bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlations among within-Channel and between-Channel Auditory Gap-Detection Thresholds in Normal Listeners
Authors: Phillips Dennis P , Smith Jennifer C
Published: April 2004

We obtained data on within-channel and between-channel auditory temporal gap-detection acuity in the normal population. Ninety-five normal listeners were tested for gap-detection thresholds, for conditions in which the gap was bounded by spectrally identical, and by spectrally different, acoustic markers. Separate thresholds were obtained with the use of an adaptive tracking method, for gaps delimited by narrowband noise bursts centred on 1.0 kHz, noise bursts centred on 4.0 kHz, and for gaps bounded by a leading marker of 4.0 kHz noise and a trailing marker of 1.0 kHz noise. Gap thresholds were lowest for silent periods bounded by identical markers—‘within-channel’ stimuli. Gap thresholds were significantly longer for the between-channel stimulus—silent periods bounded by unidentical markers ( p < 0.0001). Thresholds for the two within-channel tasks were highly correlated ( R = 0.76). Thresholds for the between-channel stimulus were weakly correlated with thresholds for the within-channel stimuli (1.0 kHz, R = 0.39; and 4.0 kHz, R = 0.46). The relatively poor predictability of between-channel thresholds from the within-channel thresholds is new evidence on the separability of the mechanisms that mediate performance of the two tasks. The data confirm that the acuity difference for the tasks, which has previously been demonstrated in only small numbers of highly trained listeners, extends to a population of untrained listeners. The acuity of the between-channel mechanism may be relevant to the formation of voice-onset time-category boundaries in speech perception.
The Effect of Colour Congruency on Shape Discriminations of Novel Objects
Authors: Nicholson Karen G , Humphrey G Keith
Published: April 2004

Although visual object recognition is primarily shape driven, colour assists the recognition of some objects. It is unclear, however, just how colour information is coded with respect to shape in long-term memory and how the availability of colour in the visual image facilitates object recognition. We examined the role of colour in the recognition of novel, 3-D objects by manipulating the congruency of object colour across the study and test phases, using an old/new shape-identification task. In experiment 1, we found that participants were faster at correctly identifying old objects on the basis of shape information when these objects were presented in their original colour, rather than in a different colour. In experiments 2 and 3, we found that participants were faster at correctly identifying old objects on the basis of shape information when these objects were presented with their original part-colour conjunctions, rather than in different or in reversed part-colour conjunctions. In experiment 4, we found that participants were quite poor at the verbal recall of part-colour conjunctions for correctly identified old objects, presented as grey-scale images at test. In experiment 5, we found that participants were significantly slower at correctly identifying old objects when object colour was incongruent across study and test, than when background colour was incongruent across study and test. The results of these experiments suggest that both shape and colour information are stored as part of the long-term representation of these novel objects. Results are discussed in terms of how colour might be coded with respect to shape in stored object representations.
Bias to Experience Approaching Motion in a Three-Dimensional Virtual Environment
Authors: Lewis Clifford F , McBeath Michael K
Published: April 2004

We used two-frame apparent motion in a three-dimensional virtual environment to test whether observers had biases to experience approaching or receding motion in depth. Observers viewed a tunnel of tiles receding in depth, that moved ambiguously either toward or away from them. We found that observers exhibited biases to experience approaching motion. The strengths of the biases were decreased when stimuli pointed away, but size of the display screen had no effect. Tests with diamond-shaped tiles that varied in the degree of pointing asymmetry resulted in a linear trend in which the bias was strongest for stimuli pointing toward the viewer, and weakest for stimuli pointing away. We show that the overall bias to experience approaching motion is consi'stent with a computational strategy of matching corresponding features between adjacent foreshortened stimuli in consecutive visual frames. We conclude that there are both adaptational and geometric reasons to favor the experience of approaching motion.
Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications
Authors: Reetz Manfred T.
Published: April 2004

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the
in vitro
evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a “Darwinistic” process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted.
Asymmetric hydrogenation of α,β-unsaturated phosphonates with Rh-BisP* and Rh-MiniPHOS catalysts: Scope and mechanism of the reaction
Authors: Gridnev Ilya D. , Yasutake Masaya , Imamoto Tsuneo , Beletskaya Irina P.
Published: April 2004

Optically active 1,2-bis(alkylmethylphosphino)ethanes and bis(alkylmethylphosphino)methanes are unique diphosphine ligands combining the simple molecular structure and P-stereogenic asymmetric environment. This work shows that these ligands exhibit excellent enantioselectivity in rhodium-catalyzed asymmetric hydrogenation of α,β-unsaturated phosphonic acid derivatives. The enantioselective hydrogenation mechanism elucidated by NMR study is also described.
An efficient, palladium-catalyzed, enantioselective synthesis of (2
R
)-3-butene-1,2-diol and its use in highly selective Heck reactions
Authors: Cheeseman Natasha , Fox Martin , Jackson Mark , Lennon Ian C. , Meek Graham
Published: April 2004

A robust and scalable procedure for the palladium-catalyzed dynamic kinetic asymmetric transformation of 3,4-epoxy-1-butene into (2
R
)-3-butene-1,2-diol with water as the cosolvent is reported. Examination of the effects of solvent and temperature led to the identification of conditions that permitted use of 0.025 mol % catalyst, providing (2
R
)-3-butene-1,2-diol in 84% isolated yield and 85% enantiomeric excess. Subsequent Heck reactions with a diverse range of coupling partners are described and the influence of their electronic nature on maintaining the enantiopurity of the diol is discussed.
Highly enantioselective alkyne additions to aldehydes in the presence of 1,1′-bi-2-naphthol and hexamethylphosphoramide
Authors: Gao Ge , Xie Ru-Gang , Pu Lin
Published: April 2004

It is found that the addition of hexamethylphosphoramide to the solution of an alkyne, Et
2
Zn, and (
S
)-1,1′-bi-2-naphthol in methylene chloride allows the generation of an alkynylzinc at room temperature and shows highly enantioselective additions to aldehydes. The mild condition for the formation of the alkynylzinc reagent enables the use of functional alkynes in this asymmetric reaction with excellent enantioselectivity. It avoids the reflux of the toluene solutions of the alkynes and Et
2
Zn as previously reported.
Enantioselective syntheses and biological studies of aeruginosin 298-A and its analogs: Application of catalytic asymmetric phase-transfer reaction
Authors: Fukuta Yuhei , Ohshima Takashi , Gnanadesikan Vijay , Shibuguchi Tomoyuki , Nemoto Tetsuhiro , Kisugi Takaya , Okino Tatsufumi , Shibasaki Masakatsu
Published: April 2004

Aeruginosin 298-A was isolated from the freshwater cyanobacterium
Microcystis aeruginosa
(NIES-298) and is an equipotent thrombin and trypsin inhibitor. A variety of analogs were synthesized to gain insight into the structure–activity relations. We developed a versatile synthetic process for aeruginosin 298-A as well as several attractive analogs, in which all stereocenters were controlled by catalytic asymmetric phase-transfer reaction promoted by two-center asymmetric catalysts and catalytic asymmetric epoxidation promoted by a lanthanide–BINOL complex. Furthermore, serine protease inhibitory activities of aeruginosin 298-A and its analogs were examined.
Palladium-catalyzed carbocyclization of 1,6-enynes leading to six-membered rings or oxidized five-membered trifluoroacetates
Authors: Mikami Koichi , Hatano Manabu
Published: April 2004

We describe palladium-catalyzed carbocyclization of 1,6-enynes leading to six-membered rings by water-originated hydride addition. Mechanistic features of this anomalous carbocyclization and isolation of a chiral five-membered C-Pd intermediate as an oxidized form of trifluoroacetate are also reported.
Mechanistic studies on the catalytic cycle of rhodium-catalyzed asymmetric 1,4-addition of aryltitanate reagents to α,β-unsaturated ketones
Authors: Tokunaga Norihito , Yoshida Kazuhiro , Hayashi Tamio
Published: April 2004

Addition of lithium aryl(tetraisopropoxy)titanates [ArTi(OPr-i)4–Li+] to α,β-unsaturated ketones proceeded with high enantioselectivity (up to 99% ee) in the presence of an excess amount of chlorotrimethylsilane and a rhodium catalyst (3 mol % Rh), generated from [RhCl(C2H4)2]2and (S)-binap, in tetrahydrofuran at 20°C to give high yields of the corresponding silyl enolates as 1,4-addition products. The presence of chlorotrimethylsilane is essential for the 1,4-addition to take place.31P NMR spectroscopic studies revealed that the catalytic cycle consists of three transformations, that is, (i) insertion of an enone into arylrhodium species forming (oxa-π-allyl)rhodium intermediate, (ii) silylation of the (oxa-π-allyl)rhodium with chlorotrimethylsilane giving silyl enolate and a chloro-rhodium complex, and (iii) transmetalation of aryl group from aryltitanate to the chloro-rhodium regenerating the aryl-rhodium.
An efficient asymmetric synthesis of an estrogen receptor modulator by sulfoxide-directed borane reduction
Authors: Song Zhiguo J. , King Anthony O. , Waters Marjorie S. , Lang Fengrui , Zewge Daniel , Bio Matthew , Leazer Johnnie L. , Javadi Gary , Kassim Amude , Tschaen David M. , Reamer Robert A. , Rosner Thorsten , Chilenski Jennifer R. , Mathre David J. , Volante R. P. , Tillyer Richard
Published: April 2004

An efficient asymmetric synthesis of a selective estrogen receptor modulator (SERM) that has a dihydrobenzoxathiin core structure bearing two stereogenic centers is reported. The stereogenic centers were established by an unprecedented chiral sulfoxide-directed stereospecific reduction of an α,β-unsaturated sulfoxide to the saturated sulfide in one step. Studies to elucidate the mechanism for this reduction are reported. Highly efficient Cu(I)-mediated ether formation was used to install the ether side chain, and selective debenzylation conditions were developed to remove the benzyl protecting groups on the phenols.
Identification of GAPDH as a protein target of the saframycin antiproliferative agents
Authors: Xing Chengguo , LaPorte Jacob R. , Barbay Joseph K. , Myers Andrew G.
Published: April 2004

Saframycin A (SafA) is a member of a class of natural products with potent antiproliferative effects in leukemia- and tumor-derived cells. This activity is frequently conjectured to derive from the ability of saframycins to covalently modify duplex DNA. We used a DNA-linked affinity purification technique to identify GAPDH as a protein target of DNA–small molecule adducts of several members of the saframycin class. Nuclear translocation of GAPDH occurs upon treatment of cancer cells with saframycins, and depletion of cellular GAPDH levels by small interfering RNA transfection confers drug resistance. Roeder and coworkers have recently suggested that GAPDH is a key transcriptional coactivator necessary for entry into S phase. Our data suggest that GAPDH is also capable of forming a ternary complex with saframycin-related compounds and DNA that induces a toxic response in cells. These studies implicate a previously unknown molecular mechanism of antiproliferative activity and, given that one member of the saframycin class has shown efficacy in cancer treatment, suggest that GAPDH may be a potential target for chemotherapeutic intervention.
Solid-phase synthesis of chiral 3,4-diazaphospholanes and their application to catalytic asymmetric allylic alkylation
Authors: Landis Clark R. , Clark Thomas P.
Published: April 2004

Functionalized chiral diazaphospholanes ligate to a variety of transition metals, yielding chiral, catalytically active, metal complexes. Previous work has established that amino acid derivatization of the carboxyl groups of (
R,R
)-
N,N
′-phthaloyl-2,3-(2-carboxyphenyl)-phenyl-3,4-diazaphospholane (
1
) yields phosphines that are excellent ligands for palladium-catalyzed asymmetric allylic alkylation reactions. Alanine functionalization is particularly effective for allylic alkylation of 1,3-dimethylallyl acetate. Standard Merrifield resins and amino acid coupling methods are used to synthesize the bead-attached phosphine having the topology bead-linker-
l
Ala-(
R,R
)-1-
l
Ala-OMe, as a 1:1 mixture of linkage isomers. Use of this supported phosphine in Pd-catalyzed asymmetric allylic alkylation yields 92% enantiomeric excess, matching prior solution-phase results. A 20-member collection of amino acid-functionalized phosphines on beads with the topology bead-linker-AA
2
-AA
1
-1-AA
1
-AA
2
was synthesized by using parallel solid-state methods and screened for efficacy in allylic alkylation. Resulting enantioselectivities indicate that the AA
1
position has the strongest effect on the reaction. Catalyst activities can vary widely with the nature of the phosphine ligand and the reaction conditions. Meaningful analysis of intrinsic catalytic activities awaits identification of the structure and abundance of the active catalyst.
Asymmetric catalysis in complex target synthesis
Authors: Taylor Mark S. , Jacobsen Eric N.
Published: April 2004

This article describes three distinct strategies by which stereochemically complex molecules are synthesized and the ways asymmetric catalysis can impact on all three. The development of general methods to prepare synthetically useful building blocks leads to an expanded “chiral pool” of potential starting materials for asymmetric synthesis. The possibility of discovering new reactions to access new types of building blocks is particularly attractive and serves to help define the frontiers of the field. Asymmetric catalysis can also be applied to diastereoselective synthesis such that the stereochemistry of the catalyst, and not that of the substrate, determines the relative configuration of the product. Finally, in reactions where multiple stereocenters are generated simultaneously or in tandem, catalyst and substrate control can operate in a complementary manner to achieve one of many possible stereochemical outcomes selectively.
Long-term effect of
in vitro
culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior
Authors: Fernández-Gonzalez Raúl , Moreira Pedro , Bilbao Ainhoa , Jiménez Adela , Pérez-Crespo Miriam , Ramírez Miguel Angel , De Fonseca Fernando Rodríguez , Pintado Belén , Gutiérrez-Adán Alfonso
Published: April 2004

The long-term developmental and behavioral consequences of mammalian embryo culture are unknown. By altering the culture medium with the addition of FCS, we wanted to determine whether mouse embryos cultured under suboptimal conditions develop aberrant mRNA expression of imprinting genes at the blastocyst stage and whether fetal development, growth, and behavior of adult mice are affected. One-cell embryos obtained from superovulated female B6CBAF
1
mice were cultured for 4 days in K
+
-modified simplex optimized medium in the presence of either 10% FCS or 1 g/liter BSA. After embryo transfer, born animals were submitted to several developmental and behavior tests. The mRNA expression of some imprinting genes was significantly affected in blastocysts cultured in the presence of FCS. Two of the eight measures of preweaning development and some specific measures of neuromotor development, such as the walking activity, were delayed in the group originated with FCS. After 34 weeks, the weight of female mice cultured
in vitro
in the presence of FCS was significantly higher than controls. In addition, the locomotion activity of mice was altered at 5 and 15 months. Anatomopathological and histological analysis of animals at 20 months of age showed some large organs and an increase in pathologies. We have found that mice derived from embryos cultured with FCS exhibited specific behavioral alterations in anxiety and displayed deficiencies in implicit memories. Our data indicate that long-term programming of postnatal development, growth, and physiology can be affected irreversibly during the preimplantation period of embryo development by suboptimal
in vitro
culture.
PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues
Authors: Yoo Seung-Hee , Yamazaki Shin , Lowrey Phillip L. , Shimomura Kazuhiro , Ko Caroline H. , Buhr Ethan D. , Siepka Sandra M. , Hong Hee-Kyung , Oh Won Jun , Yoo Ook Joon , Menaker Michael , Takahashi Joseph S.
Published: April 2004

Mammalian circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), and current dogma holds that the SCN is required for the expression of circadian rhythms in peripheral tissues. Using a PERIOD2::LUCIFERASE fusion protein as a real-time reporter of circadian dynamics in mice, we report that, contrary to previous work, peripheral tissues are capable of self-sustained circadian oscillations for >20 cycles in isolation. In addition, peripheral organs expressed tissue-specific differences in circadian period and phase. Surprisingly, lesions of the SCN in

mPer2
Luciferase

knockin mice did not abolish circadian rhythms in peripheral tissues, but instead caused phase desynchrony among the tissues of individual animals and from animal to animal. These results demonstrate that peripheral tissues express self-sustained, rather than damped, circadian oscillations and suggest the existence of organ-specific synchronizers of circadian rhythms at the cell and tissue level.
A chimeric adenovirus vector encoding reovirus attachment protein σ1 targets cells expressing junctional adhesion molecule 1
Authors: Mercier George T. , Campbell Jacquelyn A. , Chappell James D. , Stehle Thilo , Dermody Terence S. , Barry Michael A.
Published: April 2004

The utility of adenovirus (Ad) vectors for gene transduction can be limited by receptor specificity. We developed a gene-delivery vehicle in which the potent Ad5 vector was genetically reengineered to display the mucosal-targeting σ1 protein of reovirus type 3 Dearing (T3D). A σ1 construct containing all but a small virion-anchoring domain was fused to the N-terminal 44 aa of Ad5 fiber. This chimeric attachment protein Fibtail-T3Dσ1 forms trimers and assembles onto Ad virions. Fibtail-T3Dσ1 was recombined into the Ad5 genome, replacing sequences encoding wild-type fiber. The resulting vector, Ad5-T3Dσ1, expresses Fibtail-T3Dσ1 and infects Chinese hamster ovary cells transfected with human or mouse homologs of the reovirus receptor, junctional adhesion molecule 1 (JAM1), but not the coxsackievirus and Ad receptor. Treatment of Caco-2 intestinal epithelial cells with either JAM1-specific antibody or neuraminidase reduced transduction by Ad5-T3Dσ1, and their combined effect decreased transduction by 95%. Ad5-T3Dσ1 transduces primary cultures of human dendritic cells substantially more efficiently than does Ad5, and this transduction depends on expression of JAM1. These data provide strong evidence that Ad5-T3Dσ1 can be redirected to cells expressing JAM1 and sialic acid for application as a vaccine vector.
Small interfering RNA production by enzymatic engineering of DNA (SPEED)
Authors: Luo Biao , Heard Amanda D. , Lodish Harvey F.
Published: April 2004

Small interfering RNAs (siRNAs) potently silence expression of target genes. In principle siRNA libraries can be used to perform effective genome-scale functional genetic screens in mammalian cells, but their development has been hampered by the need to chemically synthesize thousands of oligonucleotides and to incorporate them into expression vectors. We have developed a technology to efficiently convert a double-stranded cDNA library into a retroviral siRNA library in which 21-base siRNAs are produced in infected cells at high levels and efficiently block expression of their target genes. The key steps are the generation of random cDNA fragments that are fused to a hairpin linker, cleavage with the
Mme
I endonuclease that creates 20- to 21-bp cDNA fragments, conversion to a double-stranded DNA that contains two copies of the cDNA insert in a head-to-head palindrome, and insertion of the construct downstream of a polymerase III promoter. We constructed a siRNA library with 3 × 10
6
clones from a mouse embryo cDNA library; siRNAs were found against many different genes; and multiple siRNAs can be generated from a single mRNA. We further showed that specific siRNAs were efficiently produced in stably infected mammalian cells and resulted in significant and specific reduction of their target mRNAs. Because no prior knowledge about target transcripts is needed, a cDNA-derived siRNA library will generate siRNAs against unknown transcripts and genes. Finally, cDNA-derived siRNA libraries can be readily generated from any cell type or species, enabling genome-wide functional screens in many biological systems.
Tobacco mosaic virus infection spreads cell to cell as intact replication complexes
Authors: Kawakami Shigeki , Watanabe Yuichiro , Beachy Roger N.
Published: April 2004

Plant viruses encode movement proteins (MPs) that facilitate cell–cell transport of infection through plasmodesmata. Intracellular and intercellular spread of virus replication complexes (VRCs) of tobacco mosaic virus was followed in intact leaf tissue from 12 to 36 h post infection (hpi) by using confocal microscopy. From 12 hpi, VRCs in primary infected cells were associated with cortical endoplasmic reticulum, and at 14 hpi, exhibited high intracellular mobility (≈160 nm/sec); mobility was slowed between 14 and 16 hpi (≈40 nm/sec), and by 18 hpi, VRCs were stationary, adjacent to plasmodesmata. VRCs traversed the plasmodesmata between 18 and 20 hpi. The process of formation and movement of VRCs was repeated in adjacent cells in 3–4 h vs. 20 h from primary infected cells. The rapid intracellular movement of the VRCs and the spread to adjacent cells was blocked by inhibitors of filamentous actin and myosin, but not by inhibitors of microtubules. We propose a model whereby cell–cell spread of tobamovirus infection is accomplished by subviral replication complexes that initiate TMV replication immediately after entry to adjacent cells.
Bim is a suppressor of Myc-induced mouse B cell leukemia
Authors: Egle Alexander , Harris Alan W. , Bouillet Philippe , Cory Suzanne
Published: April 2004

Impaired apoptosis is now recognized to be central to tumor development. Bcl2, activated by chromosomal translocation in human follicular lymphoma, promotes oncogenesis by inhibiting apoptosis. Bim, a distant proapoptotic relative, is emerging as a major physiologic antagonist of Bcl2. Here, we show that loss of Bim is oncogenic. Bim protein levels were elevated in the apoptosis-prone B lymphoid cells of Eμ-
Myc
-transgenic mice, and
Bim
-mutant Eμ-
Myc
mice had increased numbers of IgM-bearing B cells. Eμ-
Myc
-expressing B lymphoid cells deficient in Bim were refractory to apoptosis induced
in vitro
by cytokine deprivation or antigen receptor cross-linking. Thus, Bim is induced by Myc in B cells and mediates apoptosis. Remarkably, inactivation of even a single allele of
Bim
accelerated Myc-induced development of tumors, particularly acute B cell leukemia. None of the primary tumors from
Bim
+/-
Eμ-
Myc
mice displayed loss of the second allele of
Bim
. These findings indicate that Bim is a tumor suppressor, at least in B lymphocytes, and is haploinsufficient. Whereas the p19Arf/p53 pathway is frequently mutated in tumors arising in
Bim

+
/
+

Eμ-
Myc
mice, it was unaffected in most Bim-deficient tumors, indicating that Bim reduction is an effective alternative to loss of p53 function.
Shear waves in the diamond-anvil cell reveal pressure-induced instability in (Mg,Fe)O
Authors: Jacobsen Steven D. , Spetzler Hartmut , Reichmann Hans J. , Smyth Joseph R.
Published: April 2004

The emerging picture of Earth's deep interior from seismic tomography indicates more complexity than previously thought. The presence of lateral anisotropy and heterogeneity in Earth's mantle highlights the need for fully anisotropic elasticity data from mineral physics. A breakthrough in high-frequency (gigahertz) ultrasound has resulted in transmission of pure-mode elastic shear waves into a high-pressure diamond-anvil cell using a P-to-S elastic-wave conversion. The full elastic tensor (
c
ij
) of high-pressure minerals or metals can be measured at extreme conditions without optical constraints. Here we report the effects of pressure and composition on shear-wave velocities in the major lower-mantle oxide, magnesiowüstite-(Mg,Fe)O. Magnesiowüstite containing more than ≈50% iron exhibits pressure-induced
c
44
shear-mode softening, indicating an instability in the rocksalt structure. The oxide closer to expected lower-mantle compositions (≈20% iron) shows increasing shear velocities more similar to MgO, indicating that it also should have a wide pressure-stability field. A complete sign reversal in the
c
44
pressure derivative points to a change in the topology of the (Mg,Fe)O phase diagram at ≈50–60% iron. The relative stability of Mg-rich (Mg,Fe)O and the strong compositional dependence of shear-wave velocities (and ∂
c
44
/∂
P
) in (Mg,Fe)O implies that seismic heterogeneity in Earth's lower mantle may result from compositional variations rather than phase changes in (Mg,Fe)O.
Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco
Authors: Mukhopadhyay Arnab , Vij Shubha , Tyagi Akhilesh K.
Published: April 2004

Stress perception and signal transduction leading to tolerance involve a complex interplay of different gene products. We describe here the isolation and characterization of an intronless gene (
OSISAP1
) from rice encoding a zinc-finger protein that is induced after different types of stresses, namely cold, desiccation, salt, submergence, and heavy metals as well as injury. The gene is also induced by stress hormone abscisic acid. Overexpression of the gene in transgenic tobacco conferred tolerance to cold, dehydration, and salt stress at the seed-germination/seedling stage as reflected by the percentage of germination/green seedlings, the fresh weight of seedlings, and their developmental pattern. Thus,
OSISAP1
seems to be an important determinant of stress response in plants.
Bystander suppression of allergic airway inflammation by lung resident memory CD8+ T cells
Authors: Marsland Benjamin J. , Harris Nicola L. , Camberis Mali , Kopf Manfred , Hook Sarah M. , Le Gros Graham
Published: April 2004

CD8+ memory T cells have recently been recognized as playing a key role in natural immunity against unrelated viral infections, a phenomenon referred to as “heterologous antiviral immunity.” We now provide data that the cellular immunological interactions that underlie such heterologous immunity can play an equally important role in regulating T helper 2 immune responses and protecting mucosal surfaces from allergen-induced inflammation. Our data show that CD8+ T cells, either retained in the lung after infection with influenza virus, or adoptively transferred via the intranasal route can suppress allergic airway inflammation. The suppression is mediated by IFN-γ, which acts to reduce the activation level, T helper 2 cytokine production, airways hyperresponsiveness, and migration of allergen-specific CD4+ T cells into the lung, whereas the systemic and draining lymph node responses remain unchanged. Of note, adoptive transfer of previously activated transgenic CD8+ T cells conferred protection against allergic airway inflammation, even in the absence of specific-antigen. Airway resident CD8+ T cells produced IFN-γ when directly exposed to conditioned media from activated dendritic cells or the proinflammatory cytokines IL-12 and IL-18. Taken together these data indicate that effector/memory CD8+ T cells present in the airways produce IFN-γ after inflammatory stimuli, independent of specific-antigen, and as a consequence play a key role in modifying the degree and frequency of allergic responses in the lung.
A duplicated motif controls assembly of zona pellucida domain proteins
Authors: Jovine Luca , Qi Huayu , Williams Zev , Litscher Eveline S. , Wassarman Paul M.
Published: April 2004

Many secreted eukaryotic glycoproteins that play fundamental roles in development, hearing, immunity, and cancer polymerize into filaments and extracellular matrices through zona pellucida (ZP) domains. ZP domain proteins are synthesized as precursors containing C-terminal propeptides that are cleaved at conserved sites. However, the consequences of this processing and the mechanism by which nascent proteins assemble are unclear. By microinjection of mutated DNA constructs into growing oocytes and mammalian cell transfection, we have identified a conserved duplicated motif [EHP (external hydrophobic patch)/IHP (internal hydrophobic patch)] regulating the assembly of mouse ZP proteins. Whereas the transmembrane domain (TMD) of ZP3 can be functionally replaced by an unrelated TMD, mutations in either EHP or IHP do not hinder secretion of full-length ZP3 but completely abolish its assembly. Because mutants truncated before the TMD are not processed, we conclude that the conserved TMD of mammalian ZP proteins does not engage them in specific interactions but is essential for C-terminal processing. Cleavage of ZP precursors results in loss of the EHP, thereby activating secreted polypeptides to assemble by using the IHP within the ZP domain. Taken together, these findings suggest a general mechanism for assembly of ZP domain proteins.
Receptor Editing and Marginal Zone B Cell Development Are Regulated by the Helix-Loop-Helix Protein, E2A
Authors: Quong Melanie W. , Martensson Annica , Langerak Anton W. , Rivera Richard R. , Nemazee David , Murre Cornelis
Published: April 2004

Previous studies have indicated that the E2A gene products are required to initiate B lineage development. Here, we demonstrate that E2A+/− B cells that express an autoreactive B cell receptor fail to mature due in part to an inability to activate secondary immunoglobulin (Ig) light chain gene rearrangement. Both RAG1/2 gene expression and RS deletion are severely defective in E2A+/− mice. Additionally, we demonstrate that E2A+/− mice show an increase in the proportion of marginal zone B cells with a concomitant decrease in the proportion of follicular B cells. In contrast, Id3-deficient splenocytes show a decline in the proportion of marginal zone B cells. Based on these observations, we propose that E-protein activity regulates secondary Ig gene rearrangement at the immature B cell stage and contributes to cell fate determination of marginal zone B cells. Additionally, we propose a model in which E-proteins enforce the developmental checkpoint at the immature B cell stage.
Inhibition and Redistribution of NHE3, the Apical Na+/H+ Exchanger, byClostridium difficileToxin B
Authors: Hayashi Hisayoshi , Szászi Katalin , Coady-Osberg Natasha , Furuya Wendy , Bretscher Anthony P. , Orlowski John , Grinstein Sergio
Published: April 2004

NHE3, the apical isoform of the Na+/H+ exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.
Molecular Basis of pH and Ca2+ Regulation of Aquaporin Water Permeability
Authors: Németh-Cahalan Karin L. , Kalman Katalin , Hall James E.
Published: April 2004

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca2+ increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can “tune” the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca2+ sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca2+ and pH modulation are separable and arise from processes on opposite sides of the membrane.
The Adenomatous Polyposis Coli Protein Is Required for the Formation of Robust Spindles Formed in CSFXenopusExtracts
Authors: Dikovskaya Dina , Newton Ian P. , Näthke Inke S.
Published: April 2004

Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correlate with chromosomal instability. Here, we show that depletion of APC from cystostatic factor (CSF) Xenopus extracts leads to a decrease in microtubule density and changes in tubulin distribution in spindles and asters formed in such extracts. Addition of full-length APC protein or a large, N-terminally truncated APC fragment to APC-depleted extracts restored normal spindle morphology and the intact microtubule-binding site of APC was necessary for this rescue. These data indicate that the APC protein plays a role in the formation of spindles that is dependent on its effect on microtubules. Spindles formed in cycled extracts were not sensitive to APC depletion. In CSF extracts, spindles predominantly formed from aster-like intermediates, whereas in cycled extracts chromatin was the major site of initial microtubule polymerization. These data suggest that APC is important for centrosomally driven spindle formation, which was confirmed by our finding that APC depletion reduced the size of asters nucleated from isolated centrosomes. We propose that lack of microtubule binding in cancer-associated mutations of APC may contribute to defects in the assembly of mitotic spindles and lead to missegregation of chromosomes.
Src SH3/2 Domain-mediated Peripheral Accumulation of Src and Phospho-myosin Is Linked to Deregulation of E-cadherin and the Epithelial-Mesenchymal Transition
Authors: Avizienyte Egle , Fincham Valerie J. , Brunton Valerie G. , Frame Margaret C.
Published: April 2004

Elevated Src kinase in epithelial cancer cells induces adhesion changes that are associated with a mesenchymal-like state. We recently showed that Src induces dynamic integrin adhesions in KM12C colon cancer cells, whereas E-cadherin–dependent cell-cell contacts become disorganized. This promotes a fibroblastic-like morphology and expression of the mesenchymal marker vimentin. Furthermore, Src-induced deregulation of E-cadherin, and the associated mesenchymal transition, is dependent on integrin signaling (Avizienyte et al., Nat. Cell Biol. 2002, 4, 632–638), although the nature of downstream signals that mediate these Src- and integrin-dependent effects are unknown. Here we show that the SH2 and SH3 domains of Src mediate peripheral accumulation of phospho-myosin, leading to integrin adhesion complex assembly, whereas loss of SH2 or SH3 function restores normal regulation of E-cadherin and inhibits vimentin expression. Inhibitors of MEK, ROCK, or MLCK also suppress peripheral accumulation of phospho-myosin and Src-induced formation of integrin-dependent adhesions, whereas at the same time restoring E-cadherin redistribution to regions of cell-cell contact. Our data therefore implicate peripheral phospho-myosin activity as a point of convergence for upstream signals that regulate integrin- and E-cadherin–mediated adhesions. This further implicates spatially regulated contractile force as a determinant of epithelial cell plasticity, particularly in cancer cells that can switch between epithelial and mesenchymal-like states.
Endocytic Trafficking Routes of Wild Type and ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator
Authors: Gentzsch Martina , Chang Xiu-Bao , Cui Liying , Wu Yufeng , Ozols Victor V. , Choudhury Amit , Pagano Richard E. , Riordan John R.
Published: April 2004

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. ΔF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and ΔF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16°C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations ΔF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.
Constructing an enzyme-centric view of metabolism
Authors: Horne A. B. , Hodgman T. C. , Spence H. D. , Dalby A. R.
Published: April 2004

Motivation: The current paradigm for viewing metabolism, such as the Boehringer Chart or KEGG, takes a metabolite-centric view that is not ideal for genomics analysis because the same enzyme can appear in multiple places. Therefore an enzyme-centric view is also required.

Results: We have eliminated synonymous compound names taken from the ENZYME database ensuring that it is computationally parseable at all levels. Based on these results, we have written a software to create enzyme-centric graphs from reaction data, and we have created a second dataset with hub molecules removed, allowing a greater depth of information to be extracted from these graphs. We also present a detailed analysis of the various stages of the reconditioning process and the characteristics of the subgraphs resulting from the application of our software to the revised datasets.

Availability: Complete datasets and supplementary material may be downloaded from http://helix.ex.ac.uk/metabolism. The software for the creation of enzyme-centric graphs from reaction data is available on request from the authors.
Web-based kinetic modelling using JWS Online
Authors: Olivier Brett G. , Snoep Jacky L.
Published: April 2004

Summary: JWS Online is a repository of kinetic models, describing biological systems, which can be interactively run and interrogated over the Internet. It is implemented using a client–server strategy where the clients, in the form of web browser based Java applets, act as a graphical interface to the model servers, which perform the required numerical computations.

Availability: The JWS Online website is publicly accessible at http://jjj.biochem.sun.ac.za/ with mirrors at http://www.jjj.bio.vu.nl/ and http://jjj.vbi.vt.edu/
Speeding up whole-genome alignment by indexing frequency vectors
Authors: Kahveci Tamer , Ljosa Vebjorn , Singh Ambuj K.
Published: April 2004

Motivation: Many biological applications require the comparison of large genome strings. Current techniques suffer from high computational and I/O costs.

Results: We propose an efficient technique for local alignment of large genome strings. A space-efficient index is computed for one string, and the second string is compared with this index in order to prune substring pairs that do not contain similar regions. The remaining substring pairs are handed to a hash-table-based tool, such as BLAST, for alignment. A dynamic strategy is employed to optimize the number of disk seeks needed to access the hash table. Additionally, our technique provides the user with a coarse-grained visualization of the similarity pattern, quickly and before the actual search. The experimental results show that our technique aligns genome strings up to two orders of magnitude faster than BLAST. Our technique can be used to accelerate other search tools as well.

Availability: A web-based demo can be found at http://bioserver.cs.ucsb.edu/. Source code is available from the authors on request.
GeConT: gene context analysis
Authors: Ciria R. , Abreu-Goodger C. , Morett E. , Merino E.
Published: April 2004

Summary: The fact that adjacent genes in bacteria are often functionally related is widely known. GeConT (Gene Context Tool) is a web interface designed to visualize genome context of a gene or a group of genes and their orthologs in all the completely sequenced genomes. The graphical information of GeConT can be used to analyze genome annotation, functional ortholog identification or to verify the genomic context congruence of any set of genes that share a common property.

Availability:  http://www.ibt.unam.mx/biocomputo/gecont.html
An examination of the conservation of surface patch polarity for proteins
Authors: Shanahan H. P. , Thornton J. M.
Published: April 2004

Motivation: The solubility of a protein is crucial for its function and is therefore an evolutionary constraint. As the solubility of a protein is related to the distribution of polar and hydrophobic residues on its solvent accessible surface, such a constraint should provide a valuable insight into the evolution of protein surfaces. We examine how the surfaces of proteins have evolved by considering how the average hydrophobicities of patches of surface residues vary across homologous proteins. We derive distributions for the average hydrophobicity/philicity of surface patches at a residue-based level—which we refer to as the residue hydrophobic density. This is computed for a set of 28 monomeric proteins and their homologues. The resulting distributions are compared with a set of randomized sequences, with the same residue content.

Results: We find that the patches, involving typically more than 10 residues, maintain a more hydrophilic surface than one would expect from a random substitution model, indicating a cooperative behaviour for these surfaces residues in terms of this single variable.

Supplementary information: Additional plots for all of the proteins examined in this paper can be found at: http://www.ebi.ac.uk/~shanahan/PCon/index.html
Probabilistic representation of gene regulatory networks
Authors: Mao Linyong , Resat Haluk
Published: April 2004

Motivation: Recent experiments have established unambiguously that biological systems can have significant cell-to-cell variations in gene expression levels even in isogenic populations. Computational approaches to studying gene expression in cellular systems should capture such biological variations for a more realistic representation.

Results: In this paper, we present a new fully probabilistic approach to the modeling of gene regulatory networks that allows for fluctuations in the gene expression levels. The new algorithm uses a very simple representation for the genes, and accounts for the repression or induction of the genes and for the biological variations among isogenic populations simultaneously. Because of its simplicity, introduced algorithm is a very promising approach to model large-scale gene regulatory networks. We have tested the new algorithm on the synthetic gene network library bioengineered recently. The good agreement between the computed and the experimental results for this library of networks, and additional tests, demonstrate that the new algorithm is robust and very successful in explaining the experimental data.

Availability: The simulation software is available upon request.

Supplementary information: Supplementary material will be made available on the OUP server.
An interactive triple-helical collagen builder
Authors: Rainey Jan K. , Goh M. Cynthia
Published: April 2004

Summary: To facilitate the building of triple-helical polypeptide structures, we provide an interactive, platform-independent Tcl/Tk script. User specified amino acid sequences are parsed to obtain localized melting temperature estimates corresponding to helical propensity. These propensities may be graphically examined prior to their automated use in combination with two separate statistical parameter sets to build the complete polypeptide backbone and, if desired, predict side-chain terminal atom locations.

Availability: The Tcl/Tk Triple Helical collagen Building Script (THe BuScr) and full documentation is freely available under the terms of the GNU General Public License at www.pence.ca/~jrainey/ or www.chem.utoronto.ca/staff/MCG/

Supplementary information:  www.pence.ca/~jrainey