bluuebunny/crossref_metadata_2025 huggingface dataset:
Preamble computation in automated test case generation using constraint logic programming
Authors: Colin Séverine , Legeard Bruno , Peureux Fabien
Published: September 2004

BZ‐Testing‐Tools (BZ‐TT) is a tool‐set for automated model‐based test case generation from B abstract machines and Z specifications. BZ‐TT uses boundary testing as well as cause–effect testing on the basis of the formal model. It has been used and validated on several industrial case studies in the domain of critical software: in particular for smart card applications and automotive embedded systems. The main idea of BZ‐TT is to compute a boundary goal for each effect of the operations of the model and then to compute a preamble sequence of operations to place the system under test in such a state that satisfies the goal.

In this paper, the preamble computation search strategies used in BZ‐TT are presented. More precisely, two algorithms based respectively on forward chaining and backward chaining are compared. These algorithms both use a customized set constraint solver, which is able to animate the formal model. These algorithms differ, however, in their capacity to reach the boundary goals efficiently. The results of applying the tools to an industrial windscreen wiper controller application are presented. Copyright © 2004 John Wiley & Sons, Ltd.
Discursive identity: Assimilation into the culture of science and its implications for minority students
Authors: Brown Bryan A.
Published: September 2004

This study examined how, in some instances, participation in the cultural practices of high school science classrooms created intrapersonal conflict for ethnic minority students. Discourse analysis of videotaped science classroom activities, lectures, and laboratories was the primary methodology employed for analyzing students' discursive identity development. This analysis demonstrated differential appropriation of science discourse as four significant domains of discursive identities emerged: Opposition status, Maintenance status, Incorporation status, and Proficiency status. Students characterized as Opposition Status avoided use of science discourse. Students who exhibited Maintenance Status illustrated a commitment to maintaining their normative discourse behavior, despite a demonstrated ability to appropriate science discourse. Students characterized as Incorporation Status made active attempts to incorporate science discourse into their normative speech patterns, while Proficiency Status students demonstrated a fluency in applying scientific discursive. Implications for science education emerging from the study include the illumination of the need to make the use of specific scientific discourse an explicit component of classroom curriculum. © 2004 Wiley Periodicals, Inc. J Res Sci Teach 41: 810–834, 2004
Interaction between halogenated aromatic compounds in the Ah receptor signal transduction pathway
Authors: Chen Guosheng , Bunce Nigel J.
Published: September 2004

Many toxic and biochemical responses to halogenated aromatic compounds (HACs) such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo‐p‐dioxins (PCDDs) are mediated through the aryl hydrocarbon receptor (AhR), which is an intracellular cytosolic target for HACs. Environmental exposure to HACs almost always involves complex mixtures of congeners, some of which can antagonize the action of potent HACs such as 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). In this work we studied TCDD and representative PCB congeners, alone and in mixture, for their effect on CYP1A gene transcription and protein levels in primary rat hepatocytes. Together with our previous work, our results suggest that formation of the Ah receptor‐ligand‐DRE (dioxin response element) complex is the principal point of divergence in the mechanism between an AhR agonist and an AhR antagonist. The coplanar PCBs 77 and 126 and the mono‐ortho PCB 156 were full agonists toward CYP1A1 gene transcription and CYP1A protein levels, showing typical additive behavior with TCDD to the target molecule AhR. In contrast, the nonplanar PCB 153 antagonized the action of TCDD, even at concentrations that occupied a significant fraction of AhR molecules. Competitive inhibition explains the commonly reported decrease of ethoxyresorufin‐O‐deethylase (EROD) activity when PCBs are present in high concentrations and the antagonism of PCBs to the EROD activity of TCDD. The result is that Western blotting offers a much more reliable measure of CYP1A protein concentration than does the EROD assay, despite the greater convenience of the latter. © 2004 Wiley Periodicals, Inc. Environ Toxicol 19: 480–489, 2004.
Higher order chromatin architecture in the cell nucleus: on the way from structure to function
Authors: Cremer Thomas , Küpper Katrin , Dietzel Steffen , Fakan Stanislav
Published: September 2004

Abstract The cell nucleus is a highly compartmentalized structure. In this review we describe controversial views on higher order chromatin organization from the level of higher order chromatin domains built up from folded chromatin fibers to the level of chromosome territories and the interchromatin compartment (IC), which harbors non‐chromatin nuclear domains, such as interchromatin granule clusters (IGCs visualized in the electron microscope) or splicing factor‐containing speckles (visualized by fluorescence microscopy). Emphasis is laid on the definition and functional importance of a nuclear compartment located at the periphery of chromatin domains in direct contact with the IC, termed the perichromatin region (PR). Ongoing experiments to elucidate the topological relationships between PR and IC have provided new insights into the functional interplay between transcription and splicing. As an example, we discuss the structure and nuclear topology of perichromatin fibrils (FPs) contained in the PR and their functional interplay with IGCs/speckles. In addition we discuss the advantages and drawbacks of experimental approaches currently used to study nuclear architecture and function in fixed and living cells.
Ostreolysin, a pore‐forming protein from the oyster mushroom, interacts specifically with membrane cholesterol‐rich lipid domains
Authors: Sepčić Kristina , Berne Sabina , Rebolj Katja , Batista Urška , Plemenitaš Ana , Šentjurc Marjeta , Maček Peter
Published: September 2004

Ostreolysin, a 15 kDa pore‐forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono‐ or di‐unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysin's activity. It appears that the protein recognizes specifically a cholesterol‐rich lipid phase, probably the liquid‐ordered phase.
Evidence that haem iron in the malaria parasite is not needed for the antimalarial effects of artemisinin
Authors: Parapini Silvia , Basilico Nicoletta , Mondani Monica , Olliaro Piero , Taramelli Donatella , Monti Diego
Published: September 2004

The role of haem iron (II) and oxidative stress in the activation and antimalarial activity of artemisinin is unclear. Thus, we submitted malaria parasite to modified culture conditions: artemisinin activity increased by 20–30% under an oxygen‐rich atmosphere (20% O2 instead of “standard” 1% O2), and by 40–50% in the presence of carboxy‐haemoglobin, and 2% carbon monoxide, conditions which inhibit haem iron (II) reactivity. In all cases, parasite growth and chloroquine activity were unaffected. We conclude that in the malaria parasite artemisinin is not activated by haem iron and that free radicals are not needed for its toxicity.
Insertion of spliceosomal introns in proto‐splice sites: the case of secretory signal peptides
Authors: Tordai Hedvig , Patthy László
Published: September 2004

Analysis of the exon–intron structures of 2208 human genes has revealed that there is a statistically highly significant excess of phase 1 introns in the vicinity of the signal peptide cleavage sites. It is suggested that amino acid sequences surrounding signal peptide cleavage sites are significantly enriched in phase 1 proto‐splice sites and this has favored insertion of spliceosomal introns in these sites.
Nek2A kinase regulates the localization of numatrin to centrosome in mitosis
Authors: Yao Jianhui , Fu Chuanhai , Ding Xia , Guo Zhen , Zenreski Arzhang , Chen Yong , Ahmed Kashif , Liao Jian , Dou Zhen , Yao Xuebiao
Published: September 2004

Chromosome segregation in mitosis is orchestrated by the kinetochore and spindle microtubules stemming from two centrosomes. Our recent studies demonstrated the importance of Nek2A in faithful chromosome segregation during mitosis. Here, we report that Nek2A regulates the function of numatrin in mitosis. The biochemical interaction between Nek2A and numatrin in mitotic cells was revealed by a set of reciprocal immunoprecipitation experiments using Nek2A and numatrin antibodies, respectively. The interaction is validated by a pull‐down assay using recombinant Nek2A and numatrin proteins. Moreover, our immunofluorescence studies demonstrate that numatrin becomes centrosome‐associated as the cell enters into mitosis and depart from the centrosome after sister chromatid separation in anaphase. The co‐localization of numatrin and Nek2A to the centrosome suggests their interaction with and involvement in centrosome function. Indeed, elimination of Nek2A kinase by siRNA diminished its association with the centrosome. Furthermore, we show that numatrin is phosphorylated by wild type but not kinase‐death Nek2A. Our studies suggest that the Nek2A kinase cascade is essential for the localization of numatrin to the centrosome.
Exip, a splicing variant of p38α, participates in interleukin‐1 receptor proximal complex and downregulates NF‐κB pathway
Authors: Yagasaki Yuki , Sudo Tatsuhiko , Osada Hiroyuki
Published: September 2004

Here we report that Exip, but not p38α, binds to Toll interacting protein which is involved in interleukin‐1 (IL‐1) signaling pathway as a component of the receptor proximal complex and impaired NF‐κB activity. Moreover, Exip binds to another component of the complex, IL‐1 associating kinase. Exogenous‐expression of Exip resulted in downregulation of NF‐κB activities both in HeLa and HEK293T cells. Together, these results demonstrate that Exip can be a new component of NF‐κB pathway, and contribute to a comprehensive understanding of the signal transduction pathway in the inflammatory responses.
Identification of an essential sequence for dihydroceramide C‐4 hydroxylase activity of mouse DES2
Authors: Omae Fumio , Miyazaki Masao , Enomoto Ayako , Suzuki Akemi
Published: September 2004

Although the amino acid sequences of mouse DES1 (MDES1) and DES2 (MDES2) have 63% sequence identity, their enzymatic characteristics are quite different. MDES1 exhibits high dihydroceramide Δ4‐desaturase activity and very low C‐4 hydroxylase activity, while MDES2 is similarly active as both a dihydroceramide Δ4‐desaturase and a C‐4 hydroxylase. We constructed several chimeras of MDES1 and MDES2 and identified a region important for C‐4 hydroxylase activity in MDES2. This region contains the sequence XAFGY (X=T or A or V; Y=T or N) and occurs on the C‐terminal side of the first His‐box of MDES2. We confirmed the conservation of this region in DES2 family members sequenced from humans, pigs, rats, chickens, zebrafish, and Xenopus.
Management of Pseudomeningocele Following Neurotologic Procedures
Authors: Mehendale Neelesh H. , Samy Ravi N. , Roland Peter S.
Published: September 2004

OBJECTIVES

A pseudomeningocele results from the escape of cerebrospinal fluid through a dural defect with trapping of subarachnold fluid in surrounding soft tissue. The present study evaluates the incidence of pseudomeningocele following neurotologic procedures and delineates an algorithm for management.

METHODS

A retrospective review of 375 consecutive patients undergoing neurotologic procedures at a single institution identified 17 patients with the postoperative complication of pseudomeningocele.

RESULTS

The incidence of pseudomeningocele formation was 4.5% in the present study. Fourteen pseudomeningoceles resolved with nonoperative management including pressure dressing, bed rest, and lumbar spinal drainage. Three patients failed nonoperative management and required surgical procedures for resolution. All patients ultimately had resolution of their pseudomeningocele.

CONCLUSION

Skull‐base pseudomeningoceles occur as a complication following neurotologic procedures and can cause complications as they enlarge. The majority of these cases can be dealt with in a nonsurgical manner, but those failing to respond to conservative management should be considered for surgical intervention.
Cortical Measures of Anticipation
Authors: van Boxtel Geert J.M. , Böcker Koen B.E.
Published: September 2004

Abstract Anticipation increases the efficiency of cognitive processes by partial advance activation of the neural substrate involved in those processes. In the case of perceptual anticipation, a slow cortical potential named Stimulus-Preceding Negativity (SPN) has been identified. The SPN has been observed preceding four types of stimuli: (1) stimuli providing knowledge-of-results (KR) about past performance, (2) stimuli conveying an instruction about a future task, (3) probe stimuli against which the outcome of a previous task has to be matched, and (4) affective stimuli. The morphology and scalp distribution of the SPN is different in each of these cases, suggesting the presence of separable components. This article reviews more than 15 years of SPN research. Possible neurophysiological generators are considered, as well as models that may describe the generation of the SPN. Suggestions for future research into anticipatory processes and the associated psychophysiological measures are made.
Slip history of the 2003 San Simeon earthquake constrained by combining 1‐Hz GPS, strong motion, and teleseismic data
Authors: Ji Chen , Larson Kristine M. , Tan Ying , Hudnut Kenneth W. , Choi Kyuhong
Published: September 2004

The slip history of the 2003 San Simeon earthquake is constrained by combining strong motion and teleseismic data, along with GPS static offsets and 1‐Hz GPS observations. Comparisons of a 1‐Hz GPS time series and a co‐located strong motion data are in very good agreement, demonstrating a new application of GPS. The inversion results for this event indicate that the rupture initiated at a depth of 8.5 km and propagated southeastwards with a speed ∼3.0 km/sec, with rake vectors forming a fan structure around the hypocenter. We obtained a peak slip of 2.8 m and total seismic moment of 6.2 × 1018 Nm. We interpret the slip distribution as indicating that the hanging wall rotates relative to the footwall around the hypocenter, in a sense that appears consistent with the shape of the mapped fault trace.
Stochastic analysis of macrodispersion in heterogeneous bimodal variably saturated formations: The effect of water saturation
Authors: Russo David
Published: September 2004

The effect of water saturation on macrodispersion in bimodal, heterogeneous, variably saturated formations under steady state, gravity‐dominated flow conditions was analyzed here. The present study was motivated by the inconsistency between the results of the first‐order analysis of Russo [2002] and those of the simulations of Russo et al. [2001]. The latter results suggested that near saturation, increasing mean pressure head H (i.e., decreasing water saturation) may decrease log conductivity variance, σy2, and concurrently, may decrease macrodispersion, while the former results suggested the opposite. Results of the present first‐order analysis suggest that this inconsistency stems from the fact that σy2 derived by Russo [2002] is valid only for H = 0. The σy2 derived in the present study, valid for H ≥ 0, is a concave function of H, which exhibits a minimum at H = Hm, (Hm > 0), and may exceed its counterpart in saturated flow when H > Hc (Hc > Hm). The latter σy2 leads to a time‐dependent macrodispersion tensor whose principal components decrease with increasing H when 0 < H < Hm′ (Hm′ < Hm) and increase with increasing H when H > Hm′ and may exceed their counterparts in saturated flow when H > Hc′, (Hc′ < Hc), consistent with the results of the simulations of Russo et al. [2001]. Results of the present analysis suggest that for a formation of given statistics and for relatively small inclusions volume fraction P* when the formation is relatively wet, i.e., when 0 < H < Hc′, the macrodispersion is larger in formations in which the texture of the embedded soil is finer than that of the background soil, while the converse is true when the formation becomes less saturated, i.e., when H > Hc′. When P* is relatively large, however, the reverse situation occurs.
Conformational effects on vibronic spectra and excited state dynamics of 3-fluorobenzoic acid dimer
Authors: Nandi Chayan K. , Hazra Montu K. , Chakraborty Tapas
Published: September 2004

Two conformational isomers of 3-fluorobenzoic acid dimer (3-FBA2) have been identified in a supersonic jet expansion by use of laser-induced fluorescence excitation (FE), UV-UV hole-burning, and dispersed fluorescence (DF) spectroscopic methods. In the FE spectrum, the S1 origins of the two isomeric species appear at a frequency gap of only 24 cm−1, and the vibronic intensities of the redshifted dimer (dimer I) are about two times weaker than those of dimer II. However, ab initio quantum chemistry calculations at the MP2/6-31G** level of theory predict that all the isomeric species of 3-FBA2 have almost the same binding energy (∼17 kcal/mol) in the ground state. Furthermore, unlike benzoic acid dimer, the present system shows intense activity for a low-frequency mode in both the FE and DF spectra. With the aid of DFT (B3LYP/6-311G**) predicted normal mode frequencies, we have assigned the mode to the in-plane gear (cogwheel) vibration of the cyclic hydrogen-bonded frame of the dimer. The Franck-Condon profiles for vibronic excitation of the mode indicate that the distortion of the cyclic hydrogen bond frame as a result of S1←S0 excitation is larger for dimer I than dimer II. Moreover, the fluorescence lifetime at the S1 zero-point level of the former is also significantly smaller than the latter. Using the predictions of configuration interaction singles calculations, we have proposed that the spectral and dynamical differences between the two isomeric species observed in this study are manifestations of the different characteristics of their S1 surfaces. By measuring FE, DF, and hole-burning spectra of a mixed dimer between 3-fluobenzoic acid and benzoic acid we have shown that the isomeric features in the homodimer spectra are due to two locally excited rotamers of the 3-fluorobenzoic acid moiety.
Product multiplet branching in the O(1D)+H2→OH(2Π)+H reaction
Authors: Alexander Millard H. , Rackham Edward J. , Manolopoulos David E.
Published: September 2004

The statistical model of atom-diatom insertion reactions is combined with coupled-states capture theory and used to calculate product multiplet-resolved integral cross sections for the title reaction. This involves an ab initio determination of the four electronic potential energy surfaces that correlate with the products (1,3A′ and A″1,3), and an accurate description of the electronic and spin-orbit couplings between them. The dependence of the resulting cross sections on the final-state rotational quantum number shows a statistical behavior similar to that observed in earlier studies of the reaction in which only the lowest (1A′) potential was retained. In addition, however, the present calculations provide information on the branching between the OH(2Π) multiplet levels. Although the two spin-orbit manifolds are predicted to be equally populated, we find a strong propensity for the formation of the Π(A′) Λ-doublet states. These two predictions confirm the experimental results of Butler, Wiesenfeld, Gericke, Brouard, and their co-workers. The nonstatistical population of the OH Λ-doublet levels is a consequence of the bond breaking in the intermediate H2O complex and is preserved through the multiple curve crossings as the products separate. This exit-channel coupling is correctly described by the present theory.
Bridge mediated two-electron transfer reactions: On the influence of intersite Coulomb interactions
Authors: Petrov E. G. , Teslenko V. I. , May V.
Published: September 2004

Donor-acceptor two-electron transfer (TET) mediated by a linear molecular bridge is described theoretically. The particular case is considered where the TET takes place in the presence of a strong electronic intersite coupling within the bridge and against the background of fast vibrational relaxation processes. For such a situation the coarse-grained description of bridge-assisted electron transfer in molecular systems can be utilized [Petrov et al., J. Phys. Chem. B 106, 3092 (2002)]. In the present case it leads to kinetic equations and rate expression for TET reactions. Our recent treatment of completely nonadiabtic TET reactions [Petrov et al., J. Chem. Phys. 120, 4441 (2004)] including a reduction to single-exponential kinetics (with overall transfer rate KTET) is generalized here to the case of strong intrabridge coupling and the presence of intersite Coulomb interactions. The dependence of KTET on the bridge length which is determined by a separate stepwise and concerted contribution is discussed in detail. It is found that the intersite Coulomb interaction favors the TET if the donor and the acceptor are uncharged in their completely reduced states (with two excess electrons present).
Calculating expectations with time-dependent perturbations in quantum Monte Carlo
Authors: Kalos M. H. , de Saavedra F. Arias
Published: September 2004

We show that a small perturbation periodic in imaginary time can be used to compute expectation values of nondifferential operators that do not commute with the Hamiltonian within the framework of quantum diffusion Monte Carlo. Some results for the harmonic oscillator and the helium atom are presented showing the validity of the proposed method.
New hybrid method for reactive systems from integrating molecular orbital or molecular mechanics methods with analytical potential energy surfaces
Authors: Espinosa-Garcı́a Joaquı́n , Rangel Cipriano , Navarrete Marta , Corchado José C.
Published: September 2004

A computational approach to calculating potential energy surfaces for reactive systems is presented and tested. This hybrid approach is based on integrated methods where calculations for a small model system are performed by using analytical potential energy surfaces, and for the real system by using molecular orbital or molecular mechanics methods. The method is tested on a hydrogen abstraction reaction by using the variational transition-state theory with multidimensional tunneling corrections. The agreement between the calculated and experimental information depends on the quality of the method chosen for the real system. When the real system is treated by accurate quantum mechanics methods, the rate constants are in excellent agreement with the experimental measurements over a wide temperature range. When the real system is treated by molecular mechanics methods, the results are still good, which is very encouraging since molecular mechanics itself is not at all capable of describing this reactive system. Since no experimental information or additional fits are required to apply this method, it can be used to improve the accuracy of molecular orbital methods or to extend the molecular mechanics method to treat any reactive system with the single constraint of the availability of an analytical potential energy surface that describes the model system.
An improved renormalization group theory for real fluids
Authors: Mi Jianguo , Zhong Chongli , Li Yi-Gui , Tang Yiping
Published: September 2004

On the basis of White’s theory, an improved renormalization group (RG) theory is developed for chain bonding fluids inside the critical region. Outside the critical region, the statistical associating fluid theory based on the first-order mean sphere approximation [Fluid Phase Equilibria 171, 27 (2000)] is adopted and all the microscopic parameters are taken directly from its earlier application of real fluids. Inside the critical region, the RG transformation for long-range density fluctuation is derived in the k space, which illustrates explicitly the contributions from the mean-field term, the local density fluctuation, and the nonlocal density fluctuation. The RG theory is applied to describe physical behavior of ten n alkanes (C1–C10) both near to and far from the critical point. With no additional parameters for chain bonding fluids, good results are obtained for critical specific heat and phase coexistence curves and the resulting critical exponents are in good agreement with the reported nonclassic values.
The Silicon-Directed Tiffeneau - Demjanov Reaction: Some Theoretical Studies
Authors: McClure Melanie , Schiesser Carl , White Jonathan
Published: September 2004

The Tiffeneau–Demjanov chain extension reaction was modelled using the B3LYP/6-31G* basis set for both diazonium and water leaving groups. The chain extension of 12 with the diazonium leaving group occurs with an early transition state, a low activation barrier, and very little participation by the silicon substituent. Chain extension of 15, involving displacement of the less reactive leaving group water, occurs with a later transition state, a significantly higher activation barrier, and with greater participation by the silicon substituent.
A Chemoenzymatic and Enantioselective Route to the Tricyclic Frameworks Associated with the Protoilludane and Marasmane Classes of Sesquiterpene
Authors: Banwell Martin G. , Harfoot Gwion J.
Published: September 2004

The enantiomerically pure cis-1,2-dihydrocatechol 3, which is obtained in quantity by microbial dihydroxylation of toluene, has been converted over ten steps, including an initial Diels–Alder cycloaddition reaction, into the tricyclic ketone 12. Direct irradiation of a benzene solution of the latter compound affords a mixture of compounds 13 and 14 which embody the tricyclic frameworks of the sesquiterpene natural products tsugicoline A (1) and isovelleral (2), respectively.
Mesoveliidae, Hebridae, and Hydrometridae of Australia (Hemiptera : Heteroptera : Gerromorpha), with a reanalysis of the phylogeny of semiaquatic bugs
Authors: Andersen Nils Møller , Weir Tom A.
Published: September 2004

The semiaquatic bugs (Hemiptera : Heteroptera, infraorder Gerromorpha), comprising water striders and their allies, are familiar inhabitants of water surfaces in all continents. Currently, the world fauna has more than 1900 described species classified in eight families and 165 genera. A phylogenetic analysis using maximum parsimony was performed on a dataset comprising 56 morphological characters scored for 24 exemplar genera covering all families and subfamilies of Gerromorpha. The phylogenetic relationships found concur with those presented by Andersen (1982) except that the relationships between some subfamilies of Veliidae and Gerridae are unresolved. The Australian fauna of Gerromorpha comprises six families, 30 genera, and 123 species. One-third of the genera and more than 80% of the species are endemic to Australia. Previously, we have covered all Australian species of the families Gerridae, Hermatobatidae and Veliidae. The present paper deals with the families Hebridae, Hydrometridae, and Mesoveliidae. We offer redescriptions or descriptive notes on all previously described species, describe Mesovelia ebbenielseni, sp. nov. (Mesoveliidae), Austrohebrus apterus, gen. et sp. nov., and Hebrus pilosus, sp. nov. (Hebridae), and synonymise Hebrus woodwardi Lansbury, syn. nov. (Hebridae) and Hydrometra halei Hungerford and Evans, syn. nov. (Hydrometridae). We present keys for the identification of genera and species, and map the distribution of all species. We also give a key for the identification of the families of Gerromorpha known from Australia.
Targeted knockout in
Physcomitrella
reveals direct actions of phytochrome in the cytoplasm
Authors: Mittmann Franz , Brücker Gerhard , Zeidler Mathias , Repp Alexander , Abts Thomas , Hartmann Elmar , Hughes Jon
Published: September 2004

The plant photoreceptor phytochrome plays an important role in the nucleus as a regulator of transcription. Numerous studies imply, however, that phytochromes in both higher and lower plants mediate physiological reactions within the cytoplasm. In particular, the tip cells of moss protonemal filaments use phytochrome to sense light direction, requiring a signaling system that transmits the directional information directly to the microfilaments that direct tip growth. In this work we describe four canonical phytochrome genes in the model moss species
Physcomitrella patens
, each of which was successfully targeted via homologous recombination and the distinct physiological functions of each gene product thereby identified. One homolog in particular mediates positive phototropism, polarotropism, and chloroplast movement in polarized light. This photoreceptor thus interacts with a cytoplasmic signal/response system. This is our first step in elucidating the cytoplasmic signaling function of phytochrome at the molecular level.
Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells
Authors: Cohen David , Rodriguez-Boulan Enrique , Müsch Anne
Published: September 2004

Simple (i.e., nonstratified) epithelial cells use two different routes to target their newly synthesized luminal plasma membrane proteins to the cell surface: a direct route from the Golgi complex, as in the kidney-derived MDCK cell line, or an indirect route that involves a intermediate stop at the ab-luminal (basolateral) membrane, as in hepatocytes. The mechanisms or proteins responsible for these different protein targeting strategies are not known. Here, we show that increased expression of EMK1, a mammalian ortholog of
Caenorhabditis elegans
Par-1, in MDCK cells promotes a switch from a direct to a transcytotic mode of apical protein delivery and other trafficking changes typical of hepatocytes. These results, together with our recent demonstration that PAR-1 promotes morphological features of hepatocytes in MDCK cells, indicate that Par-1 modulates the developmental decision to build a columnar versus a hepatic epithelial cell. To our knowledge, Par-1 is the first gene assigned to this task in epithelial morphogenesis.
The magnetic polarity stratigraphy of the Mauch Chunk Formation, Pennsylvania
Authors: Opdyke Neil D. , DiVenere Victor J.
Published: September 2004

Three sections of Chesterian Mauch Chunk Formation in Pennsylvania have been studied paleomagnetically to determine a Late Mississippian magnetic polarity stratigraphy. The upper section at Lavelle includes a conglomerate with abundant red siltstone rip-up clasts that yielded a positive conglomerate test. All samples were subjected to progressive thermal demagnetization to temperatures as high as 700°C. Two components of magnetization were isolated: a synfolding “B” component and the prefolding “C” component. The conglomerate test is positive, indicating that the C component was acquired very early in the history of the sediment. A coherent pattern of magnetic polarity reversals was identified. Five magnetozones were identified in the upper Lavelle section, which yields a pattern that is an excellent match with the pattern of reversals obtained from the upper Mauch Chunk at the original type section of the Mississippian/Pennsylvanian boundary at Pottsville, PA. The frequency of reversals in the upper Mississippian, as identified in the Mauch Chunk Formation, is approximately one to two per million years, which is an average for field reversal through time.
Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of
Streptomyces
telomere complex
Authors: Bao Kai , Cohen Stanley N.
Published: September 2004

Replication of
Streptomyces
linear chromosomes and plasmids proceeds bidirectionally from a central origin, leaving recessed 5′ termini that are extended by a telomere binding complex. This complex contains both a telomere-protecting terminal protein (Tpg) and a telomere-associated protein that interacts with Tpg and the DNA ends of linear
Streptomyces
replicons. By using histidine-tagged telomere-associated protein (Tap) as a scaffold, we identified DNA polymerase (PolA) and topoisomerase I (TopA) proteins as other components of the
Streptomyces
telomere complex. Biochemical characterization of these proteins indicated that both PolA and TopA exhibit highly efficient reverse transcriptase (RT) activity in addition to their predicted functions. Although RT activity innate to other DNA-dependent DNA polymerases has been observed previously, its occurrence in a topoisomerase is unprecedented. Deletion mapping and sequence analysis showed that the RT activity of
Streptomcyces
TopA resides in a peptide region containing motifs that are absent from most bacterial topoisomerases but are highly conserved in a novel subfamily of eubacterial topoisomerases found largely in Actinobacteria. Within one of these motifs, and essential to the RT function of
Streptomyces
TopA, is an Asp-Asp doublet sequence required also for the RT activities of human immunodeficiency virus and eukaryotic cell telomerases.
The inositol polyphosphate 5-phosphatase Ocrl associates with endosomes that are partially coated with clathrin
Authors: Ungewickell Alexander , Ward Michael E. , Ungewickell Ernst , Majerus Philip W.
Published: September 2004

The subcellular localization of Ocrl, the inositol polyphosphate 5-phosphatase that is mutated in Lowe syndrome, was investigated by fluorescence microscopy. Ocrl was localized to endosomes and Golgi membranes along with clathrin, giantin, the mannose 6-phosphate receptor, transferrin, and the early endosomal antigen 1 endosomal marker in fixed cells. The endosomal localization of Ocrl was confirmed by live-cell time-lapse microscopy in which we monitored the dynamics of Ocrl on endosomes. GST binding assays show that Ocrl interacts with the clathrin terminal domain and the clathrin adaptor protein AP-2. Our findings suggest a role for Ocrl in endosomal receptor trafficking and sorting.
Structural basis for the interaction of
Escherichia coli
NusA with protein N of phage λ
Authors: Bonin Irena , Mühlberger Rene , Bourenkov Gleb P. , Huber Robert , Bacher Adelbert , Richter Gerald , Wahl Markus C.
Published: September 2004

The C terminus of transcription factor NusA from
Escherichia coli
comprises two repeat units, which bind during antitermination to protein N from phage λ. To delineate the structural basis of the NusA–λN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a λN peptide (residues 34–47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single λN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the α-helical conformation of the λN N terminus in complex with
boxB
RNA, residues 34–40 of λN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA–λN interaction modes is biologically significant, supporting an equimolar ratio of NusA and λN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and λN. Contrary to the RNA polymerase α subunit, λN binding does not stimulate RNA interaction of NusA. The results demonstrate that λN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.
De novo
synthesis and development of an RNA enzyme
Authors: Ikawa Yoshiya , Tsuda Kentaro , Matsumura Shigeyoshi , Inoue Tan
Published: September 2004

Arbitrary manipulation of molecular recognition at the atomic level has many applications. However, systematic design and
de novo
synthesis of an artificial enzyme based on such manipulation has been a long-standing challenge in the field of chemistry and biotechnology. In this report, we developed an artificial RNA ligase by implementing a synthetic strategy that fuses a series of 3D molecular modelings based on naturally occurring RNA–RNA recognition motifs with a small-scale combinatorial synthesis of a modular catalytic unit. The resulting ligase produces a 3′–5′ linkage in a template-directed manner for any combinations of two nucleotides at the reaction site. The reaction rate is 10
6
-fold over that of the uncatalyzed reaction with a yield higher than those of previously reported ligase ribozymes. The strategy may be applicable to the synthesis and development of a variety of nonnatural functional RNAs with defined 3D structures.
Higher offspring survival among Tibetan women with high oxygen saturation genotypes residing at 4,000 m
Authors: Beall Cynthia M. , Song Kijoung , Elston Robert C. , Goldstein Melvyn C.
Published: September 2004

Here we test the hypothesis that high-altitude native resident Tibetan women with genotypes for high oxygen saturation of hemoglobin, and thus less physiological hypoxic stress, have higher Darwinian fitness than women with low oxygen saturation genotypes. Oxygen saturation and genealogical data were collected from residents of 905 households in 14 villages at altitudes of 3,800–4,200 m in the Tibet Autonomous Region along with fertility histories from 1,749 women. Segregation analysis confirmed a major gene locus with an autosomal dominant mode of inheritance for high oxygen saturation levels, associated with a 10% higher mean. Oxygen saturation genotypic probability estimators were then used to calculate the effect of the inferred oxygen saturation locus on measures of fertility, in a subsample of 691 women (20–59 years of age and still married to their first husbands, those with the highest exposure to the risk of pregnancy). The genotypic probability estimators were not significantly associated with the number of pregnancies or live births. The high oxygen saturation genotypic mean offspring mortality was significantly lower, at 0.48 deaths compared with 2.53 for the low oxygen saturation homozygote, because of lower infant mortality. Tibetan women with a high likelihood of possessing one to two alleles for high oxygen saturation had more surviving children. These findings suggest that high-altitude hypoxia is acting as an agent of natural selection on the locus for oxygen saturation of hemoglobin by the mechanism of higher infant survival of Tibetan women with high oxygen saturation genotypes.
Including the Community in Local Regeneration? The Case of Greater Pollok Social Inclusion Partnership
Authors: McWilliams Chris
Published: September 2004

This paper seeks to evaluate critically the extent to which there is community involvement in the construction and delivery of urban policy under New Labour, through a case study of the Greater Pollok Social Inclusion Partnership. The paper concentrates on the Scottish experience, but its lessons resonate more widely. Its key finding is that, for all the rhetoric of New Labour and community involvement, the degree to which it has occurred in practice is little different to the level of involvement under Conservative urban policy initiatives. The paper argues that the processes of community consultation and participation during the early stages of the Greater Pollok Social Inclusion Partnership were woefully inadequate. At best it was tokenistic, and at worst, local people were being ‘exploited’ to legitimise the policy process.
Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells
Authors: Leung Anthony Kar Lun , Gerlich Daniel , Miller Gail , Lyon Carol , Lam Yun Wah , Lleres David , Daigle Nathalie , Zomerdijk Joost , Ellenberg Jan , Lamond Angus I.
Published: September 2004

One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.
Visualization of early chromosome condensation
Authors: Kireeva Natashe , Lakonishok Margot , Kireev Igor , Hirano Tatsuya , Belmont Andrew S.
Published: September 2004

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”
Decreased Survival of B Cells of HIV-viremic Patients Mediated by Altered Expression of Receptors of the TNF Superfamily
Authors: Moir Susan , Malaspina Angela , Pickeral Oxana K. , Donoghue Eileen T. , Vasquez Joshua , Miller Natalie J. , Krishnan Surekha R. , Planta Marie A. , Turney John F. , Justement J. Shawn , Kottilil Shyamasundaran , Dybul Mark , Mican JoAnn M. , Kovacs Colin , Chun Tae-Wook , Birse Charles E. , Fauci Anthony S.
Published: September 2004

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21low B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21low B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21low B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.
Determination of the Critical Concentration of Neutrophils Required to Block Bacterial Growth in Tissues
Authors: Li Yongmei , Karlin Arthur , Loike John D. , Silverstein Samuel C.
Published: September 2004

We showed previously that the competition between bacterial killing by neutrophils and bacterial growth in stirred serum-containing suspensions could be modeled as the competition between a first-order reaction (bacterial growth) and a second-order reaction (bacterial killing by neutrophils). The model provided a useful parameter, the critical neutrophil concentration (CNC), below which bacterial concentration increased and above which it decreased, independent of the initial bacterial concentration. We report here that this model applies to neutrophil killing of bacteria in three-dimensional fibrin matrices and in rabbit dermis. We measured killing of 103–108 colony forming units/ml Staphylococcus epidermidis by 105–108 human neutrophils/ml in fibrin gels. The CNC was ∼4 × 106 neutrophils/ml gel in the presence of normal serum and ∼1.6 × 107 neutrophils/ml gel in the presence of C5-deficient serum. Application of our model to published data of others on killing of ∼5 × 107 to 2 × 108 E. coli/ml rabbit dermis yielded CNCs from ∼4 × 106 to ∼8 × 106 neutrophils/ml dermis. Thus, in disparate tissues and tissuelike environments, our model fits the kinetics of bacterial killing and gives similar lower limits (CNCs) to the neutrophil concentration required to control bacterial growth.
Non‐immunostimulatory nonviral vectors
Authors: Liu Feng , Shollenberger Lisa M. , Huang Leaf
Published: September 2004

The vectors for gene delivery are usually classified as viral and nonviral vectors. While the viral vectors are very efficient in transducing cells, safety concerns regarding their use in humans make nonviral vectors an attractive alternative. Among the nonviral vectors, the lipoplexes (complexes of cationic liposome/pDNA) are the most studied and represent the most promising approaches for human clinical trials. However, an inflammatory response is invariably associated with administration of the lipoplexes, which must be avoided in the clinical application. Here, we have successfully developed a nonimmunostimulatory vector for gene therapy. The vector possesses dual functions of: 1) efficiently delivering a gene to target cells and 2) codelivering DNA and inflammatory suppressors into the immune cells where the released suppressor can inhibit cytokine production. The inflammatory suppressors successfully delivered by the vector included glucocorticoids, a nonsteroidal anti‐inflammatory drug (NSAID), an NF‐κB inhibitor, and a natural compound from an herbal medicine. Intravenous injection of the vector dramatically suppressed the cytokine production induced by CpG motif pDNA, including TNF‐α, IL‐12 and IFN‐γ. This new gene vector has a great potential in clinical gene therapy. Another potential use of the vector is codelivery of an enhancer candidate, acting at the transcriptional and translational levels to improve the efficiency of gene transfer by the nonviral vector. Moreover, the unique feature of this vector is that it can be used as an easy and powerful tool for in vivo screening of anti‐inflammatory drugs.
Food for thought ‐ What mad cows have wrought with respect to food safety regulation in the EU and UK
Authors: Shears Peter , Zollers Fran , Hurd Sandy
Published: September 2004

An in‐depth examination of the evolution of legislation, practice and thinking in food safety in the UK and Europe in general. Explores the reduced levels of confidence exhibited by consumers.