies of rising rates of labour-market participation among women in Greater London. We conclude by outlining a new research agenda.
Identification of viral genes essential for replication of murine γ-herpesvirus 68 using signature-tagged mutagenesis
Authors: Song Moon Jung , Hwang Seungmin , Wong Wendy H. , Wu Ting-Ting , Lee Sangmi , Liao Hsiang-I , Sun Ren
Published: March 2005

γ-Herpesviruses, Epstein–Barr virus, and Kaposi's sarcoma-associated herpesvirus are important human pathogens, because they are involved in tumor development. Murine γ-herpesvirus-68 (MHV-68 or γHV-68) has emerged as a small animal model system for the study of γ-herpesvirus pathogenesis and host–virus interactions. To identify the genes required for viral replication
in vitro
and
in vivo
, we generated 1,152 mutants using signature-tagged transposon mutagenesis on an infectious bacterial artificial chromosome of MHV-68. Almost every ORF was mutated by random insertion. For each ORF, a mutant with an insertion proximal to the N terminus of each ORF was examined for the ability to grow in fibroblasts. Our results indicate that 41 genes are essential for
in vitro
growth, whereas 26 are nonessential and 6 attenuated. Replication-competent mutants were pooled to infect mice, which led to the discovery of ORF 54 being important for MHV-68 to replicate in the lung. This genetic analysis of a tumor-associated herpesvirus at the whole genome level validates signature-tagged transposon mutagenesis screening as an effective genetic system to identify important virulent genes
in vivo
and define interactions with the host immune system.
Nuclear processing and export of microRNAs in
Arabidopsis
Authors: Park Mee Yeon , Wu Gang , Gonzalez-Sulser Alfredo , Vaucheret Hervé , Poethig R. Scott
Published: March 2005

In mammalian cells, the nuclear export receptor, Exportin 5 (Exp5), exports pre-microRNAs (pre-miRNAs) as well as tRNAs into the cytoplasm. In this study, we examined the function of HASTY (HST), the
Arabidopsis
ortholog of Exp5, in the biogenesis of miRNAs and tRNAs. In contrast to mammals, we found that miRNAs exist as single-stranded 20- to 21-nt molecules in the nucleus in
Arabidopsis
. This observation is consistent with previous studies indicating that proteins involved in miRNA biogenesis are located in the nucleus in
Arabidopsis
. Although miRNAs exist in the nucleus, a majority accumulate in the cytoplasm. Interestingly, loss-of-function mutations in HST reduced the accumulation of most miRNAs but had no effect on the accumulation of tRNAs and endogenous small interfering RNAs, or on transgene silencing. In contrast, a mutation in PAUSED (PSD), the
Arabidopsis
ortholog of the tRNA export receptor, Exportin-t, interfered with the processing of tRNA-Tyr but did not affect the accumulation or nuclear export of miRNAs. These results demonstrate that HST and PSD do not share RNA cargos in nuclear export and strongly suggest that there are multiple nuclear export pathways for these small RNAs in
Arabidopsis
.
Theta oscillations and sensorimotor performance
Authors: Kay Leslie M.
Published: March 2005

Performance and cognitive effort in humans have recently been related to amplitude and multisite coherence of alpha (7-12 Hz) and theta (4-7 Hz) band electroencephalogram oscillations. I examined this phenomenon in rats by using theta band oscillations of the local field potential to signify sniffing as a sensorimotor process. Olfactory bulb (OB) theta oscillations are coherent with those in the dorsal hippocampus (HPC) during odor sniffing in a two-odor olfactory discrimination task. Coherence is restricted to the high-frequency theta band (6-12 Hz) associated with directed sniffing in the OB and type 1 theta in the HPC. Coherence and performance fluctuate on a time scale of several minutes. Coherence magnitude is positively correlated with performance in the two-odor condition but not in extended runs of single odor conditional-stimulus-positive trials. Simultaneous with enhanced OB-HPC theta band coherence during odor sniffing is a significant decrease in lateral entorhinal cortex (EC)-HPC and OB-EC coherence, suggesting that linkage of the olfactory and hippocampal theta rhythms is not through the synaptic relay from OB to HPC in the lateral EC. OB-HPC coupling at the sniffing frequency is proposed as a mechanism underlying olfactory sensorimotor effort as a cognitive process.
Real-time characterization of intermediates in the pathway to open complex formation by
Escherichia coli
RNA polymerase at the T7A1 promoter
Authors: Sclavi Bianca , Zaychikov Evgeny , Rogozina Anastasia , Walther Ferdinand , Buckle Malcolm , Heumann Hermann
Published: March 2005

We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by
Escherichia coli
RNA polymerase on the T7A1 promoter at 37°C. Three sets of intermediates, corresponding to two major conformational changes, are resolved as a function of time; multiple conformations equilibrate amongst each other before the next large structural change. Analysis of these data in the context of published structural models indicates that initial recognition involves interaction of the UP element with the α-subunit C-terminal domain and binding of the σ subunit to the -35 sequence. In the subsequent isomerization step, two complexes with footprints extending into the -10 region can be differentiated as the DNA becomes distorted during nucleation of strand separation. During the final isomerization step, the downstream double helix becomes embedded in the β/β′ jaws, leading to a transcriptionally active complex.
Small-molecule-mediated stabilization of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants against unfolding and aggregation
Authors: Ray Soumya S. , Nowak Richard J. , Brown Robert H. , Lansbury Peter T.
Published: March 2005

Familial amyotrophic lateral sclerosis (FALS) is a fatal motor neuron disease that is caused by mutations in the gene encoding superoxide dismutase-type 1 (SOD1). The affected regions of the FALS brain are characterized by aggregated SOD1, and the mutations that destabilize SOD1 appear to promote its aggregation
in vitro
. Because dissociation of the native SOD1 dimer is required for its
in vitro
aggregation, we initiated an
in silico
screening program to find drug-like molecules that would stabilize the SOD1 dimer. A potential binding site for such molecules at the SOD1 dimer interface was identified, and its importance was validated by mutagenesis. About 1.5 million molecules from commercial databases were docked at the dimer interface. Of the 100 molecules with the highest predicted binding affinity, 15 significantly inhibited
in vitro
aggregation and denaturation of A4V, a FALS-linked variant of SOD1. In the presence of several of these molecules, A4V and other FALS-linked SOD1 mutants such as G93A and G85R behaved similarly to wild-type SOD1, suggesting that these compounds could be leads toward effective therapeutics against FALS.
Genome-wide transcriptional analysis of flagellar regeneration in
Chlamydomonas reinhardtii
identifies orthologs of ciliary disease genes
Authors: Stolc Viktor , Samanta Manoj Pratim , Tongprasit Waraporn , Marshall Wallace F.
Published: March 2005

The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in
Chlamydomonas reinhardtii
suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in
Chlamydomonas
by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes
Qilin
,
Reptin
, and
Pontin
, as well as the testis-expressed tubby-like protein TULP2.
Differential influence of dynamic processes on forward and reverse electron transfer across a protein-protein interface
Authors: Hoffman Brian M. , Celis Laura M. , Cull Deborah A. , Patel Ami D. , Seifert Jennifer L. , Wheeler Korin E. , Wang Jingyun , Yao Jiang , Kurnikov Igor V. , Nocek Judith M.
Published: March 2005

We propose that the forward and reverse halves of a flash-induced protein-protein electron transfer (ET) photocycle should exhibit differential responses to dynamic interconversion of configurations when the most stable configuration is not the most reactive, because the reactants exist in different initial configurations: the flash-photoinitiated forward ET process begins with the protein partners in an equilibrium ensemble of configurations, many of which have little or no reactivity, whereas the reactant of the thermal back ET (the charge-separated intermediate) is formed in a nonequilibrium, “activated” protein configuration. We report evidence for this proposal in measurements on (
i
) mixed-metal hemoglobin hybrids, (
ii
) the complex between cytochrome
c
peroxidase and cytochrome
c
, and (
iii
and
iv
) the complexes of myoglobin and isolated hemoglobin α-chains with cytochrome
b
5
. For all three systems, forward and reverse ET does respond differently to modulation of dynamic processes; further, the response to changes in viscosity is different for each system.
Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers
Authors: Reeves M. B. , MacAry P. A. , Lehner P. J. , Sissons J. G. P. , Sinclair J. H.
Published: March 2005

Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their
ex vivo
differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.
Protein dynamics and electron transfer: Electronic decoherence and non-Condon effects
Authors: Skourtis Spiros S. , Balabin Ilya A. , Kawatsu Tsutomu , Beratan David N.
Published: March 2005

We compute the autocorrelation function of the donor-acceptor tunneling matrix element 〈

T
DA

(
t
)

T
DA

(0)〉 for six Ru-azurin derivatives. Comparison of this decay time to the decay time of the time-dependent Franck-Condon factor {computed by Rossky and coworkers [Lockwood, D. M., Cheng, Y.-K. & Rossky, P. J. (2001)
Chem. Phys. Lett.
345, 159-165]} reveals the extent to which non-Condon effects influence the electron-transfer rate. 〈

T
DA

(
t
)

T
DA

(0)〉 is studied as a function of donor-acceptor distance, tunneling pathway structure, tunneling energy, and temperature to explore the structural and dynamical origins of non-Condon effects. For azurin, the correlation function is remarkably insensitive to tunneling pathway structure. The decay time is only slightly shorter than it is for solvent-mediated electron transfer in small organic molecules and originates, largely, from fluctuations of valence angles rather than bond lengths.
Disrupting integrin transmembrane domain heterodimerization increases ligand binding affinity, not valency or clustering
Authors: Luo Bing-Hao , Carman Christopher V. , Takagi Junichi , Springer Timothy A.
Published: March 2005

Residues important in the interaction between the 23-residue transmembrane (TM) domains of the integrin α
IIb
- and β
3
-subunits were identified by mutating each non-Leu residue to Leu. Leu substitutions of α
IIb
at G972, G976, and T981, and of β
3
at I693 and G708, increased ligand binding. Substitutions with other amino acids at α
IIb
G972 and β
3
G708 could also increase ligand binding. The results are consistent with and extend the helical interface between the integrin α- and β-subunit TM domains previously defined by cysteine scanning and disulfide bond formation. We differentiated between affinity- and valency-based modes of activation by TM domain mutations. The mutant α
IIb
W967C forms disulfide-linked α
IIb
-subunits within an (α
IIb
β
3
)
2
tetramer. This tetramer behaved as an ideal model for the valency mode of regulation, because it exhibited significantly increased binding to multivalent but not monovalent ligands and basally retained the bent conformation. By contrast, the activating Leu mutants showed increased binding to the monovalent, ligand-mimetic PAC-1 Fab and increased exposure of ligand-induced binding site (LIBS) epitopes, suggesting that they partially adopt an extended conformation. Furthermore, the previously described β
3
G708N mutation in Chinese hamster ovary cells enhanced ligand binding affinity, not valency, and did not alter cell-surface clustering as defined by confocal microscopy. Our studies provide evidence that disrupting the integrin heterodimeric TM helix–helix interface activates ligand binding mainly by increasing the monomeric affinity for ligand, but not the receptor valency, i.e., clustering.
Diagnosis of human prion disease
Authors: Safar Jiri G. , Geschwind Michael D. , Deering Camille , Didorenko Svetlana , Sattavat Mamta , Sanchez Henry , Serban Ana , Vey Martin , Baron Henry , Giles Kurt , Miller Bruce L. , DeArmond Stephen J. , Prusiner Stanley B.
Published: March 2005

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt–Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP
Sc
) used limited proteolysis to digest the precursor cellular PrP (PrP
C
). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP
Sc
, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP
Sc
in all 10–24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, ≈90% of the total PrP
Sc
was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP
C
. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.
Automated microscope system for determining factors that predict neuronal fate
Authors: Arrasate Montserrat , Finkbeiner Steven
Published: March 2005

Unraveling cause-and-effect relationships in the nervous system is challenging because some biological processes begin stochastically, take a significant amount of time to unfold, and affect small neuronal subpopulations that can be difficult to isolate and measure. Single-cell approaches are slow, subject to user bias, and sometimes too laborious to achieve sample sizes large enough to detect important effects. Here, we describe an automated imaging and analysis system that enables us to follow the fates of individual cells and intracellular proteins over time. Observations can be quantified in a high-throughput manner with minimal user bias. We have adapted survival analysis methods to determine whether and how factors measured during longitudinal analysis predict a particular biological outcome. The ability to monitor complex processes at single-cell resolution quickly, quantitatively, and over long intervals should have wide applications for biology.
Remodeling of the intestine during metamorphosis of
Xenopus laevis
Authors: Schreiber Alex M. , Cai Liquan , Brown Donald D.
Published: March 2005

Thyroid hormone controls remodeling of the tadpole intestine during the climax of amphibian metamorphosis. In 8 days, the
Xenopus laevis
tadpole intestine shortens in length by 75%. Simultaneously, the longitudinal muscle fibers contract by about the same extent. The radial muscle fibers also shorten as the diameter narrows. Many radial fibers undergo programmed cell death. We conclude that muscle remodeling and contraction play key roles in the shortening process. Shortening is accompanied by a temporary “heaping” of the epithelial cells into many layers at climax. Cells that face the lumen undergo apoptosis. By the end of metamorphosis, when the epithelium is folded into crypts and villi, the epithelium is a single-cell layer once again. Throughout this remodeling, DNA replication occurs uniformly throughout the epithelium, as do changes in gene expression. The larval epithelial cells as a whole, rather than a subpopulation of stem cells, are the progenitors of the adult epithelial cells.
Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock
Authors: Allemand Eric , Guil Sònia , Myers Michael , Moscat Jorge , Cáceres Javier F. , Krainer Adrian R.
Published: March 2005

Heterogenous nuclear ribonucleoprotein (hnRNP) A1 is an alternative splicing factor that is mainly nuclear, although it shuttles rapidly between nuclear and cytoplasmic compartments. Cells stressed by osmotic shock (OSM) activate the mitogen-activated protein kinase kinase3/6-p38 signaling pathway, which in turn results in accumulation of hnRNP A1 in the cytoplasm. This effect modulates alternative splicing regulationin vivoand correlates with increased hnRNP A1 phosphorylation. We have characterized the molecular mechanism involved in the cytoplasmic accumulation of hnRNP A1 in NIH 3T3 cells subjected to OSM. This treatment results in serine-specific phosphorylation within a C-terminal peptide, dubbed the “F-peptide,” which is adjacent to the M9 motif that mediates bidirectional transport of hnRNP A1. Analysis of mutants in which the F-peptide serines were replaced by aspartic acids or alanines showed that F-peptide phosphorylation is required for the subcellular redistribution of hnRNP A1 in cells subjected to OSM. Furthermore, F-peptide phosphorylation modulates the interaction of hnRNP A1 with transportin Trn1. Our findings suggest that the phosphorylation of F-peptide by cell-signaling pathways regulates the rate of hnRNP A1 nuclear import.
Culturing at atmospheric oxygen levels impacts lymphocyte function
Authors: Atkuri Kondala R. , Herzenberg Leonard A. , Herzenberg Leonore A.
Published: March 2005

To determine whether culturing peripheral blood mononuclear cells at atmospheric oxygen levels skews responses in comparison with culturing lymphocytes at physiologic oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen levels influenced lymphocyte proliferation stimulated by two commonly used stimuli: Con A and antibodies that crosslink surface CD3 and CD28 to mimic antigen presentation. In both cases, proliferation increased as gas-phase oxygen levels increased. In contrast, oxygen levels did not influence proliferation stimulated by phytohemagglutinin, another commonly used mitogen. Similarly, oxygen levels did not impact cell viability in unstimulated cultures. Thus, we conclude that the influence of oxygen levels on proliferation depends on the stimulus, and, most importantly from the standpoint of immune responses, culturing cells at atmospheric rather than physiologic oxygen levels results in significantly increased proliferation responses to the CD3/CD28 crosslinking, a proliferation stimulus commonly used to mimic T cell antigen receptor signaling.
Cortical processing of a brightness illusion
Authors: Roe Anna Wang , Lu Haidong D. , Hung Chou P.
Published: March 2005

Several brightness illusions indicate that borders can affect the perception of surfaces dramatically. In the Cornsweet illusion, two equiluminant surfaces appear to be different in brightness because of the contrast border between them. Here, we report the existence of cells in monkey visual cortex that respond to such an “illusory” brightness. We find that luminance responsive cells are located in color-activated regions (cytochrome oxidase blobs and bridges) of primary visual cortex (V1), whereas Cornsweet responsive cells are found preferentially in the color-activated regions (thin stripes) of second visual area (V2). This colocalization of brightness and color processing within V1 and V2 suggests a segregation of contour and surface processing in early visual pathways and a hierarchy of brightness information processing from V1 to V2 in monkeys.
Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors
Authors: Peart Melissa J. , Smyth Gordon K. , van Laar Ryan K. , Bowtell David D. , Richon Victoria M. , Marks Paul A. , Holloway Andrew J. , Johnstone Ricky W.
Published: March 2005

Histone deacetylase inhibitors (HDACis) inhibit tumor cell growth and survival, possibly through their ability to regulate the expression of specific proliferative and/or apoptotic genes. However, the HDACi-regulated genes necessary and/or sufficient for their biological effects remain undefined. We demonstrate that the HDACis suberoylanilide hydroxamic acid (SAHA) and depsipeptide regulate a highly overlapping gene set with at least 22% of genes showing altered expression over a 16-h culture period. SAHA and depsipeptide coordinately regulated the expression of several genes within distinct apoptosis and cell cycle pathways. Multiple genes within the Myc, type β TGF, cyclin/cyclin-dependent kinase, TNF, Bcl-2, and caspase pathways were regulated in a manner that favored induction of apoptosis and decreased cellular proliferation.APAF-1, a gene central to the intrinsic apoptotic pathway, was induced by SAHA and depsipeptide and shown to be important, but not essential, for HDACi-induced cell death. Overexpression of p16INK4Aand arrest of cells in G1can suppress HDACi-mediated apoptosis. Although p16INK4Adid not affect the genome-wide transcription changes mediated by SAHA, a small number of apoptotic genes, includingBCLXLandB-MYB, were differentially regulated in a manner consistent with attenuated HDACi-mediated apoptosis in arrested cells. We demonstrate that different HDACi alter transcription of a large and common set of genes that control diverse molecular pathways important for cell survival and proliferation. The ability of HDACi to target multiple apoptotic and cell proliferation pathways may provide a competitive advantage over other chemotherapeutic agents because suppression/loss of a single pathway may not confer resistance to these agents.
MET
and
MYC
cooperate in mammary tumorigenesis
Authors: Welm Alana L. , Kim Suwon , Welm Bryan E. , Bishop J. Michael
Published: March 2005

In human breast cancer, overexpression of the protooncogene
MET
is strongly associated with poor prognosis and high risk of metastasis. It stands out as a reliable prognostic indicator of survival and defines a set of tumors exclusive of those that express
HER2
or hormone receptors. Studies have shown that overexpression of mutant forms of
MET
cause cancer in mice. However,
MET
mutations have not been found in human breast cancer, and the consequences of overexpression of normal
MET
are unknown. To investigate the role of
MET
and other putative oncogenes in breast cancer, we developed an experimental system that involves retroviral delivery of genes into primary mammary epithelial cells, followed by transplantation of the transduced cells into mammary fat pads. Using this approach, we found that overexpression of wild-type
MET
leads to the development of nonprogressive neoplasms. The lesions progressed to mammary adenocarcinoma when a second protooncogene,
MYC
, was overexpressed, indicating that
MET
and
MYC
cooperate in mammary tumorigenesis. Both the nonprogressive neoplasms and adenocarcinomas display characteristics consistent with transformation and expansion of mammary progenitor cells. The approach described here should provide a useful model with which to efficiently test effects of various genes on tumor development in the breast.
Accumulation of miR-155 and
BIC
RNA in human B cell lymphomas
Authors: Eis Peggy S. , Tam Wayne , Sun Liping , Chadburn Amy , Li Zongdong , Gomez Mario F. , Lund Elsebet , Dahlberg James E.
Published: March 2005

We show that the microRNA miR-155 can be processed from sequences present in
BIC
RNA, a spliced and polyadenylated but non-protein-coding RNA that accumulates in lymphoma cells. The precursor of miR-155 is likely a transient spliced or unspliced nuclear
BIC
transcript rather than accumulated
BIC
RNA, which is primarily cytoplasmic. By using a sensitive and quantitative assay, we find that clinical isolates of several types of B cell lymphomas, including diffuse large B cell lymphoma (DLBCL), have 10- to 30-fold higher copy numbers of miR-155 than do normal circulating B cells. Similarly, the quantities of
BIC
RNA are elevated in lymphoma cells, but ratios of the amounts of the two RNAs are not constant, suggesting that the level of miR-155 is controlled by transcription and processing. Significantly higher levels of miR-155 are present in DLBCLs with an activated B cell phenotype than with the germinal center phenotype. Because patients with activated B cell-type DLBCL have a poorer clinical prognosis, quantification of this microRNA may be diagnostically useful.
Architecture of the bacteriophage T4 primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)
Authors: Norcum Mona T. , Warrington J. Anthony , Spiering Michelle M. , Ishmael Faoud T. , Trakselis Michael A. , Benkovic Stephen J.
Published: March 2005

Replication of DNA requires helicase and primase activities as part of a primosome assembly. In bacteriophage T4, helicase and primase are separate polypeptides for which little structural information is available and whose mechanism of association within the primosome is not yet understood. Three-dimensional structural information is provided here by means of reconstructions from electron microscopic images. Structures have been calculated for complexes of each of these proteins with ssDNA in the presence of MgATPγS. Both the helicase (gp41) and primase (gp61) complexes are asymmetric hexagonal rings. The gp41 structure suggests two distinct forms that have been termed “open” and “closed.” The gp61 structure is clearly a six-membered ring, which may be a trimer of dimers or a traditional hexamer of monomers. This structure provides conclusive evidence for an oligomeric primase-to-ssDNA stoichiometry of 6:1.
Function and regulation of Tumbleweed (RacGAP50C) in neuroblast proliferation and neuronal morphogenesis
Authors: Goldstein Ann Y. N. , Jan Yuh-Nung , Luo Liqun
Published: March 2005

Drosophila
RacGAP50C and its homologues act as part of a complex with a kinesin-like protein (Pavarotti/Zen-4) that is essential for the formation of the central spindle and completion of cytokinesis [Mishima, M., Kaitna, S. & Glotzer, M. (2002)
Dev. Cell
2, 41–54; Somers, W. G. & Saint, R. (2003)
Dev. Cell
4, 29–39; Jantsch-Plunger
et al.
(2000)
J. Cell Biol.
149, 1391–1404]. We report here that RacGAP50C corresponds to the
tumbleweed
(
tum
) gene previously identified based on its defects in dendrite development of sensory neurons [Gao, F. B., Brenman, J. E., Jan, L. Y. & Jan, Y. N. (1999)
Genes Dev.
13, 2549–2561]. Using mushroom body neurogenesis and morphogenesis as a model, we show that Tumbleweed (Tum), Pavarotti, and their association are required for neuroblast proliferation. Tum with a mutation predicted to disrupt the GTPase-activating protein (GAP) activity still largely retains its activity in regulating cell division but is impaired in its activity to limit axon growth. We also provide evidence that Tum and Pavarotti regulate the subcellular localization of each other in postmitotic neurons and that cytoplasmic accumulation of both proteins disrupts axon development in a GAP-dependent manner. Taken together with previous studies of RacGAP50C in regulating cytokinesis, we propose that Tum serves as a scaffolding protein in regulating cell division but acts as a GAP to limit axon growth in postmitotic neurons.
Incorporating structure to predict microRNA targets
Authors: Robins Harlan , Li Ying , Padgett Richard W.
Published: March 2005

MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including
Caenorhabditis elegans
,
Drosophila
, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. (2001)
Science
294, 853–858; Lai, E. C., Tomancak, P., Williams, R. W. & Rubin, G.M. (2003)
Genome Biol.
4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. (2001)
Science
294, 858–862; Lee, R. C. & Ambros, V. (2001)
Science
294, 862-8644; and Lee, R. C., Feinbaum, R. L. & Ambros, V. (1993)
Cell
115, 787–798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3′ UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using
Drosophila
miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is
mad
as a target for
bantam
. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported.
Independence of replisomes in
Escherichia coli
chromosomal replication
Authors: Breier Adam M. , Weier Heinz-Ulrich G. , Cozzarelli Nicholas R.
Published: March 2005

In
Escherichia coli
DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding
oriC
in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple
E. coli
strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.