bluuebunny/crossref_metadata_2025 huggingface dataset:
Compact medical fluorosensor for minimally invasive tissue characterization
Authors: af Klinteberg Claes , Andreasson Markus , Sandström Ola , Andersson-Engels Stefan , Svanberg Sune
Published: March 2005

A compact fiber-optic point-measuring fluorosensor fully adapted to clinical studies is described. The system can use two excitation wavelengths, 337 and 405nm, obtained from a nitrogen laser directly, or after dye laser conversion, respectively. The image intensifier used in the spectrometer can be gated with a variable time delay, allowing also time-resolved spectra to be extracted, with a time resolution of about 4ns. Moreover, diffusely scattered white light can be spectrally recorded. The system is fully computer controlled enabling short recording times in clinical application, which are illustrated.
Shape recovery of nanoscale imprints in a thermoset “shape memory” polymer
Authors: Nelson Brent A. , King William P. , Gall Ken
Published: March 2005

This letter reports temperature-dependent recovery of atomic force microscope tip-formed indentations in a thermoset shape memory polymer. The indentations are made at both room temperature and 69°C, and then recovered at temperatures between 40°C and 70°C. The shape recovery is more complete for higher anneal temperatures, and is relatively independent of time for 102–104s. The experiments show shape memory in the 1–100nm size scale.
Monolithic integration of vertical-cavity surface-emitting lasers with in-plane waveguides
Authors: Witzens Jeremy , Scherer Axel , Pickrell Gregory , Louderback Duane , Guilfoyle Peter
Published: March 2005

The ability to couple light from a vertical-cavity surface-emitting laser into a planar, on-chip waveguide creates new opportunities for achieving higher levels of integration and functionality. Here we propose to use a strong grating etched into a waveguide defined into the top layer of the epitaxially grown structure, so that epitaxial regrowth is not required. By introducing a defect mode into the cavity we were able to achieve a 40% coupling efficiency even though light is coupled through a ninety degrees bend. We also show that polarization control of the vertical-cavity surface-emitting laser is enhanced by coupling to the defect mode. Calculations were performed using the finite-difference time-domain method.
Self-assembly of faceted Ni nanodots on Si(111)
Authors: Aurongzeb D. , Patibandla S. , Holtz M. , Temkin H.
Published: March 2005

We report the formation of Ni nanodots on Si(111). Island density is varied by annealing temperature and time and is studied using atomic force microscopy (AFM) and magnetic force microscopy. Activation energies of 0.09±0.02 and 0.31±0.05eV are observed for the formation of these islands. These are associated with Ni surface self-diffusion across the (111) and (110) Ni facets, respectively. For brief 500°C anneals, regular nanodots are observed with self-limiting sizes of height ∼16nm and area 180nm×260nm, while density exhibits a power-law time dependence with exponent 1.13±0.12. AFM analysis reveals a “truncated hut” shape consistent with (110) top and (111) sidewall surfaces.
Modelling High-Intensity Crime Areas: Comparing Police Perceptions with Offence/Offender Data in Sheffield
Authors: Craglia Massimo , Haining Robert , Signoretta Paola
Published: March 2005

High-intensity crime areas are areas where high levels of violent crime coexist with large numbers of offenders, thereby creating an area that may present significant policing problems. In an earlier paper, the authors analysed police perceptions of high-intensity crime areas, and now extend that earlier work by comparing the police's perception of where such areas are located with offence/offender data. They also report on the construction of predictive models that identify the area-specific attributes that explain the distribution of such areas. By focusing on the city of Sheffield, the authors draw on a wider range of local area data than was possible in the original paper, and also question how widespread such areas may be in Sheffield.
Extinction dynamics in experimental metapopulations
Authors: Molofsky Jane , Ferdy Jean-Baptiste
Published: March 2005

Metapopulation theory provides a framework for understanding population persistence in fragmented landscapes and as such has been widely used in conservation biology to inform management of fragmented populations. However, classical metapopulation theory [Levins, R. (1970)
Lect. Notes Math.
2, 75-107] ignores metapopulation structure and local population dynamics, both of which may affect extinction dynamics. Here, we investigate metapopulation dynamics in populations that are subject to different migration rates by using experimental metapopulations of the annual plant
Cardamine pensylvanica
. As predicted by classical metapopulation theory, connected populations persisted longer than did isolated populations, but the relationship between migration and persistence time was nonlinear. Extinction risk sharply increased as the distance between local populations increased above a threshold value that was consistent with stochastic simulations and calculation of metapopulation capacity [Hanski, I. & Ovaskainen, O. (2000)
Nature
404, 755-758]. In addition, the most connected metapopulations did not have the highest persistence levels. Stochastic simulations indicated an increase in extinction risk with the highest migration rates. Moreover, calculation of population coherence [Earn, D. J. D., Levin, S. A. & Rohani, P. (2000)
Science
290, 1360-1364], a metric that predicts synchronous cycles, indicated that continuous populations should cycle in phase, resulting in an increased extinction risk. Determining empirically the optimal migration level to improve survival chances will be challenging for any natural population. Migration rates that would not increase migration above the threshold value would be ineffectual, but migration rates that would homogenize local densities could increase the risk of coherent oscillations and enhance extinction risk.
Chemical genomic profiling to identify intracellular targets of a multiplex kinase inhibitor
Authors: Kung Charles , Kenski Denise M. , Dickerson Scott H. , Howson Russell W. , Kuyper Lee F. , Madhani Hiten D. , Shokat Kevan M.
Published: March 2005

The identification of the kinase or kinases targeted by protein kinase inhibitors is a critical challenge in validating their use as therapeutic agents or molecular probes. Here, to address this problem, we describe a chemical genomics strategy that uses a direct comparison between microarray transcriptional signatures elicited by an inhibitor of unknown specificity and those elicited by highly specific pharmacological inhibition of engineered candidate kinase targets. By using this approach, we have identified two cyclin-dependent kinases, Cdk1 and Pho85, as the targets of the inhibitor GW400426 in
Saccharomyces cerevisiae
. We demonstrate that simultaneous inhibition of Cdk1 and Pho85, and not inhibition of either kinase alone, by GW400426 controls the expression of specific transcripts involved in polarized cell growth, thus revealing a cellular process that is uniquely sensitive to the multiplex inhibition of these two kinases. Our results suggest that the cellular responses induced by multiplex protein kinase inhibitors may be an emergent property that cannot be understood fully by considering only the sum of individual inhibitor–kinase interactions.
Long-range electron transfer
Authors: Gray Harry B. , Winkler Jay R.
Published: March 2005

Recent investigations have shed much light on the nuclear and electronic factors that control the rates of long-range electron tunneling through molecules in aqueous and organic glasses as well as through bonds in donor–bridge–acceptor complexes. Couplings through covalent and hydrogen bonds are much stronger than those across van der Waals gaps, and these differences in coupling between bonded and nonbonded atoms account for the dependence of tunneling rates on the structure of the media between redox sites in Ru-modified proteins and protein–protein complexes.
Structural and mechanistic analysis of two prolyl endopeptidases: Role of interdomain dynamics in catalysis and specificity
Authors: Shan Lu , Mathews Irimpan I. , Khosla Chaitan
Published: March 2005

Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing insights into the mode of action of these enzymes. The structure of the
Sphingomonas capsulata
PEP, solved and refined to 1.8-Å resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound PEP from
Myxococcus xanthus
was crystallized (1.5-Å resolution) in a closed form. Comparative analysis of the two structures highlights a critical role for the domain interface in regulating interdomain dynamics and substrate specificity. Structure-based mutagenesis of the
M. xanthus
PEP confirms an important role for several interfacial residues. A salt bridge between Arg-572 and Asp-196/Glu-197 appears to act as a latch for opening or closing the didomain enzyme, and Arg-572 and Ile-575 may also help secure the incoming peptide substrate to the open form of the enzyme. Arg-618 and Asp-145 are responsible for anchoring the invariant proline residue in the active site of this postproline-cleaving enzyme. A model is proposed for the docking of a representative substrate PQPQLPYPQPQLP in the active site, where the N-terminal substrate residues interact extensively with the catalytic domain, and the C-terminal residues stretch into the propeller domain. Given the promise of the
M. xanthus
PEP as an oral therapeutic enzyme for treating celiac sprue, our results provide a strong foundation for further optimization of the PEP's clinically useful features.
Regulation of platelet granule exocytosis by S-nitrosylation
Authors: Morrell Craig N. , Matsushita Kenji , Chiles Kelly , Scharpf Robert B. , Yamakuchi Munekazu , Mason Rebecca J. A. , Bergmeier Wolfgang , Mankowski Joseph L. , Baldwin William M. , Faraday Nauder , Lowenstein Charles J.
Published: March 2005

Nitric oxide (NO) regulates platelet activation by cGMP-dependent mechanisms and by mechanisms that are not completely defined. Platelet activation includes exocytosis of platelet granules, releasing mediators that regulate interactions between platelets, leukocytes, and endothelial cells. Exocytosis is mediated in part by
N
-ethylmaleimide-sensitive factor (NSF), an ATPase that disassembles complexes of soluble NSF attachment protein receptors. We now demonstrate that NO inhibits exocytosis of dense granules, lysosomal granules, and α-granules from human platelets by S-nitrosylation of NSF. Platelets lacking endothelial NO synthase show increased rolling on venules, increased thrombosis in arterioles, and increased exocytosis
in vivo
. Regulation of exocytosis is thus a mechanism by which NO regulates thrombosis.
Ultrasensitivity and noise propagation in a synthetic transcriptional cascade
Authors: Hooshangi Sara , Thiberge Stephan , Weiss Ron
Published: March 2005

The precise nature of information flow through a biological network, which is governed by factors such as response sensitivities and noise propagation, greatly affects the operation of biological systems. Quantitative analysis of these properties is often difficult in naturally occurring systems but can be greatly facilitated by studying simple synthetic networks. Here, we report the construction of synthetic transcriptional cascades comprising one, two, and three repression stages. These model systems enable us to analyze sensitivity and noise propagation as a function of network complexity. We demonstrate experimentally steady-state switching behavior that becomes sharper with longer cascades. The regulatory mechanisms that confer this ultrasensitive response both attenuate and amplify phenotypical variations depending on the system's input conditions. Although noise attenuation allows the cascade to act as a low-pass filter by rejecting short-lived perturbations in input conditions, noise amplification results in loss of synchrony among a cell population. The experimental results demonstrating the above network properties correlate well with simulations of a simple mathematical model of the system.
Wire-like charge transport at near constant bridge energy through fluorene oligomers
Authors: Goldsmith Randall H. , Sinks Louise E. , Kelley Richard F. , Betzen Laura J. , Liu Wenhao , Weiss Emily A. , Ratner Mark A. , Wasielewski Michael R.
Published: March 2005

The study of photoinitiated electron transfer in donor–bridge–acceptor molecules has helped elucidate the distance dependence of electron transfer rates and behavior of various electron transfer mechanisms. In all reported cases, the energies of the bridge electronic states involved in the electron transfer change dramatically as the length of the bridge is varied. We report here, in contrast, an instance in which the length of the bridge, and therefore the distance over which the electron is transferred, can be varied without significantly changing the energies of the relevant bridge states. A series of donor–bridge–acceptor molecules having phenothiazine (PTZ) donors, 2,7-oligofluorene (FL

n

) bridges, and perylene-3,4:9,10-bis(dicarboximide) (PDI) acceptors was studied. Photoexcitation of PDI to its lowest excited singlet state results in oxidation of PTZ via the FL

n

bridge. In toluene, the rate constants for both charge separation and recombination as well as the energy levels of the relevant

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\begin{equation*}{\mathrm{FL}}_{n}^{+{\bullet}}\end{equation*}\end{document}

bridge states for
n
= 1–4 are only weakly distance dependent. After the initial photo-generation of
1
(PTZ
+·
–FL

n

–PDI
-·
), radical pair intersystem crossing results in formation of
3
(PTZ
+·
–FL

n

–PDI
-·
) that recombines to give
3*
PDI. The dependence of the
3*
PDI yield on an applied magnetic field shows a resonance, which gives the singlet–triplet splitting, 2
J
, of the radical ion pair. The magnitude of 2
J
directly monitors the contribution of coherent charge transfer (superexchange) to the overall electron transfer rate. These data show that charge recombination through FL

n

is dominated by incoherent hopping at long distances.
Protein–DNA charge transport: Redox activation of a DNA repair protein by guanine radical
Authors: Yavin Eylon , Boal Amie K. , Stemp Eric D. A. , Boon Elizabeth M. , Livingston Alison L. , O'Shea Valerie L. , David Sheila S. , Barton Jacqueline K.
Published: March 2005

DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S]
2+
cluster of MutY to [4Fe-4S]
3+
and its decomposition product [3Fe-4S]
1+
. Flash/quench experiments monitored by EPR spectroscopy reveal spectra with
g
= 2.08, 2.06, and 2.02, characteristic of the oxidized clusters. Transient absorption spectra of poly(dGC) and [Ru(phen)
2
dppz]
3+
(dppz = dipyridophenazine), generated
in situ
, show an absorption characteristic of the guanine radical that is depleted in the presence of MutY with formation instead of a long-lived species with an absorption at 405 nm; we attribute this absorption also to formation of the oxidized [4Fe-4S]
3+
and [3Fe-4S]
1+
clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5′-G of a 5′-GG-3′ doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With ruthenium-tethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair.
Three-dimensional reconstruction of the dynactin complex by single-particle image analysis
Authors: Hodgkinson J. L. , Peters C. , Kuznetsov S. A. , Steffen W.
Published: March 2005

Dynactin is a large complex of at least nine distinct proteins that co-complexes with cytoplasmic dynein within cells, where it plays a major role as a regulator of the motor's function. Owing to its large size and complexity, relatively little is known about dynactin's 3D structure or the structural basis of its function. Use of single-particle image analysis techniques has enabled us to produce the first 3D reconstruction of the dynactin complex, to a resolution of 3 nm. The actin-related protein (Arp) backbone of the filament has been clearly visualized. Fitting of models of the Arp backbone showed that it consists of 10 subunits. Additional mass, not part of the Arp backbone, was also seen. A preliminary fitting of the capping protein CapZ structure into our 3D reconstruction of the dynactin complex suggests that it is optimally placed to perform its proposed function as a stabilizer of the Arp1 backbone and gives clues as to likely interaction points between the capping protein and Arp subunits. The results provide the first detailed visualization of the dynactin complex and shed light on the mode of interaction between several of its constituent proteins and their possible functions.
Interprotein electron transfer from cytochromec2to photosynthetic reaction center: Tunneling across an aqueous interface
Authors: Miyashita Osamu , Okamura Melvin Y. , Onuchic José N.
Published: March 2005

Interprotein electron transfer (ET) reactions play an important role in biological energy conversion processes. One of these reactions, the ET between cytochromec2(cyt) and reaction center from photosynthetic bacteria, is the focus of this theoretical study. The changes in the ET rate constant at fixed distances during the association process were calculated as the cyt moved from the electrostatically stabilized encounter complex to the bound state having short range van der Waals contacts in the tunneling region. Multiple conformations of the protein were generated by molecular dynamics simulations including explicit water molecules. For each of these conformations, the ET rate was calculated by using the Pathways model. The ET rate increased smoothly as the cyt approached from the encounter complex to the bound state, with a tunneling decay factor β = 1.1 Å-1. This relatively efficient coupling between redox centers is due to the ability of interfacial water molecules to form multiple strong hydrogen bonding pathways connecting tunneling pathways on the surfaces of the two proteins. The ET rate determined for the encounter complex ensemble of states is only about a factor of 100 slower than that of the bound state (τ = 100 μs, compared with 1 μs), because of fluctuations of the cyt within the encounter complex ensemble through configurations having strong tunneling pathways. The ET rate for the encounter complex is in agreement with rates observed in mutant reaction centers modified to remove shortrange hydrophobic interactions, suggesting that in this case, ET occurs within the solvent-separated, electrostatically stabilized encounter complex.
Perinatal exposure to nicotine causes deficits associated with a loss of nicotinic receptor function
Authors: Cohen Gary , Roux Jean-Christophe , Grailhe Régis , Malcolm Girvan , Changeux Jean-Pierre , Lagercrantz Hugo
Published: March 2005

We investigated the role played by β2-containing neuronal nicotinic receptors [nicotinic acetylcholine receptors (nAChRs)] in mediating nicotine's side effects in the fetus and newborn. Pregnant WT and mutant mice lacking the β2 nAChR subunit were implanted with osmotic minipumps that delivered either water or a controlled dose of nicotine. Subsequently, we compared the development of the sympathoadrenal system and breathing and arousal reflexes of offspring shortly after birth, a period of increased vulnerability to nicotine exposure. Newborn WT pups exposed to nicotine exhibited all of the deficits associated with maternal tobacco and nicotine use, and linked to poor neonatal outcome: growth restriction, unstable breathing, and impaired arousal and catecholamine biosynthesis. Remarkably similar deficits were detected in pups lacking β2-containing nAChRs. Loss-of-function of these nAChRs consequently reproduces with astonishing fidelity many of the abnormalities caused by perinatal nicotine exposure. We propose that the underlying mechanisms of nicotine's detrimental side effects on a range of crucial defensive reflexes involve loss of function of nAChR subtypes, possibly via activity-dependent desensitization.
Comparison of the protein–protein interfaces in the p53–DNA crystal structures: Towards elucidation of the biological interface
Authors: Ma Buyong , Pan Yongping , Gunasekaran K. , Venkataraghavan R. Babu , Levine Arnold J. , Nussinov Ruth
Published: March 2005

p53, the tumor suppressor protein, functions as a dimer of dimers. However, how the tetramer binds to the DNA is still an open question. In the crystal structure, three copies of the p53 monomers (containing chains A, B, and C) were crystallized with the DNA-consensus element. Although the structure provides crucial data on the p53–DNA contacts, the active oligomeric state is unclear because the two dimeric (A–B and B–C) interfaces present in the crystal cannot both exist in the tetramer. Here, we address the question of which of these two dimeric interfaces may be more biologically relevant. We analyze the sequence and structural properties of the p53–p53 dimeric interfaces and carry out extensive molecular dynamics simulations of the crystal structures of the human and mouse p53 dimers. We find that the A–B interface residues are more conserved than those of the B–C. Molecular dynamics simulations show that the A–B interface can provide a stable DNA-binding motif in the dimeric state, unlike B–C. Our results indicate that the interface between chains A–B in the p53–DNA complex constitutes a better candidate for a stable biological interface, whereas the B–C interface is more likely to be due to crystal packing. Thus, they have significant implications toward our understanding of DNA binding by p53 as well as p53-mediated interactions with other proteins.
Climate-driven regime shifts in the biological communities of arctic lakes
Authors: Smol John P. , Wolfe Alexander P. , Birks H. John B. , Douglas Marianne S. V. , Jones Vivienne J. , Korhola Atte , Pienitz Reinhard , Rühland Kathleen , Sorvari Sanna , Antoniades Dermot , Brooks Stephen J. , Fallu Marie-Andrée , Hughes Mike , Keatley Bronwyn E. , Laing Tamsin E. , Michelutti Neal , Nazarova Larisa , Nyman Marjut , Paterson Andrew M. , Perren Bianca , Quinlan Roberto , Rautio Milla , Saulnier-Talbot Émilie , Siitonen Susanna , Solovieva Nadia , Weckström Jan
Published: March 2005

Fifty-five paleolimnological records from lakes in the circumpolar Arctic reveal widespread species changes and ecological reorganizations in algae and invertebrate communities since approximately
anno Domini
1850. The remoteness of these sites, coupled with the ecological characteristics of taxa involved, indicate that changes are primarily driven by climate warming through lengthening of the summer growing season and related limnological changes. The widespread distribution and similar character of these changes indicate that the opportunity to study arctic ecosystems unaffected by human influences may have disappeared.
Transdifferentiation of corneal epithelium into epidermis occurs by means of a multistep process triggered by dermal developmental signals
Authors: Pearton David J. , Yang Ying , Dhouailly Danielle
Published: March 2005

Differentiated cells of the corneal epithelium are converted to hair, along with their associated stem cells, then interfollicular epidermis, by means of a multistep process triggered by dermal developmental signals. The committed basal cells of the adult corneal epithelium dedifferentiate under the control of signals from an associated embryonic hair-forming dermis, likely Wnts, and revert to a limbal basal cell phenotype. This initial process involves the down-regulation of Pax6 and the loss of expression of corneal-specific keratins and the induction of basal keratinocyte markers. These dedifferentiated cells are able to reinduce dermal condensations, which in turn induce the formation of hair follicles from cells that have lost Pax6 expression, by means of a Noggin-dependent mechanism. An epidermis is subsequently formed by cells derived from the newly segregated hair stem cells.
Proton binding within a membrane protein by a protonated water cluster
Authors: Garczarek Florian , Brown Leonid S. , Lanyi Janos K. , Gerwert Klaus
Published: March 2005

Proton transfer is crucial for many enzyme reactions. Here, we show that in addition to protonatable amino acid side chains, water networks could constitute proton-binding sites in proteins. A broad IR continuum absorbance change during the proton pumping photocycle of bacteriorhodopsin (bR) indicates most likely deprotonation of a protonated water cluster at the proton release site close to the surface. We investigate the influence of several mutations on the proton release network and the continuum change, to gain information about the location and extent of the protonated water network and to reveal the participating residues necessary for its stabilization. We identify a protonated water cluster consisting in total of one proton and about five water molecules surrounded by six side chains and three backbone groups (Tyr-57, Arg-82, Tyr-83, Glu-204, Glu-194, Ser-193, Pro-77, Tyr-79, and Thr-205). The observed perturbation of proton release by many single-residue mutations is now explained by the influence of numerous side chains on the protonated H bonded network.
In situ
hydrogen/deuterium exchange Fourier transform IR measurements of the bR ground state, show that the proton of the release group becomes localized on Glu-204 and Asp-204 in the ground state of the mutants E194D and E204D, respectively, even though it is delocalized in the ground state of wild-type bR. Thus, the release mechanism switches between the wild-type and mutated proteins from a delocalized to a localized proton-binding site.
Hereditary juvenile cobalamin deficiency caused by mutations in the intrinsic factor gene
Authors: Tanner Stephan M. , Li Zhongyuan , Perko James D. , Öner Cihan , Çetin Mualla , Altay Çiğdem , Yurtsever Zekiye , David Karen L. , Faivre Laurence , Ismail Essam A. , Gräsbeck Ralph , de la Chapelle Albert
Published: March 2005

Hereditary juvenile megaloblastic anemia due to vitamin B12(cobalamin) deficiency is caused by intestinal malabsorption of cobalamin. In Imerslund–Gräsbeck syndrome (IGS), cobalamin absorption is completely abolished and not corrected by the administration of intrinsic factor (IF); if untreated, the disease is fatal. Biallelic mutations either in the cubilin (CUBN) or amnionless (AMN) gene cause IGS. In a series of families clinically diagnosed with likely IGS, at least six displayed no evidence of mutations inCUBNorAMN. A genome-wide search for linkage followed by mutational analysis of candidate genes was performed in five of these families. A region in chromosome 11 showed evidence of linkage in four families. The gastric IF (GIF) gene located in this region harbored homozygous nonsense and missense mutations in these four families and in three additional families. The disease in these cases therefore should be classified as hereditary IF deficiency. Clinically, these patients resembled those with typical IGS; radiocobalamin absorption tests had been inconclusive regarding the nature of the defect. In the diagnosis of juvenile cobalamin deficiency, mutational analysis of theCUBN,AMN, andGIFgenes provides a molecular characterization of the underlying defect and may be the diagnostic method of choice.
A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization
Authors: Zhao Xiaolan , Blobel Günter
Published: March 2005

Through a genetic screen using myosin-like protein strains
mlp1
Δ
mlp2
Δ and biochemical purification, we identified a complex of eight proteins, each required for growth and DNA repair in
Saccharomyces cerevisiae
. Among the subunits are Mms21 that contains a putative Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain characteristic of small ubiquitin-like m
o
difier (SUMO) ligases, two structural-maintenance-of-chromosome (Smc) proteins, Smc5 and Smc6, and a protein that contains an ubiquitin ligase signature domain. We show that these proteins colocalized to several distinct nuclear foci. Biochemical and genetic data demonstrated that Mms21 indeed functions as a SUMO ligase and that this activity requires the Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain. The substrates for this SUMO ligase include a subunit of the octameric complex, Smc5, and the DNA repair protein Yku70. We further show that the abolition of the SUMO E3 activity of Mms21 leads to such disparate phenotypes as DNA damage sensitivity, defects in nucleolar integrity and telomere clustering, silencing, and length regulation. We propose that Mms21 sumoylates proteins involved in these diverse processes and that the other members of the complex, particularly Smc5/6, facilitate proper substrate sumoylation by localizing Mms21 to specific chromosomal regions.
Inactivation of liver X receptor β leads to adult-onset motor neuron degeneration in male mice
Authors: Andersson Sandra , Gustafsson Nina , Warner Margaret , Gustafsson Jan-Åke
Published: March 2005

Male mice with inactivated liver X receptor (LXR) β suffer from adult-onset motor neuron degeneration. By 7 months of age, motor coordination is impaired, and this condition is associated with lipid accumulation and loss of motor neurons in the spinal cord, together with axonal atrophy and astrogliosis. Several of these features are reminiscent of the neuropathological signs of chronic motor neuron disease such as amyotrophic lateral sclerosis. Because the LXRs are important for cholesterol and lipid metabolism, we speculate that absence of LXRβ leads to pathological accumulation of sterols and lipids that may themselves be neurotoxic or may modulate intracellular pathways and thereby predispose motor neurons to degeneration.
Liver-specific inactivation of the Nrf1 gene in adult mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia
Authors: Xu Zhenrong , Chen Linyun , Leung Laura , Yen T. S. Benedict , Lee Candy , Chan Jefferson Y.
Published: March 2005

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. We report here that mice with somatic inactivation ofnrf1in the liver developed hepatic cancer. Before cancer development, mutant livers exhibited steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer.
Structure of human carboxypeptidase A4 with its endogenous protein inhibitor, latexin
Authors: Pallarès Irantzu , Bonet Roman , García-Castellanos Raquel , Ventura Salvador , Avilés Francesc X. , Vendrell Josep , Gomis-Rüth F. Xavier
Published: March 2005

The only endogenous protein inhibitor known for metallocarboxypeptidases (MCPs) is latexin, a 25-kDa protein discovered in the rat brain. Latexin, alias endogenous carboxypeptidase inhibitor, inhibits human CPA4 (hCPA4), whose expression is induced in prostate cancer cells after treatment with histone deacetylase inhibitors. hCPA4 is a member of the A/B subfamily of MCPs and displays the characteristic α/β-hydrolase fold. Human latexin consists of two topologically equivalent subdomains, reminiscent of cystatins, consisting of an α-helix enveloped by a curved β-sheet. These subdomains are packed against each other through the helices and linked by a connecting segment encompassing a third α-helix. The enzyme is bound at the interface of these subdomains. The complex occludes a large contact surface but makes rather few contacts, despite a nanomolar inhibition constant. This low specificity explains the flexibility of latexin in inhibiting all vertebrate A/B MCPs tested, even across species barriers. In contrast, modeling studies reveal why the N/E subfamily of MCPs and invertebrate A/B MCPs are not inhibited. Major differences in the loop segments shaping the border of the funnel-like access to the protease active site impede complex formation with latexin. Several sequences ascribable to diverse tissues and organs have been identified in vertebrate genomes as being highly similar to latexin. They are proposed to constitute the latexin family of potential inhibitors. Because they are ubiquitous, latexins could represent for vertebrate A/B MCPs the counterparts of tissue inhibitors of metalloproteases for matrix metalloproteinases.
Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients
Authors: Muraro Paolo A. , Douek Daniel C. , Packer Amy , Chung Katherine , Guenaga Francisco J. , Cassiani-Ingoni Riccardo , Campbell Catherine , Memon Sarfraz , Nagle James W. , Hakim Frances T. , Gress Ronald E. , McFarland Henry F. , Burt Richard K. , Martin Roland
Published: March 2005

Clinical trials have indicated that autologous hematopoietic stem cell transplantation (HSCT) can persistently suppress inflammatory disease activity in a subset of patients with severe multiple sclerosis (MS), but the mechanism has remained unclear. To understand whether the beneficial effects on the course of disease are mediated by lympho-depletive effects alone or are sustained by a regeneration of the immune repertoire, we examined the long-term immune reconstitution in patients with MS who received HSCT. After numeric recovery of leukocytes, at 2-yr follow-up there was on average a doubling of the frequency of naive CD4+ T cells at the expense of memory T cells. Phenotypic and T cell receptor excision circle (TREC) analysis confirmed a recent thymic origin of the expanded naive T cell subset. Analysis of the T cell receptor repertoire showed the reconstitution of an overall broader clonal diversity and an extensive renewal of clonal specificities compared with pretherapy. These data are the first to demonstrate that long-term suppression of inflammatory activity in MS patients who received HSCT does not depend on persisting lymphopenia and is associated with profound qualitative immunological changes that demonstrate a de novo regeneration of the T cell compartment.
An immunoglobulin Cκ-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system
Authors: Ait-Azzouzene Djemel , Verkoczy Laurent , Peters Jorieke , Gavin Amanda , Skog Patrick , Vela José Luis , Nemazee David
Published: March 2005

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igκ–reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igκ-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.
Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor–induced Nur77 expression and apoptosis
Authors: Dequiedt Franck , Van Lint Johan , Lecomte Emily , Van Duppen Viktor , Seufferlein Thomas , Vandenheede Jackie R. , Wattiez Ruddy , Kettmann Richard
Published: March 2005

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH2 terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.
Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product
Authors: Pasquier Benoit , Yin Luo , Fondanèche Marie-Claude , Relouzat Francis , Bloch-Queyrat Coralie , Lambert Nathalie , Fischer Alain , de Saint-Basile Geneviève , Latour Sylvain
Published: March 2005

SAP is an adaptor protein expressed in T cells and natural killer cells. It plays a critical role in immunity, as it is mutated in humans with X-linked lymphoproliferative syndrome (XLP), a fatal immunodeficiency characterized by an abnormal response to Epstein-Barr virus (EBV) infection. SAP interacts with the SLAM family receptors and promotes transduction signal events by these receptors through its capacity to recruit and activate the Src kinase FynT. Because it has been previously established that FynT is selectively required for the development of NKT cells, we examined NKT cells in SAP-deficient mice and in humans with XLP. In the absence of SAP, the development of NKT cells is severely impaired both in mice and in humans. These results imply that SAP is a potent regulator of NKT cell development. They also identify for the first time a defect in NKT cells associated with a human primary immunodeficiency, revealing a potential role of NKT cells in the immune response to EBV.
Coordinated group response to nest intruders in social shrimp
Authors: Tóth Eva , Duffy J. Emmett
Published: March 2005

A key characteristic of highly social animals is collective group response to important stimuli such as invasion by enemies. The marine societies of social snapping shrimp share many convergences with terrestrial eusocial animals, including aggressive reaction to strangers, but no group actions have yet been observed in shrimp. Here we describe ‘coordinated snapping’, during which a sentinel shrimp reacts to danger by recruiting other colony members to snap in concert for several to tens of seconds. This distinctive behaviour is a specific response to intrusion by strange shrimp into the colony's sponge and is highly successful at repelling these intruders. Although coordinated snapping apparently functions analogously to alarm responses in other social animals, colony members in social shrimp do not rush to the site of the attack. Coordinated snapping appears instead to be a warning signal to would-be intruders that the sponge is occupied by a cooperative colony ready to defend it. This is the first evidence for coordinated communication in social shrimp and represents yet another remarkable convergence between social shrimp, insects and vertebrates.
Herd-level risk factors associated with tuberculosis breakdowns among cattle herds in England before the 2001 foot‐and‐mouth disease epidemic
Authors: Johnston W. T , Gettinby G , Cox D. R , Donnelly C. A , Bourne J , Clifton-Hadley R , Le Fevre A. M , McInerney J. P , Mitchell A , Morrison W. I , Woodroffe R
Published: March 2005

A case–control study of the factors associated with the risk of a bovine tuberculosis (TB) breakdown in cattle herds was undertaken within the randomized badger culling trial (RBCT). TB breakdowns occurring prior to the 2001 foot-and-mouth disease epidemic in three RBCT triplets were eligible to be cases; controls were selected from the same RBCT area. Data from 151 case farms and 117 control farms were analysed using logistic regression. The strongest factors associated with an increased TB risk were movement of cattle onto the farm from markets or farm sales, operating a farm over multiple premises and the use of either covered yard or ‘other’ housing types. Spreading artificial fertilizers or farmyard manure on grazing land were both associated with decreased risk. These first case–control results from the RBCT will be followed by similar analyses as more data become available.
Perspective: machines for RNAi
Authors: Tomari Yukihide , Zamore Phillip D.
Published: March 2005

RNA silencing pathways convert the sequence information in long RNA, typically double-stranded RNA, into ∼21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs and miRNAs provide specificity to protein effector complexes that repress mRNA transcription or translation, or catalyze mRNA destruction. Here, we review our current understanding of how small RNAs are produced, how they are loaded into protein complexes, and how they repress gene expression.
Inactivation of TGFβ signaling in neural crest stem cells leads to multiple defects reminiscent of DiGeorge syndrome
Authors: Wurdak Heiko , Ittner Lars M. , Lang Karl S. , Leveen Per , Suter Ueli , Fischer Jan A. , Karlsson Stefan , Born Walter , Sommer Lukas
Published: March 2005

Specific inactivation of TGFβ signaling in neural crest stem cells (NCSCs) results in cardiovascular defects and thymic, parathyroid, and craniofacial anomalies. All these malformations characterize DiGeorge syndrome, the most common microdeletion syndrome in humans. Consistent with a role of TGFβ in promoting non-neural lineages in NCSCs, mutant neural crest cells migrate into the pharyngeal apparatus but are unable to acquire non-neural cell fates. Moreover, in neural crest cells, TGFβ signaling is both sufficient and required for phosphorylation of CrkL, a signal adaptor protein implicated in the development of DiGeorge syndrome. Thus, TGFβ signal modulation in neural crest differentiation might play a crucial role in the etiology of DiGeorge syndrome.
Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception
Authors: Warnecke Marei , Oster Henrik , Revelli Jean-Pierre , Alvarez-Bolado Gonzalo , Eichele Gregor
Published: March 2005

The orphan nuclear receptor Ear2 (Nr2f6) is transiently expressed in the rostral part of the rhombic lip in which the locus coeruleus (LC) arises. LC development, regulated by a signaling cascade (Mash1 → Phox2b → Phox2a), is disrupted in Ear2-/- embryos as revealed by an approximately threefold reduction in the number of Phox2a- and Phox2b-expressing LC progenitor cells. Mash1 expression in the rhombic lip, however, is unaffected, placing Ear2 in between Mash1 and Phox2a/b. Dopamine-β-hydroxylase and tyrosine hydroxylase staining demonstrate that >70% of LC neurons are absent in the adult with agenesis affecting primarily the dorsal division of the LC. Normally, this division projects noradrenergic efferents to the cortex that appear to be diminished in Ear2-/- since the cortical concentration of noradrenaline is four times lower in these mice. The rostral region of the cortex is known to contain a circadian pacemaker regulating adaptability to light- and restricted food-driven entrainment. In situ hybridization establishes that the circadian expression pattern of the clock gene Period1 is abolished in the Ear2-/- forebrain. Behavioral experiments reveal that Ear2 mutants have a delayed entrainment to shifted light-dark cycles and adapt less efficiently to daytime feeding schedules. We propose that neurons in the dorsal division of LC contribute to the regulation of the forebrain clock, at least in part, through targeted release of noradrenaline into the cortical area.
A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube
Authors: Stamataki Despina , Ulloa Fausto , Tsoni Stavroula V. , Mynett Anita , Briscoe James
Published: March 2005

During development, many signaling factors behave as morphogens, long-range signals eliciting different cellular responses according to their concentration. In ventral regions of the spinal cord, Sonic Hedgehog (Shh) is such a signal and controls the emergence, in precise spatial order, of distinct neuronal subtypes. The Gli family of transcription factors plays a central role in this process. Here we demonstrate that a gradient of Gli activity is sufficient to mediate, cell-autonomously, the full range of Shh responses in the neural tube. The incremental two- to threefold changes in Shh concentration, which determine alternative neuronal subtypes, are mimicked by similar small changes in the level of Gli activity, indicating that a gradient of Gli activity represents the intracellular correlate of graded Shh signaling. Moreover, our analysis suggests that cells integrate the level of signaling over time, consistent with the idea that signal duration, in addition to signal strength, is an important parameter controlling dorsal-ventral patterning. Together, these data indicate that Shh signaling is transduced, without amplification, into a gradient of Gli activity that orchestrates patterning of the ventral neural tube.