bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of metastables on a sustaining mechanism in inductively coupled plasma in Ar
Authors: Sato Toshikazu , Makabe Toshiaki
Published: December 2005

We numerically predicted the spatial distribution of Ar metastables in an inductively coupled plasma (ICP) source; this distribution may be an indicator of the behavior of long-lived neutral radicals in a reactive plasma. We investigated the effect of metastables on the sustaining mechanism in ICP in Ar. The predicted two-dimensional profile of Ar metastables agreed reasonably well with experimental results. The transition of the sustaining mechanism from direct ionization to stepwise ionization is found as a function of input power at 50 mTorr. In addition, a strong hysteresis of plasma density is predicted between the increasing and decreasing phases of the input power based on the stepwise ionization of Ar metastables in the ICP.
Counterstreaming magnetized plasmas. I. Parallel wave propagation
Authors: Tautz R. C. , Schlickeiser R.
Published: December 2005

The properties of longitudinal and transverse oscillations in magnetized counterstreaming Maxwellian plasmas of arbitrary composition and different thermal velocities for waves propagating parallel to the stream direction are investigated on the basis of the Maxwell equations and the relativistic Vlasov equation. These dispersion relations describe the linear response of the system to the initial perturbations and thus define all existing linear parallel propagating plasma modes in the system. The dispersion relations hold for nonrelativistic values of the plasma temperatures, streaming velocities, and for any values of the relative intensities of the two streams and for all wave-number and frequency values of the oscillations. All dispersion relations are expressed in terms of the well-documented Fried and Conte plasma dispersion function and are thus valid for the whole complex-frequency plane, which assures the validity of growth and damping rates.
Exciton quenching in poly(phenylene vinylene) polymer light-emitting diodes
Authors: Markov D. E. , Blom P. W. M.
Published: December 2005

The quenching of excitons at the metallic cathode of a polymer light-emitting diode (PLED) has been investigated by time-resolved photoluminescence. The decay of the luminescence is analyzed including both nonradiative energy transfer to the metallic cathode and exciton diffusion. Incorporation of the resulting exciton density profiles into a PLED device model consistently describes the reduction of the quantum efficiency at low bias voltage.
Cross focusing of two laser beams and plasma wave excitation
Authors: Gupta M. K. , Sharma R. P. , Gupta V. L.
Published: December 2005

This article presents the cross focusing of two high power laser beams in a plasma when relativistic and ponderomotive nonlinearities are operative. The effect of electron density modification changes the critical power significantly in contrast to (only) relativistic case. The plasma wave generation at the difference frequency and particle acceleration has also been studied. In a typical case when laser wavelengths are 1047 and 1064nm and electron density 1.9×1019cm−3, the maximum electron plasma wave power flux comes out to be 6×1017W∕cm2 (laser power P1=3.6×1018W∕cm2 and P2=3.2×1018W∕cm2).
Stealth communication: Zero-power classical communication, zero-quantum quantum communication and environmental-noise communication
Authors: Kish Laszlo B.
Published: December 2005

An alternative physical way of communication, communication by the inherent background noise, is proposed which does not need net energy transfer in the information channel. The communicator devices do dissipate energy; however, they do not emit net energy into the channel, instead of that, they modulate the parameters of inherent spontaneous fluctuations in the channel. The method can use two different mechanisms, thermal noise (Johnson–Nyquist noise) for classical communication, and vacuum fluctuations/zero-point energy (quantum uncertainty noise) for quantum communication. The strongest advantage of the method is that it is apparently the most hidden (stealth) way of communication, because it is using the inherent background noise for communication. Therefore, it is extremely difficult or impossible to discover its presence. With proper wave-based arrangements and specific conditions, the sender and the receiver can easily detect eavesdropper activities, so that the eavesdropper is detected as soon as she extracts a single bit of information, thus the security of the method is comparable to the security of quantum communication/quantum key distribution schemes. Finally, concerning practical applications, environmental noise, out of the fundamental/inherent fluctuations, can also be used for this kind of communication provided that is sufficiently stationary.
Application of a downstream calorimetric probe to reactive plasma
Authors: Chen Ing-Shin , Neuner Jeffery W. , Chen Philip S. H. , Welch James J. , DiMeo Frank
Published: December 2005

We explore the application of calorimetric probe located downstream from the plasma zone. By positioning the probe in a downstream location, the probe signal can be correlated with the integral energy flux carried by the effluent species as they propagate downstream from the plasma zone. Because the integral energy flux channeled downstream depends on the plasma conditions, it is possible to infer the plasma conditions from the probe response even though the probe is located away from the plasma zone. A calorimetric probe, based on resistance-temperature detector principle, adequate for fluorine plasma exposure is constructed. Probe operation is demonstrated for NF3–Ar plasmas.
Quantum efficiencies of PtSi∕p-Si composites in the 3–5μm wavelength range
Authors: Bates Clayton W. , Hendricks Alphonso
Published: December 2005

A formula for the quantum efficiency of Schottky barrier metal-semiconductor composite photon detectors is derived. It includes the two factors that completely describe the quantum efficiency of a planar Schottky barrier detector. It has in addition a third factor, that can be larger or smaller than one depending on the ratio of the mean-free path of the photoexcited carriers to the dispersed metal particle sizes. Using this formula, quantum efficiencies, due to photoexcited holes in experimentally prepared PtSi∕p-Si composite films, were calculated in the 3–5μm micron wavelength range using measured absorption and particle size data. The results provide evidence for the contribution of particle size effects to the efficiencies in PtSi∕p-Si composite detectors and are consistent with previous particle size effects in other metal-semiconductor composite materials.
Vegetation state change and consequent carbon dynamics in savanna woodlands of Australia in response to grazing, drought and fire: a scenario approach using 113 years of synthetic annual fire and grassland growth
Authors: Hill Michael J. , Roxburgh Stephen H. , Carter John O. , McKeon Gregory M.
Published: December 2005

A spatially explicit state and transition model for assessing the interactive effects of grazing, fire and climate on carbon dynamics in Australian savannas is described. The model runs on a yearly time step. It is based on a sequential treatment of events within each year, involving, in order, growth of biomass, consumption of biomass by livestock and burning of the remaining fuel (growth minus consumption). The major drivers are 113 years of annual rainfall data, annual modelled rangeland growth, synthetic fire incidence and timing data, and data describing stocking rates in dry sheep equivalents. Baseline carbon stocks are derived from pre-settlement estimates from the VAST steady-state carbon model. State and transition models for nine vegetation zones define vegetation condition and consequent carbon stock. Change is mediated by key indices of driver variables such as grazing, growth and fire, a series of parameters and default thresholds, and a set of rules. The model is run for three 113-year spin-up cycles to establish a set of initial condition grids that represent a plausible synthetic current state. Sensitivity analyses on selected parameters and index thresholds showed that fire and growth thresholds were most important for woodland zones, and utilisation rates and degradation and recovery periods were most important for grassland zones. The model was used to examine a series of scenarios involving changes to grazing pressure and suppression of fire for different climate sequences. Changes to a few parameters enabled simulation of low and high levels of encroachment of woody weeds, and hence carbon accumulation, in the Astrebla (Mitchell) grasslands; and effective capture of the climate-induced variation in grassland condition, and hence potential for soil carbon loss, within Heteropogon spp.-dominated grasslands in northern Queensland. The scenario results suggest that this simple state and transition model with an annual time step provides a potentially useful and flexible scenario model framework for exploration of vegetation and consequent carbon dynamics of the Australian tropical savanna region.
Viewpoint: social and economic dimensions of involving savanna communities in carbon management systems
Authors: Vella Karen J. , Williams Richard J. , Walker Daniel H. , Smajgl Alex , Kirschbaum Miko U. F. , Greiner Romy
Published: December 2005

In this paper we discuss the social, economic and institutional aspects of the development of carbon management systems within Australia’s tropical savannas. Land-use values in savanna landscapes are changing as a result of changing economic markets, greater recognition of native title, and growing social demands and expectations for tourism, recreation and conservation. In addition, there is increasing interest in developing markets and policy arrangements for greenhouse gas abatement, carbon sequestration and carbon trade in savannas. We argue that for carbon management to lead to national greenhouse outcomes, attention must be paid to social, economic and institutional issues in environmental planning and policy arrangements. From an economic perspective, the financial impact of carbon management on savanna enterprises will depend on appropriate and available policy mechanisms, unit price for carbon, landscape condition, existing management strategies and abatement measurements used. Local social and cultural features of communities and regions may enhance or constrain the implementation of carbon abatement strategies, depending on how they are perceived. In terms of institutional arrangements, policies and plans must support and enable carbon management. We identify three areas that require priority investigation and adjustment: regional planning arrangements, property rights, and rules for accounting at enterprise and regional scales. We conclude that the best potential for managing for carbon will be achieved while managing for range of other natural resource management outcomes, especially where managing for carbon delivers collateral benefits to enterprises.
Viewpoint: Is the European attitude to GM products suffocating African development?
Authors: Bodulovic Greg
Published: December 2005

Currently, parts of Southern Africa are experiencing the third major drought in five years. The previous two droughts greatly affected food production, resulting in food shortages, which necessitated the provision of food aid to the region by developed nations. However, some of the food aid included genetically modified (GM) crops, the supply of which triggered hostile reactions by southern African governments, and in one case resulted in food aid being withheld from people on the verge of starvation. This article will examine the background and reasons behind the condemnation of GM crops by southern African nations, and will consider whether the lack of support of agricultural biotechnology by European nations has contributed to this situation. Furthermore, the necessity of agricultural biotechnology in future African development will be considered.
Seasonal courses of maximum hydraulic conductance in shoots of six temperate deciduous tree species
Authors: Aasamaa Krõõt , Sõber Anu
Published: December 2005

The seasonal course of maximum hydraulic conductance of leaf laminae (K lamina) of shoots correlated strongly with the seasonal course of the maximum hydraulic conductance of leaf laminae of HgCl2-treated shoots (K lamina(HgCl2)), and with the seasonal course of the difference (dK lamina) between K lamina and K lamina(HgCl2). However, it did not correlate strongly with the seasonal course of the hydraulic conductance of stem and petioles of the shoot (K stpt) in six temperate deciduous tree species. The species ranked according to K lamina as follows: Populus tremula L. > Salix caprea L. > Padus avium Mill. > Quercus robur L. > Tilia cordata Mill. > Acer platanoides L. The species-specific maxima of K lamina correlated positively with the simultaneous values of K lamina(HgCl2), dK lamina and K stpt; the correlation was strongest with K lamina(HgCl2). It was concluded that the seasonal dynamics of maximum hydraulic conductance of leaf laminae was determined almost equally by the seasonal dynamics of the hydraulic conductance of foliar protoplasts and apoplast, but the inter-specific differences in K lamina were mainly caused by the different apoplastic hydraulic conductance in leaves of these species. The relative contribution of dK lamina (in K lamina) was highest in slow-growing species (~55% in A. platanoides) and the lowest in fast-growers (~30% in S. caprea).
Molecular tuning of fast gating in pentameric ligand-gated ion channels
Authors: Grutter Thomas , de Carvalho Lia Prado , Dufresne Virginie , Taly Antoine , Edelstein Stuart J. , Changeux Jean-Pierre
Published: December 2005

Neurotransmitters such as acetylcholine (ACh) and glycine mediate fast synaptic neurotransmission by activating pentameric ligand-gated ion channels (LGICs). These receptors are allosteric transmembrane proteins that rapidly convert chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with an extracellular agonist-binding domain (ECD), triggering a tertiary/quaternary conformational change in the protein that results in the fast opening of an ion pore domain (IPD). However, the molecular mechanism that determines the fast opening of LGICs remains elusive. Here, we show by combining whole-cell and single-channel recordings of recombinant chimeras between the ECD of α7 nicotinic receptor (nAChR) and the IPD of the glycine receptor (GlyR) that only two GlyR amino acid residues of loop 7 (Cys-loop) from the ECD and at most five α7 nAChR amino acid residues of the M2-M3 loop (2-3L) from the IPD control the fast activation rates of the α7/Gly chimera and WT GlyR. Mutual interactions of these residues at a critical pivot point between the agonist-binding site and the ion channel fine-tune the intrinsic opening and closing rates of the receptor through stabilization of the transition state of activation. These data provide a structural basis for the fast opening of pentameric LGICs.
Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels
Authors: Surti Toral S. , Huang Lan , Jan Yuh Nung , Jan Lily Y. , Cooper Edward C.
Published: December 2005

Neuronal potassium channel subunits of the KCNQ (Kv7) family underlie M-current (I
M
), and may also underlie the slow potassium current at the node of Ranvier, I
Ks
. I
M
and I
Ks
are outwardly rectifying currents that regulate excitability of neurons and myelinated axons, respectively. Studies of native I
M
and heterologously expressed Kv7 subunits suggest that,
in vivo
, KCNQ channels exist within heterogeneous, multicomponent protein complexes. KCNQ channel properties are regulated by protein phosphorylation, protein-protein interactions, and protein-lipid interactions within such complexes. To better understand the regulation of neuronal KCNQ channels, we searched directly for posttranslational modifications on KCNQ2/KCNQ3 channels
in vivo
by using mass spectrometry. Here we describe two sites of phosphorylation. One site, specific for KCNQ3, appears functionally silent in electrophysiological assays but is located in a domain previously shown to be important for subunit tetramerization. Mutagenesis and electrophysiological studies of the second site, located in the S4-S5 intracellular loop of all KCNQ subunits, reveal a mechanism of channel inhibition.
Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism
Authors: Wang Dai , Shenk Thomas
Published: December 2005

Human cytomegalovirus replicates in many different cell types, including epithelial cells, endothelial cells, and fibroblasts. However, laboratory strains of the virus, many of which were developed as attenuated vaccine candidates by serial passage in fibroblasts, have lost the ability to infect epithelial and endothelial cells. Their growth is restricted primarily to fibroblasts, due to mutations in the UL131-UL128 locus. We now demonstrate that two products of this locus, pUL130 and pUL128, form a complex with gH and gL, but not gO. The AD169 laboratory strain, which lacks a functional UL131 protein, produces virions containing only the gH-gL-gO complex. An epithelial and endothelial cell tropic AD169 variant in which the UL131 ORF has been repaired, termed BAD
r
UL131, produces virions that carry both gH-gL-gO and gH-gL-pUL128-pUL130 complexes. Antibodies against pUL130 and pUL128 block infection of epithelial and endothelial cells by BAD
r
UL131 and the fusion-inducing factor X clinical human cytomegalovirus isolate but do not affect the efficiency with which fibroblasts are infected.
Regulation of N-Cadherin Dynamics at Neuronal Contacts by Ligand Binding and Cytoskeletal Coupling
Authors: Thoumine Olivier , Lambert Mireille , Mège René-Marc , Choquet Daniel
Published: December 2005

N-cadherin plays a key role in axonal outgrowth and synaptogenesis, but how neurons initiate and remodel N-cadherin-based adhesions remains unclear. We addressed this issue with a semiartificial system consisting of N-cadherin coated microspheres adhering to cultured neurons transfected for N-cadherin-GFP. Using optical tweezers, we show that growth cones are particularly reactive to N-cadherin coated microspheres, which they capture in a few seconds and drag rearward. Such strong coupling requires an intact connection between N-cadherin receptors and catenins. As they move to the basis of growth cones, microspheres slow down while gradually accumulating N-cadherin-GFP, demonstrating a clear delay between bead coupling to the actin flow and receptor recruitment. Using FRAP and photoactivation, N-cadherin receptors at bead-to-cell contacts were found to continuously recycle, consistently with a model of ligand-receptor reaction not limited by membrane diffusion. The use of N-cadherin-GFP receptors truncated or mutated in specific cytoplasmic regions show that N-cadherin turnover is exquisitely regulated by catenin partners. Turnover rates are considerably lower than those obtained previously in single molecule studies, demonstrating an active regulation of cadherin bond kinetics in intact cells. Finally, spontaneous neuronal contacts enriched in N-cadherin exhibited similar turnover rates, suggesting that such dynamics of N-cadherin may represent an intrinsic mechanism underlying the plasticity of neuronal adhesions.
Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii
Authors: Sahoo Nivedita , Beatty Wandy , Heuser John , Sept David , Sibley L. David
Published: December 2005

Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.
Late Activation of Stress Kinases (SAPK/JNK) by Genotoxins Requires the DNA Repair Proteins DNA-PKcsand CSB
Authors: Fritz Gerhard , Kaina Bernd
Published: December 2005

Although genotoxic agents are powerful inducers of stress kinases (SAPK/JNK), the contribution of DNA damage itself to this response is unknown. Therefore, SAPK/JNK activation of cells harboring specific defects in DNA damage-recognition mechanisms was studied. Dual phosphorylation of SAPK/JNK by the genotoxin methyl methanesulfonate (MMS) occurred in two waves. The early response (≤2 h after exposure) was similar in cells knockout for ATM, PARP, p53, and CSB or defective in DNA-PKcscompared with wild-type cells. The late response however (≥4 h), was drastically reduced in DNA-PKcsand Cockayne's syndrome B (CSB)-deficient cells. Similar results were obtained with human cells lacking DNA-PKcsand CSB. Activation of SAPK/JNK by MMS was not affected upon inhibition of base excision repair (BER), indicating base damage itself does not signal to SAPK/JNK. Because SAPK/JNK activation was attenuated in nongrowing cells, DNA replication-dependent processing of lesions, involving DNA-PKcsand CSB, appears to be required. DNA-PKcscoprecipitates with SEK1/MKK4 and SAPK/JNK, supporting a role of DNA-PKcsin SAPK/JNK activation. In this process, Rho GTPases are involved since inhibition of Rho impairs MMS-induced signaling to SAPK/JNK. The data show that sensing of DNA damage by DNA-PKcsand CSB causes a delayed SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK.
Two Independent Pathways Traffic the Intraperoxisomal Peroxin PpPex8p into Peroxisomes: Mechanism and Evolutionary Implications
Authors: Zhang Lan , Léon Sébastien , Subramani Suresh
Published: December 2005

Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix. We show that Pichia pastoris Pex8p (PpPex8p) enters the peroxisome matrix using two redundant pathways in a Pex14p-dependent, but Pex2p-independent, manner, showing that the intact importomer and RING subcomplex are not required for its import. One pathway depends on the TPR motifs in Pex5p, the C-terminal PTS1 sequence (AKL) in PpPex8p, and the intraperoxisomal presence of this peroxin. The alternative pathway uses the PTS2 receptor, Pex7p, its accessory protein, Pex20p, and a putative PTS2 motif in PpPex8p, but does not require intraperoxisomal PpPex8p. Pex20p interaction with PpPex8p is independent of Pex7p, but the interaction of PpPex8p with Pex7p requires Pex20p. These data suggest a direct interaction between PpPex8p and Pex20p. Our studies shed light on the mechanism and evolution of the dual import pathways for PpPex8p.
Cell Cycle-dependent Recruitment of Telomerase RNA and Cajal Bodies to Human Telomeres
Authors: Jády Beáta E. , Richard Patricia , Bertrand Edouard , Kiss Tamás
Published: December 2005

Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10-40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance.
Regulated Cellular Partitioning of SR Protein-specific Kinases in Mammalian Cells
Authors: Ding Jian-Hua , Zhong Xiang-Yang , Hagopian Jonathan C. , Cruz Marissa M. , Ghosh Gourisankar , Feramisco James , Adams Joseph A. , Fu Xiang-Dong
Published: December 2005

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.
Smad transcription factors
Authors: Massagué Joan , Seoane Joan , Wotton David
Published: December 2005

Smad transcription factors lie at the core of one of the most versatile cytokine signaling pathways in metazoan biology—the transforming growth factor-β (TGFβ) pathway. Recent progress has shed light into the processes of Smad activation and deactivation, nucleocytoplasmic dynamics, and assembly of transcriptional complexes. A rich repertoire of regulatory devices exerts control over each step of the Smad pathway. This knowledge is enabling work on more complex questions about the organization, integration, and modulation of Smad-dependent transcriptional programs. We are beginning to uncover self-enabled gene response cascades, graded Smad response mechanisms, and Smad-dependent synexpression groups. Our growing understanding of TGFβ signaling through the Smad pathway provides general principles for how animal cells translate complex inputs into concrete behavior.
Targeting of cohesin by transcriptionally silent chromatin
Authors: Chang Chuang-Rung , Wu Ching-Shyi , Hom Yolanda , Gartenberg Marc R.
Published: December 2005

Eukaryotic DNA replication produces sister chromatids that are linked together until anaphase by cohesin, a ring-shaped protein complex that is thought to act by embracing both chromatids. Cohesin is enriched at centromeres, as well as discrete sites along chromosome arms where transcription positions the complex between convergent gene pairs. A relationship between cohesin and Sir-mediated transcriptional silencing has also begun to emerge. Here we used fluorescence microscopy and site-specific recombination to characterize interactions between newly replicated copies of the silent HMR mating-type locus. HMR was tagged with lac-GFP and flanked by binding sites for an inducible site-specific recombinase. Excision of the locus in cells with sister chromatids produced two chromatin circles that remained associated with one another. Pairing of the circles required silent chromatin, cohesin, and the RSC chromatin-remodeling complex. Chromatin immunoprecipitation showed that targeting of cohesin to the locus is Sir-dependent, and functional tests showed that silent chromatin acts in a continuous fashion to maintain cohesion. Remarkably, loss of silencing led to loss of cohesin from linear chromosomal templates but not from excised chromatin circles. The results are consistent with a model in which cohesin binds silent chromatin via topological linkage to individual chromatids.
Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells
Authors: Takeda David Y. , Shibata Yoshiyuki , Parvin Jeffrey D. , Dutta Anindya
Published: December 2005

Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy.
NIR is a novel INHAT repressor that modulates the transcriptional activity of p53
Authors: Hublitz Philip , Kunowska Natalia , Mayer Ulrich P. , Müller Judith M. , Heyne Kristina , Yin Na , Fritzsche Claudia , Poli Cecilia , Miguet Laurent , Schupp Ingo W. , van Grunsven Leo A. , Potiers Noëlle , van Dorsselaer Alain , Metzger Eric , Roemer Klaus , Schüle Roland
Published: December 2005

Most transcriptional repression pathways depend on the targeted deacetylation of histone tails. In this report, we characterize NIR, a novel transcriptional corepressor with inhibitor of histone acetyltransferase (INHAT) activity. NIR (Novel INHAT Repressor) is ubiquitously expressed throughout embryonic development and adulthood. NIR is a potent transcriptional corepressor that is not blocked by histone deacetylase inhibitors and is capable of silencing both basal and activator-driven transcription. NIR directly binds to nucleosomes and core histones and prevents acetylation by histone acetyltransferases, thus acting as a bona fide INHAT. Using a tandem affinity purification approach, we identified the tumor suppressor p53 as a NIR-interacting partner. Association of p53 and NIR was verified in vitro and in vivo. Upon recruitment by p53, NIR represses transcription of both p53-dependent reporters and endogenous target genes. Knock-down of NIR by RNA interference significantly enhances histone acetylation at p53-regulated promoters. Moreover, p53-dependent apoptosis is robustly increased upon depletion of NIR. In summary, our findings describe NIR as a novel INHAT that plays an important role in the control of p53 function.
Recalling an aversive experience by day-old chicks is not dependent on somatic protein synthesis
Authors: Mileusnic Radmila , Lancashire Christine L. , Rose Steven P.R.
Published: December 2005

Long-term memory is dependent on protein synthesis and inhibiting such synthesis following training results in amnesia for the task. Proteins synthesized during training must be transported to the synapse and disrupting microtubules with Colchicines, and hence, blocking transport, results in transient amnesia. Reactivating memory for a previously learned avoidance triggers a biochemical cascade analogous to that following the initial training and renders the memory labile once more to protein synthesis inhibitors. However, the reminder-induced cascade differs in certain key features from that following training. Here we show that in a one-trial passive avoidance task in chicks, in contrast with initial consolidation following training, memory following a reminder is not impaired by Colchicine. We conclude that recall after a reminder does not require synaptic access to somatically synthesized proteins in this task. Our results support the hypothesis that in the chick, a reminder may instead engage local protein synthesis at the synapse, rather than in the soma.
Sensory responsiveness and the effects of equal subjective rewards on tactile learning and memory of honeybees
Authors: Scheiner Ricarda , Kuritz-Kaiser Anthea , Menzel Randolf , Erber Joachim
Published: December 2005

In tactile learning, sucrose is the unconditioned stimulus and reward, which is usually applied to the antenna to elicit proboscis extension and which the bee can drink when it is subsequently applied to the extended proboscis. The conditioned stimulus is a tactile object that the bee can scan with its antennae. In this paper we describe the quantitative relationships between gustatory antennal stimulation, gustatory proboscis stimulation, and tactile learning and memory. Bees are 10-fold more responsive to sucrose solutions when they are applied to the antenna compared to proboscis stimulation. During tactile conditioning, the sucrose solution applied to the proboscis determines the level of acquisition, whereas antennal input is of minor importance. Bees differing in their gustatory responsiveness measured at the antenna differ strongly in their tactile acquisition and memory. We demonstrate how these differences in tactile acquisition and memory can be greatly reduced by calculating equal subjective rewards, based on individual gustatory responsiveness.
Structure of MurF from Streptococcus pneumoniae co‐crystallized with a small molecule inhibitor exhibits interdomain closure
Authors: Longenecker Kenton L. , Stamper Geoffrey F. , Hajduk Philip J. , Fry Elizabeth H. , Jakob Clarissa G. , Harlan John E. , Edalji Rohinton , Bartley Diane M. , Walter Karl A. , Solomon Larry R. , Holzman Thomas F. , Gu Yu Gui , Lerner Claude G. , Beutel Bruce A. , Stoll Vincent S.
Published: December 2005

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small‐molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X‐ray crystallography, which reveal the multidomain structure of a MurF–inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate‐binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure‐based design to explore the suitability of MurF as a pharmaceutical target.
A statistical approach to the interpretation of molecular dynamics simulations of calmodulin equilibrium dynamics
Authors: Likić Vladimir A. , Gooley Paul R. , Speed Terence P. , Strehler Emanuel E.
Published: December 2005

A sample of 35 independent molecular dynamics (MD) simulations of calmodulin (CaM) equilibrium dynamics was prepared from different but equally plausible initial conditions (20 simulations of the wild‐type protein and 15 simulations of the D129N mutant). CaM's radius of gyration and backbone mean‐square fluctuations were analyzed for the effect of the D129N mutation, and simulations were compared with experiments. Statistical tests were employed for quantitative comparisons at the desired error level. The computational model predicted statistically significant compaction of CaM relative to the crystal structure, consistent with the results of small‐angle X‐ray scattering (SAXS) experiments. This effect was not observed in several previously reported studies of (Ca2+)4‐CaM, which relied on a single MD run. In contrast to radius of gyration, backbone mean‐square fluctuations showed a distinctly non‐normal and positively skewed distribution for nearly all residues. Furthermore, the D129N mutation affected the backbone dynamics in a complex manner and reduced the mobility of Glu123, Met124, Ile125, Arg126, and Glu127 located in the adjacent α‐helix G. The implications of these observations for the comparisons of MD simulations with experiments are discussed. The proposed approach may be useful in studies of protein equilibrium dynamics where MD simulations fall short of properly sampling the conformational space, and when the comparison with experiments is affected by the reproducibility of the computational model.
The kinetic behavior of insulin fibrillation is determined by heterogeneous nucleation pathways
Authors: Librizzi Fabio , Rischel Christian
Published: December 2005

When subjected to acidic conditions and high temperature, insulin is known to produce fibrils that display the common properties of disease amyloids. Thus, clarifying the mechanisms of insulin fibrillation can help the general understanding of amyloidal aggregation. Insulin fibrillation exhibits a very sharp time dependence, with a pronounced lag phase and subsequent explosive growth of amyloidal aggregates. Here we show that the initial stages of this process can be well described by exponential growth of the fibrillated proteins. This indicates that the process is mainly controlled by a secondary nucleation pathway.
Gateway vectors for the production of combinatorially‐tagged His6‐MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli
Authors: Nallamsetty Sreedevi , Austin Brian P. , Penrose Kerri J. , Waugh David S.
Published: December 2005

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP‐tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His‐tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.
A common dimerization interface in bacterial response regulators KdpE and TorR
Authors: Toro‐Roman Alejandro , Wu Ti , Stock Ann M.
Published: December 2005

Bacterial response regulators are key regulatory proteins that function as the final elements of so‐called two‐component signaling systems. The activities of response regulators in vivo are modulated by phosphorylation that results from interactions between the response regulator and its cognate histidine protein kinase. The level of response regulator phosphorylation, which is regulated by intra‐or extracellular signals sensed by the histidine protein kinase, ultimately determines the output response that is initiated or carried out by the response regulator. We have recently hypothesized that in the OmpR/PhoB subfamily of response regulator transcription factors, this activation involves a common mechanism of dimerization using a set of highly conserved residues in the α4–β5–α5 face. Here we report the X‐ray crystal structures of the regulatory domains of response regulators TorR (1.8 Å), Ca2+‐bound KdpE (2.0 Å), and Mg2+/BeF3−‐bound KdpE (2.2 Å), both members of the OmpR/ PhoB subfamily from Escherichia coli. Both regulatory domains form symmetric dimers in the asymmetric unit that involve the α4–β5–α5 face. As observed previously in other OmpR/PhoB response regulators, the dimer interfaces are mediated by highly conserved residues within this subfamily. These results provide further evidence that most all response regulators of the OmpR/ PhoB subfamily share a common mechanism of activation by dimerization.
Evaluation of biochemical methods for the non‐destructive identification of sex in upstream migrating salmon and sea trout
Authors: Pottinger T. G. , Pulman K. G. T. , Carrick T. R. , Scott A. P.
Published: December 2005

Female‐specific markers of reproductive activity [plasma 17β‐oestradiol (E2), vitellogenin (VTG) and alkali‐labile phosphoprotein phosphorous (ALP)] were measured over 12 months in a captive population of brown trout Salmo trutta. During the early months of the reproductive season (February to May) and using the concentration of plasma E2 or plasma ALP as a marker for females the proportion of fish in which sex was misidentified was high (15–50%). The misidentification rate was considerably lower (1–8%) using plasma VTG. Preliminary evaluation of a commercial immunochromatographic VTG test system as a screen for the presence or absence of VTG in plasma from brown trout provided results that were consistent with those obtained from direct measurement of plasma VTG levels by enzyme‐linked immunosorbent assay (ELISA). These preliminary conclusions were verified by sampling upstream‐migrating anadromous brown trout, sea trout, and Atlantic salmon Salmo salar trapped over a 6 month period. Plasma E2 levels did not satisfactorily discriminate between male and female sea trout and Atlantic salmon. Plasma VTG levels in both species, however, were bimodally distributed and it was assumed that this divergence corresponded to male (plasma VTG levels <10 μg ml−1) and female (plasma VTG levels >800 μg ml−1) fishes. Plasma ALP provided a more accurate indication of sex in the wild Atlantic salmon and sea trout than was suggested by the pilot study on captive brown trout. The commercial immunochromatographic VTG test system provided results that were wholly consistent with the data obtained from the trapped fishes by direct measurement of plasma VTG.
Occurrence of dusky grouper Epinephelus marginatus (Lowe, 1834) in gulfs of northern Patagonia, Argentina
Authors: Irigoyen A. J. , Galván D. E. , Venerus L. A.
Published: December 2005

The occurrence of the dusky grouper Epinephelus marginatus in northern Patagonian Gulfs San Matías and Nuevo, Argentina, is recorded. This finding represents the southernmost record of the species for the southern hemisphere.
Parasites of Cynoscion guatucupa along South American Atlantic coasts: evidence for stock discrimination
Authors: Timi J. T. , Luque J. L. , Sardella N. H.
Published: December 2005

Two stocks of striped weakfish Cynoscion guatucupa in the south‐west Atlantic Ocean, were identified using parasites as biological tags. A total of 297 fish caught in Brazil, Uruguay and Argentina, contained 29 species of metazoan parasites. Univariate analysis on parasite populations, as well as multivariate discriminant analysis, calculated for juvenile and adult fish separately, allowed the identification of the two stocks, one from Argentina and Uruguay and the other from Brazil, to be made. Southern samples were characterized by higher prevalences and abundance of larval endohelminths, whereas in the northern stock, gastrointestinal and ectoparasitic species were common. Discriminant analyses on parasite infracommunities of Brazilian fish showed notable differences between juvenile and adult hosts; no such trend was observed in fish from Argentina and Uruguay, despite differences between juveniles and adults at the population level. Different oceanographic conditions and their influence on the distribution of parasites as well as of other hosts involved in their life cycles could be key factors for the differences observed among stocks of C. guatucupa.
Age and growth of Saurenchelys (Nettastomatidae) and Dysomma (Synaphobranchidae) leptocephali in the East China Sea
Authors: Ma T. , Miller M. J. , Shinoda A. , Minagawa G. , Aoyama J. , Tsukamoto K.
Published: December 2005

The otolith microstructures of the leptocephali of Saurenchelys stylura and Dysomma sp., collected in November and December 2000 in the East China Sea, were examined to determine their larval ages and growth rates, and the spawning times of these two species of outer shelf and slope marine eels. Leptocephali ranging in size from 8 to 48 mm total length were examined, and the nettastomatid, S. stylura, and the synaphobranchid, Dysomma sp., had estimated ages that ranged from 16 to 75 days and 17 to 66 days, respectively. The overall growth rate of S. stylura was 0·68 mm day−1(n = 21), and of Dysomma sp. was 0·44 mm day−1(n = 22). These growth rates were similar or slightly faster than those observed for anguillid leptocephali in offshore areas of the western Pacific. The backcalculated hatching dates for these two species were from September to November. The otolith increment widths of S. stylura showed an increase before 20 to 30 days that were similar to those in anguillid species, but in Dysomma sp. there were no remarkable increases.
Genetic differentiation between close eastern Mediterranean Dicentrarchus labrax (L.) populations
Authors: Castilho R. , Ciftci Y.
Published: December 2005

Differentiation at nine microsatellite loci revealed that a Levantine Basin sea bass Dicentrarchus labrax population probably represents a further subdivision of this species in the eastern Mediterranean.