bluuebunny/crossref_metadata_2025 huggingface dataset:
Gaze Stability of Observers Watching Op Art Pictures
Authors: Zanker Johannes M , Doyle Melanie , Walker Robin
Published: March 2004

It has been the matter of some debate why we can experience vivid dynamic illusions when looking at static pictures composed from simple black and white patterns. The impression of illusory motion is particularly strong when viewing some of the works of ‘Op Artists’, such as Bridget Riley's painting Fall. Explanations of the illusory motion have ranged from retinal to cortical mechanisms, and an important role has been attributed to eye movements. To assess the possible contribution of eye movements to the illusory-motion percept we studied the strength of the illusion under different viewing conditions, and analysed the gaze stability of observers viewing the Riley painting and control patterns that do not produce the illusion. Whereas the illusion was reduced, but not abolished, when watching the painting through a pinhole, which reduces the effects of accommodation, it was not perceived in flash afterimages, suggesting an important role for eye movements in generating the illusion for this image. Recordings of eye movements revealed an abundance of small involuntary saccades when looking at the Riley pattern, despite the fact that gaze was kept within the dedicated fixation region. The frequency and particular characteristics of these rapid eye movements can vary considerably between different observers, but, although there was a tendency for gaze stability to deteriorate while viewing a Riley painting, there was no significant difference in saccade frequency between the stimulus and control patterns. Theoretical considerations indicate that such small image displacements can generate patterns of motion signals in a motion-detector network, which may serve as a simple and sufficient, but not necessarily exclusive, explanation for the illusion. Why such image displacements lead to perceptual results with a group of Op Art and similar patterns, but remain invisible for other stimuli, is discussed.
Wagon-Wheel Illusion under Steady Illumination: Real or Illusory?
Authors: Pakarian Pooya , Yasamy Mohammad Taghi
Published: March 2004

Wheels turning in the movies sometimes appear to rotate backwards. This is called the wagon-wheel illusion (WWI). The mechanism of this illusion is based on the intermittent nature of light in films and other stroboscopic presentations, which renders them as a series of snapshots rather than a continuous visual data stream. However, there have been claims that this illusion is seen even in continuous light, which would suggest that the visual system itself may sample a continuous visual data stream. We examined the rate of this putative sampling and its variations across individuals while in different psychological states. We obtained two results: (i) WWI occurred in stroboscopic lights as expected, (ii) WWI was never reported by our subjects under continuous lights, such as sunlight and lamps with DC power source. Thus, WWI cannot be taken as evidence for discreteness of conscious visual perception.
Transfer of Spatial Context from Visual to Haptic Search
Authors: Nabeta Tomohiro , Ono Fuminori , Kawahara Jun-Ichiro
Published: March 2004

Under incidental learning conditions, spatial layouts can be acquired implicitly and facilitate visual search (contextual-cueing effect). We examined whether the contextual-cueing effect is specific to the visual modality or transfers to the haptic modality. The participants performed 320 (experiment 1) or 192 (experiment 2) visual search trials based on a typical contextual-cueing paradigm, followed by haptic search trials in which half of the trials had layouts used in the previous visual search trials. The visual contextual-cueing effect was obtained in the learning phase. More importantly, the effect was transferred from visual to haptic searches; there was greater facilitation of haptic search trials when the spatial layout was the same as in the previous visual search trials, compared with trials in which the spatial layout differed from those in the visual search. This suggests the commonality of spatial memory to allocate focused attention in both visual and haptic modalities.
Allozyme, chromosomal and morphological variability in the Litoria lesueuri species group (Anura : Hylidae), including a description of a new species
Authors: Donnellan S. C. , Mahony M. J.
Published: March 2004

Allozyme variation at 36 loci was screened in 179 individuals of the Litoria lesueuri species group from 33 locations from across the species' range in eastern Australia. We identified three genetic groups, with discrete and disjunct distributions, that we consider to be separate species. The northernmost group, L.�jungguy, sp. nov., had a distinctive standard and C-band karyotype, while the karyotypes of the two southern groups, L. lesueuri (Duméril & Bibron, 1841) and L. wilcoxii Gunther, 1864, differed only in minor details of their C-band patterns. Stepwise discriminant function analysis of morphometric variation among 13 meristic variables was able to discriminate L. lesueuri and L. wilcoxii in 100% of cases but discrimination between L. wilcoxii and L. jungguy was less successful, with 75% and 56% of each group respectively being correctly classified. Locations with inter-group hybrids were identified in the Australian Capital Territory, mid-east Queensland and two localities in far north Queensland. However, each of the groups has a large geographic range within which there was relative allozyme and karyotype uniformity. On the basis of the genetic data, each group is herein elevated to species status. Existing types were assigned to their respective genetic groups on the basis of a discriminant function analysis. Formal taxonomic accounts are provided. The closely related L. booroolongensis (Moore, 1961), sole member of the L.�booroolongensis species group, differed allozymically from all three species of the L. lesueuri species group but only from the northern species in its karyotype. Small genetic distances between L.�booroolongensis and members of the L. lesueuri species group indicate a close relationship for the two species groups.
CBF2/DREB1C is a negative regulator ofCBF1/DREB1BandCBF3/DREB1Aexpression and plays a central role in stress tolerance inArabidopsis
Authors: Novillo Fernando , Alonso José M. , Ecker Joseph R. , Salinas Julio
Published: March 2004

CBF/DREB1(C-repeat-binding factor/dehydration responsive element-binding factor 1) genes encode a small family of transcriptional activators that have been described as playing an important role in freezing tolerance and cold acclimation inArabidopsis. To specify this role, we used a reverse genetic approach and identified a mutant,cbf2, in which theCBF2/DREB1Cgene was disrupted. Here, we show thatcbf2plants have higher capacity to tolerate freezing than WT ones before and after cold acclimation and are more tolerant to dehydration and salt stress. All these phenotypes correlate with a stronger and more sustained expression of CBF/DREB1-regulated genes, which results from an increased expression ofCBF1/DREB1BandCBF3/DREB1Ain the mutant. In addition, we show that the expression ofCBF1/DREB1BandCBF3/DREB1Ain response to low temperature precedes that ofCBF2/DREB1C. These results indicate that CBF2/DREB1C negatively regulatesCBF1/DREB1BandCBF3/DREB1A, ensuring that their expression is transient and tightly controlled, which, in turn, guarantees the proper induction of downstream genes and the accurate development ofArabidopsistolerance to freezing and related stresses.
Protein dynamics and the immunological evolution of molecular recognition
Authors: Jimenez Ralph , Salazar Georgina , Yin Jun , Joo Taiha , Romesberg Floyd E.
Published: March 2004

While it is accepted that protein flexibility plays a role in protein folding, catalysis, and molecular recognition, few techniques are capable of the rigorous measurement of protein motions required to quantify flexibility. Three-pulse photon echo shift spectroscopy can be used to measure the time scale of protein motions, and we have used this technique, along with steady-state spectroscopy and binding and structural data, to examine the immunological evolution of protein flexibility in an anti-fluorescein antibody. Two light chain somatic mutations increase affinity for fluorescein by 12-fold but also significantly affect flexibility. Specifically, a rigidification of the protein is seen in each of three observable motions; two slower motions undergo decreased amplitudes of displacement, by 3- and 20-fold, respectively, in response to an applied force, and the distribution associated with the amplitude of a faster motion is narrowed upon somatic mutation. The somatic mutations appear to rigidify the antibody-fluorescein complex by more strongly anchoring fluorescein to the protein and by more tightly packing the complex. The data demonstrate that in addition to affinity, antibody dynamics are systematically manipulated during affinity maturation, and they imply that the evolution of protein flexibility may be a central component of the immune response. The results also reflect the type of protein rigidification that may be important for other biological interactions, such as protein-protein, protein-ligand or protein-drug, and enzyme-substrate recognition.
The scaling of frontal cortex in primates and carnivores
Authors: Bush Eliot C. , Allman John M.
Published: March 2004

Size has a profound effect on the structure of the brain. Many brain structures scale allometrically, that is, their relative size changes systematically as a function of brain size. Here we use independent contrasts analysis to examine the scaling of frontal cortex in 43 species of mammals including 25 primates and 15 carnivores. We find evidence for significant differences in scaling between primates and carnivores. Primate frontal cortex hyperscales relative to the rest of neocortex and the rest of the brain. The slope of frontal cortex contrasts on rest of cortex contrasts is 1.18 (95% confidence interval, 1.06-1.30) for primates, which is significantly greater than isometric. It is also significantly greater than the carnivore value of 0.94 (95% confidence interval, 0.82-1.07). This finding supports the idea that there are substantial differences in frontal cortex structure and development between the two groups.
CbiZ, an amidohydrolase enzyme required for salvaging the coenzyme B
12
precursor cobinamide in archaea
Authors: Woodson Jesse D. , Escalante-Semerena Jorge C.
Published: March 2004

The existence of a pathway for salvaging the coenzyme B
12
precursor dicyanocobinamide (Cbi) from the environment was established by genetic and biochemical means. The pathway requires the function of a previously unidentified amidohydrolase enzyme that converts adenosylcobinamide to adenosylcobyric acid, a bona fide intermediate of the
de novo
coenzyme B
12
biosynthetic route. The
cbiZ
gene of the methanogenic archaeon
Methanosarcina mazei
strain Göl was cloned, was overproduced in
Escherichia coli
, and the recombinant protein was isolated to homogeneity. HPLC, UV-visible spectroscopy, MS, and bioassay data established adenosylcobyric as the corrinoid product of the CbiZ-catalyzed reaction. Inactivation of the
cbiZ
gene in the extremely halophilic archaeon
Halobacterium
sp. strain NRC-1 blocked the ability of this archaeon to salvage Cbi.
cbiZ
function restored Cbi salvaging in a strain of the bacterium
Salmonella enterica
, whose Cbi-salvaging pathway was blocked. The salvaging of Cbi through the CbiZ enzyme appears to be an archaeal strategy because all of the genomes of B
12
-producing archaea have a
cbiZ
ortholog. Reasons for the evolution of two distinct pathways for Cbi salvaging in prokaryotes are discussed.
GABA-mediated giant depolarizing potentials as coincidence detectors for enhancing synaptic efficacy in the developing hippocampus
Authors: Kasyanov Alexander M. , Safiulina Victoria F. , Voronin Leon L. , Cherubini Enrico
Published: March 2004

Spontaneously occurring neuronal oscillations constitute a hallmark of developmental networks. They have been observed in the retina, neocortex, hippocampus, thalamus, and spinal cord. In the immature hippocampus, the so-called “giant depolarizing potentials” (GDPs) are network-driven synaptic events generated by γ-aminobutyric acid (GABA), which at this stage is depolarizing and excitatory. We have tested the hypothesis that during the first postnatal week, GDP-associated calcium signals may alter the properties of synaptic transmission at poorly developed mossy fiber (MF)-CA3 connections. We found that “pairing” GDPs with MF stimulation induced a persistent increase in synaptic efficacy at MF-CA3 synapses. When the interval between GDPs and MF stimulation was increased, the potentiating effect progressively declined and disappeared. The potentiation depended on activation of voltage-dependent calcium channels and calcium flux. This activity may contribute to the refinement of neuronal connectivity before the establishment of the adult neuronal circuit.
The quantal nature of Ca
2+
sparks and
in situ
operation of the ryanodine receptor array in cardiac cells
Authors: Wang Shi Qiang , Stern Michael D. , Ríos Eduardo , Cheng Heping
Published: March 2004

Intracellular Ca
2+
release in many types of cells is mediated by ryanodine receptor Ca
2+
release channels (RyRCs) that are assembled into two-dimensional paracrystalline arrays in the endoplasmic/sarcoplasmic reticulum. However, the
in situ
operating mechanism of the RyRC array is unknown. Here, we found that the elementary Ca
2+
release events, Ca
2+
sparks from individual RyRC arrays in rat ventricular myocytes, exhibit quantized Ca
2+
release flux. Analysis of the quantal property of Ca
2+
sparks provided a view of unitary Ca
2+
current and gating kinetics of the RyRC in intact cells and revealed that spark activation involves dynamic recruitment of small, variable cohorts of RyRCs. Intriguingly, interplay of RyRCs in multichannel sparks renders an unusual, thermodynamically irreversible mode of channel gating that is unshared by an RyRC acting solo, nor by RyRCs
in vitro
. Furthermore, an array-based inhibitory feedback, overriding the regenerative Ca
2+
-induced Ca
2+
release of RyRCs, provides a supramolecular mechanism for the microscopic stability of intracellular Ca
2+
signaling.
The brain of the Remipedia (Crustacea) and an alternative hypothesis on their phylogenetic relationships
Authors: Fanenbruck Martin , Harzsch Steffen , Wägele Johann Wolfgang
Published: March 2004

Remipedia are rare and ancient mandibulate arthropods inhabiting almost inaccessible submerged cave systems. Their phylogenetic position is still enigmatic and the subject of extremely controversial debates. To contribute arguments to this discussion, we analyzed the brain of
Godzilliognomus frondosus
Yager, 1989 (Remipedia, Godzilliidae) and provide a detailed 3D reconstruction of its anatomy. This reconstruction yielded the surprising finding that in comparison with the brain of other crustaceans such as representatives of the Branchiopoda and Maxillopoda the brain of
G. frondosus
is highly organized and well differentiated. It is matched in complexity only by the brain of “higher” crustaceans (Malacostraca) and Hexapoda. A phylogenetic analysis limited to brain anatomy across the Mandibulata strongly contradicts the prevailing hypothesis that the Remipedia are a basal, ancestral crustacean group but instead argues in favor of a remipede-malacostracan-hexapod clade and most likely a sister-group relationship of Remipedia and Malacostraca.
Universality and flexibility in gene expression from bacteria to human
Authors: Ueda Hiroki R. , Hayashi Satoko , Matsuyama Shinichi , Yomo Tetsuya , Hashimoto Seiichi , Kay Steve A. , Hogenesch John B. , Iino Masamitsu
Published: March 2004

Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but to explore for underlying governing principles. Recent analysis of the large-scale organization of gene expression has revealed its complex and dynamic nature. However, the underlying dynamics that generate complex gene expression and cellular organization are not yet understood. To comprehensively and quantitatively elucidate these underlying gene expression dynamics, we have analyzed genome-wide gene expression in many experimental conditions in
Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus
, and
Homo sapiens
. Here we demonstrate that the gene expression dynamics follows the same and surprisingly simple principle from
E. coli
to human, where gene expression changes are proportional to their expression levels, and show that this “proportional” dynamics or “rich-travel-more” mechanism can regenerate the observed complex and dynamic organization of the transcriptome. These findings provide a universal principle in the regulation of gene expression, show how complex and dynamic organization can emerge from simple underlying dynamics, and demonstrate the flexibility of transcription across a wide range of expression levels.
Topical DNA oligonucleotide therapy reduces UV-induced mutations and photocarcinogenesis in hairless mice
Authors: Goukassian David A. , Helms Elizabeth , van Steeg Harry , van Oostrom Conny , Bhawan Jag , Gilchrest Barbara A.
Published: March 2004

UV-induced DNA damage gives rise to mutations and skin cancer. We have previously reported that treatment of skin cells
in vitro
with thymidine dinucleotide (pTT) activates p53 and increases the ability of cells to repair subsequent UV-induced DNA damage by enhancing endogenous DNA repair capacity. Here we show that topical pTT pretreatment enhances the rate of DNA photoproduct removal, decreases UV-induced mutations, and reduces photocarcinogenesis in UV-irradiated hairless WT repair-proficient and Xpc
+/-
heterozygous partially repair-deficient mice, both transgenic for the
lacZ
/pUR288 mutation-indicator gene. These data support the existence of inducible mammalian DNA damage responses that increase DNA repair capacity after DNA damage and hence reduce the impact of future exposures to environmental carcinogens. The ability of topically applied pTT to induce protective physiologic responses that normally result from DNA damage suggests a previously undescribed means of reducing skin cancer in high-risk individuals.
Human CD34+cells differentiate into microglia and express recombinant therapeutic protein
Authors: Asheuer Muriel , Pflumio Françoise , Benhamida Sonia , Dubart-Kupperschmitt Anne , Fouquet Françoise , Imai Yoshinori , Aubourg Patrick , Cartier Nathalie
Published: March 2004

In rodents, bone marrow-derived cells enter the brain during adult life. Allogeneic bone marrow transplantation is used to treat genetic CNS diseases, but the fate of human bone marrow and CD34+cells within the brain remains to be elucidated. The present study demonstrates that cells derived from human CD34+cells, isolated from either cord blood or peripheral blood, migrate into the brain after infusion into nonobese diabetic/severe combined immunodeficient mice. Both types of CD34+-derived cells differentiate into perivascular and ramified microglia. The lentiviral transfer of genes into CD34+cells before infusion does not modify the differentiation of human CD34+cells into microglia, allowing new transgenic proteins to be expressed in these cells. The transplantation of CD34+cells could thus be used for the treatment of CNS diseases.
Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump
Authors: Dimitriadis George , Drysdale Adam , Myers Jeffrey K. , Arora Pooja , Radford Sheena E. , Oas Terence G. , Smith D. Alastair
Published: March 2004

The small size (58 residues) and simple structure of the B domain of staphylococcal protein A (BdpA) have led to this domain being a paradigm for theoretical studies of folding. Experimental studies of the folding of BdpA have been limited by the rapidity of its folding kinetics. We report the folding kinetics of a fluorescent mutant of BdpA (G29A F13W), named F13W
*
, using nanosecond laser-induced temperature jump experiments. Automation of the apparatus has permitted large data sets to be acquired that provide excellent signal-to-noise ratio over a wide range of experimental conditions. By measuring the temperature and denaturant dependence of equilibrium and kinetic data for F13W
*
, we show that thermodynamic modeling of multidimensional equilibrium and kinetic surfaces is a robust method that allows reliable extrapolation of rate constants to regions of the folding landscape not directly accessible experimentally. The results reveal that F13W
*
is the fastest-folding protein of its size studied to date, with a maximum folding rate constant at 0 M guanidinium chloride and 45°C of 249,000 s
-1
. Assuming the single-exponential kinetics represent barrier-limited folding, these data limit the value for the preexponential factor for folding of this protein to at least ≈2 × 10
6
s
-1
.
The tissue plasminogen activator-plasmin system participates in the rewarding effect of morphine by regulating dopamine release
Authors: Nagai Taku , Yamada Kiyofumi , Yoshimura Masako , Ishikawa Kazuhiro , Miyamoto Yoshiaki , Hashimoto Kazuki , Noda Yukihiro , Nitta Atsumi , Nabeshima Toshitaka
Published: March 2004

Tissue plasminogen activator (tPA) is a serine protease that catalyzes the conversion of plasminogen (plg) to plasmin, which in turn functions to degrade extracellular matrix proteins in the central nervous system. The tPA-plasmin system plays a role in synaptic plasticity and remodeling. Here we show that this protease system participates in the rewarding effects of morphine by acutely regulating morphine-induced dopamine release in the nucleus accumbens (NAcc). A single morphine treatment induced tPA mRNA and protein expression in a naloxone-sensitive manner, which was associated with an increase in the enzyme activity in the NAcc. The acute effect of morphine in inducing tPA expression was diminished after repeated administration. Morphine-induced conditioned place preference and hyperlocomotion were significantly reduced in tPA
-/-
and plg
-/-
mice, being accompanied by a loss of morphine-induced dopamine release in the NAcc. The defect of morphine-induced dopamine release and hyperlocomotion in tPA
-/-
mice was reversed by microinjections of either exogenous tPA or plasmin into the NAcc. Our findings demonstrate a previously undescribed function of the tPA-plasmin system in regulating dopamine release, which is involved in the rewarding effects of morphine.
Physiologically mediated self/non-self discrimination in roots
Authors: Gruntman Michal , Novoplansky Ariel
Published: March 2004

Recent evidence suggests that self/non-self discrimination exists among roots; its mechanisms, however, are still unclear. We compared the growth of
Buchloe dactyloides
cuttings that were grown in the presence of neighbors that belonged to the same physiological individual, were separated from each other for variable periods, or originated from adjacent or remote tillers on the same clone. The results demonstrate that
B. dactyloides
plants are able to differentiate between self and non-self neighbors and develop fewer and shorter roots in the presence of other roots of the same individual. Furthermore, once cuttings that originate from the very same node are separated, they become progressively alienated from each other and eventually relate to each other as genetically alien plants. The results suggest that the observed self/non-self discrimination is mediated by physiological coordination among roots that developed on the same plant rather than allogenetic recognition. The observed physiological coordination is based on an as yet unknown mechanism and has important ecological implications, because it allows the avoidance of competition with self and the allocation of greater resources to alternative functions.
Attractant binding alters arrangement of chemoreceptor dimers within its cluster at a cell pole
Authors: Homma Motohiro , Shiomi Daisuke , Homma Michio , Kawagishi Ikuro
Published: March 2004

Many sensory systems involve multiple steps of signal amplification to produce a significant response. One such mechanism may be the clustering of transmembrane receptors. In bacterial chemotaxis, where a stoichiometric His-Asp phosphorelay from the kinase CheA to the response regulator CheY plays a central role, the chemoreceptors (methyl-accepting chemotaxis proteins) cluster together with CheA and the adaptor CheW, at a pole of a rod-shaped cell. This clustering led to a proposal that signal amplification occurs through an interaction between chemoreceptor homodimers. Here, by using
in vivo
disulfide crosslinking assays, we examined an interdimer interaction of the aspartate chemoreceptor (Tar). Two cysteine residues were introduced into Tar: one at the subunit interface and the other at the external surface of the dimer. Crosslinked dimers and higher oligomers (especially a deduced hexamer) were detected and their abundance depended on CheA and CheW. The ligand aspartate significantly reduced the amounts of higher oligomers but did not affect the polar localization of Tar-GFP. Thus, the binding of aspartate alters the rate of collisions between Tar dimers in assembled signaling complexes, most likely due to a change in the relative positions or trajectories of the dimers. These collisions could occur within a trimer-ofdimers predicted by crystallography, or between such trimers. These results are consistent with the proposal that the interaction of chemoreceptor dimers is involved in signal transduction.
Vascularized thymic lobe transplantation in miniature swine: Thymopoiesis and tolerance induction across fully MHC-mismatched barriers
Authors: Kamano Chisako , Vagefi Parsia A. , Kumagai Naoki , Yamamoto Shin , Barth Rolf N. , LaMattina John C. , Moran Shannon G. , Sachs David H. , Yamada Kazuhiko
Published: March 2004

As the major site of self-nonself discrimination in the immune system, the thymus, if successfully transplanted, could potentially carry with it the induction of central tolerance to any other organ or tissue from the same donor. We have recently developed a technique for transplantation of an intact, vascularized thymic lobe (VTL) in miniature swine. In the present study, we have examined the ability of such VTL allografts to support thymopoiesis and induce transplantation tolerance across fully MHC-mismatched barriers. Six miniature swine recipients received fully MHC-mismatched VTL grafts with a 12-day course of tacrolimus. Three of these recipients were thymectomized before transplantation and accepted their VTL allografts long-term, with evidence of normal thymopoiesis. In contrast, three euthymic recipients rejected their VTL allografts. Donor renal allografts, matched to the donor VTL grafts, were transplanted without immunosuppression into two of the three thymectomized recipients, and one of the three euthymic recipients. These renal allografts were accepted by thymectomized recipients, but rejected by the euthymic recipient in an accelerated fashion. This study thus demonstrates that successful transplantation of a vascularized thymus across a fully MHC-mismatched barrier induces tolerance in this preclinical, large-animal model. This procedure should enable studies on the role of the thymus in transplantation immunology as well as offer a potential strategy for tolerance induction in clinical transplantation.
Fluorometric measurements of conformational changes in glutamate transporters
Authors: Larsson H. Peter , Tzingounis Anastassios V. , Koch Hans P. , Kavanaugh Michael P.
Published: March 2004

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neurotoxic levels (1). It is thought that glutamate transporters mediate glutamate transport through a reaction cycle with conformational changes between the two major access states that alternatively expose glutamate-binding sites to the extracellular or to the intracellular solution. However, there is no direct real-time evidence for the conformational changes predicted to occur during the transport cycle. In the present study, we used voltage-clamp fluorometry to measure conformational changes in the neuronal excitatory amino acid transporter (EAAT) 3 glutamate transporter covalently labeled with a fluorescent reporter group. Alterations in glutamate and cotransported ion concentrations or in the membrane voltage induced changes in the fluorescence that allowed detection of conformational rearrangements occurring during forward and reverse transport. In addition to the transition between the two major access states, our results show that there are significant Na+-dependent conformational changes preceding glutamate binding. We furthermore show that Na+and H+are cotransported with glutamate in the forward part of the transport cycle. The data further suggest that an increase in proton concentrations slows the reverse transport of glutamate, which may play a neuro-protective role during ischemia.
Modulation of rhythmic brain activity by diazepam: GABA
A
receptor subtype and state specificity
Authors: Kopp C. , Rudolph U. , Löw K. , Tobler I.
Published: March 2004

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is involved in the generation of various brain rhythmic activities that can be modulated by benzodiazepines. Here, we assessed the contribution of α
2
GABA type A (GABA
A
) receptors to the effects of benzodiazepines on sleep and waking oscillatory patterns by combining pharmacological and genetic tools. The effects of diazepam on the electroencephalogram were compared between α
2
(H101R) knock-in mice in which the α
2
GABA
A
receptor was rendered diazepam-insensitive, and their wild-type controls. The suppression of delta activity typically induced by diazepam in non-rapid eye movement (REM) sleep was significantly stronger in wild-type control mice than in α
2
(H101R) mice. Moreover, electroencephalogram frequency activity above 16-18 Hz was enhanced in wild-type mice both in non-REM sleep and waking. This effect was absent in α
2
(H101R) mice. Theta activity was enhanced after diazepam both in REM sleep and in waking in wild-type mice. In α
2
(H101R) mice, this effect was markedly reduced in REM sleep whereas it persisted in waking. These findings suggest that α
2
GABA
A
receptors, which are expressed in hypothalamic and pontine nuclei and in the hippocampus, are localized in distinct neural circuits relevant for the modulation of rhythmic brain activities by benzodiazepines.
A single amino acid of APOBEC3G controls its species-specific interaction with virion infectivity factor (Vif)
Authors: Schröfelbauer Bärbel , Chen Darlene , Landau Nathaniel R.
Published: March 2004

The virion infectivity factor (Vif) accessory protein of HIV-1 forms a complex with the cellular cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) to block its antiviral activity. The antiviral property of APOBEC3G is conserved in several mammalian species, but the ability of Vif to block this activity is species-specific. HIV-1 Vif blocks human APOBEC3G but does not block the mouse or African green monkey (AGM) enzyme. Conversely, SIV
AGM
Vif blocks the antiviral activity of AGM but not human APOBEC3G. We demonstrate that the species specificity is caused by a single amino acid difference in APOBEC3G. Replacement of Asp-128 in human APOBEC3G with the Lys-128 of AGM APOBEC3G caused the enzyme to switch its interaction, becoming sensitive to SIV
AGM
Vif and resistant to HIV-1 Vif. Conversely, the reciprocal Lys to Asp switch in AGM APOBEC3G reversed its specificity for Vif. The reversal of biological activity was accompanied by the corresponding switch in the species specificity with which the enzyme physically associated with Vif and was excluded from virions. The charge of the amino acid at position 128 was a critical determinant of species specificity. Based on the crystal structure of the distantly related
Escherichia coli
cytidine deaminase, we propose that this amino acid is positioned on a solvent-exposed loop of APOBEC3G on the same face of the protein as the catalytic site.
Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export
Authors: Thomas Joanne , Stafford Graham P. , Hughes Colin
Published: March 2004

Bacterial type III protein export underlies flagellum assembly and delivery of virulence factors into eukaryotic cells. The sequence of protein interactions underlying the export pathway are poorly characterized; in particular, it is not known how chaperoned substrates in the cytosol are engaged by the membrane-localized export apparatus. We have identified a stalled intermediate export complex in the flagellar type III export pathway of
Salmonella typhimurium
by generating dominant-negative chaperone variants that are export-defective and arrest flagellar assembly in the wild-type bacterium. These chaperone variants bound their specific export substrates strongly and severely reduced their export. They also attenuated export of other flagellar proteins, indicating that inhibition occurs at a common step in the pathway. Unlike the cytosolic wild-type chaperone, the variants localized to the inner membrane, but not in the absence of the flagellar type III export apparatus. Membrane localization persisted in
fliOPQR, flhB, flhA, fliJ
, and
fliH
null mutants lacking specific flagellar export components but depended on the presence of the membrane-associated ATPase FliI. After expression of the variant chaperones in
Salmonella
, a stalled intermediate export complex, which contained chaperone, substrate, and the FliI ATPase with its regulator FliH, was isolated. Neither chaperone nor substrate alone was able to interact with liposome-associated FliI, but the chaperone-substrate-FliI(FliH) complex was assembled when chaperone was prebound to its substrate. Our data establish a key event in the type III protein export mechanism, docking of the cytosolic chaperone-substrate complex at the ATPase of the membrane-export apparatus.
Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by α5 subunit-containing γ-aminobutyric acid type A receptors
Authors: Caraiscos Valerie B. , Elliott Erin M. , You-Ten Kong E. , Cheng Victor Y. , Belelli Delia , Newell J. Glen , Jackson Michael F. , Lambert Jeremy J. , Rosahl Thomas W. , Wafford Keith A. , MacDonald John F. , Orser Beverley A.
Published: March 2004

The principal inhibitory neurotransmitter in the mammalian brain, γ-aminobutyric acid (GABA), is thought to regulate memory processes by activating transient inhibitory postsynaptic currents. Here we describe a nonsynaptic, tonic form of inhibition in mouse CA1 pyramidal neurons that is generated by a distinct subpopulation of GABA type A receptors (GABA
A
Rs). This tonic inhibitory conductance is predominantly mediated by α5 subunit-containing GABA
A
Rs (α5GABA
A
Rs) that have different pharmacological and kinetic properties compared to postsynaptic receptors. GABA
A
Rs that mediate the tonic conductance are well suited to detect low, persistent, ambient concentrations of GABA in the extracellular space because they are highly sensitive to GABA and desensitize slowly. Moreover, the tonic current is highly sensitive to enhancement by amnestic drugs. Given the restricted expression of α5GABA
A
Rs to the hippocampus and the association between reduced α5GABA
A
R function and improved memory performance in behavioral studies, our results suggest that tonic inhibition mediated by α5GABA
A
Rs in hippocampal pyramidal neurons plays a key role in cognitive processes.
Transmembrane protein domains rarely use covalent domain recombination as an evolutionary mechanism
Authors: Liu Yang , Gerstein Mark , Engelman Donald M.
Published: March 2004

Recombination of evolutionarily unrelated domains is a mechanism often used by evolution to produce variety in soluble proteins. By using a classification of polytopic transmembrane domains into families, we examined integral membrane proteins for evidence of this mechanism. Surprisingly, we found that domain recombination is not common for the transmembrane regions of membrane proteins, a majority of integral membrane proteins containing only a single transmembrane domain. We suggest that noncovalent oligomeric associations, which are common in membrane proteins, may provide an alternative source of evolutionary diversity.
Impaired IgA class switching in APRIL-deficient mice
Authors: Castigli Emanuela , Scott Sumi , Dedeoglu Fatma , Bryce Paul , Jabara Haifa , Bhan Atul K. , Mizoguchi Emiko , Geha Raif S.
Published: March 2004

The tumor necrosis factor (TNF) family member APRIL binds to the receptors BCMA on B cells and TACI on B and T cells. To investigate the role of APRIL in immunity, we generated APRIL-deficient mice. APRIL-/-mice have normal T and B lymphocyte development, normal T and B cell proliferationin vitro, but increased numbers of CD44hiCD62LloCD4+effector/memory T cells and increased IgG responses to T-dependent antigens. Serum IgA levels were significantly decreased, and serum IgA antibody responses to mucosal immunization with TD antigens and to type 1 T-independent antigens were impaired in APRIL-/-mice. APRIL by itself induced IgA as well as IgG1 isotype switching in CD40-deficient IgM+IgD+sorted B cells. These results suggest that APRIL down-regulates T cell-dependent antibody responses and promotes IgA class switching.
Hearing loss and retarded cochlear development in mice lacking type 2 iodothyronine deiodinase
Authors: Ng Lily , Goodyear Richard J. , Woods Chad A. , Schneider Mark J. , Diamond Edward , Richardson Guy P. , Kelley Matthew W. , Germain Donald L. St. , Galton Valerie Anne , Forrest Douglas
Published: March 2004

The later stages of cochlear differentiation and the developmental onset of hearing require thyroid hormone. Although thyroid hormone receptors (TRs) are a prerequisite for this process, it is likely that other factors modify TR activity during cochlear development. The mouse cochlea expresses type 2 deiodinase (D2), an enzyme that converts thyroxine, the main form of thyroid hormone in the circulation, into 3,5,3′-triiodothyronine (T3) the major ligand for TRs. Here, we show that D2-deficient mice have circulating thyroid hormone levels that would normally be adequate to allow hearing to develop but they exhibit an auditory phenotype similar to that caused by systemic hypothyroidism or TR deletions. D2-deficient mice have defective auditory function, retarded differentiation of the cochlear inner sulcus and sensory epithelium, and deformity of the tectorial membrane. The similarity of this phenotype to that caused by TR deletions suggests that D2 controls the T3 signal that activates TRs in the cochlea. Thus, D2 is essential for hearing, and the results suggest that this hormone-activating enzyme confers on the cochlea the ability to stimulate its own T3 response at a critical developmental period.
Transcription-induced barriers to supercoil diffusion in the
Salmonella typhimurium
chromosome
Authors: Deng Shuang , Stein Richard A. , Higgins N. Patrick
Published: March 2004

Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the
Salmonella typhimurium
chromosome to analyze supercoiling dynamics of regions encoding membrane proteins. Regulation of Tn
10
-derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA. Strains deficient in TetR activity had 60-fold higher transcription levels (from

P
A

) than TetR-positive strains. High
tetA
transcription caused a 10- to 80-fold decrease in the γδ resolution efficiency for the domain that includes the Tet module. Replacing
tetA
with genes encoding cytosolic proteins LacZ and Kan also caused the appearance of supercoil diffusion barriers in a defined region of the chromosome. In strains containing a functional TetR located next to a regulated
lacZ
reporter (

P
R
tetR-P
A
lacZ

), induction of transcription with chlortetracycline caused a 5-fold drop in resolution efficiency in the test domain interval. A short half-life resolvase showed that barriers appeared and disappeared over a 10- to 20-min span. These studies demonstrate the importance of transcription in chromosome structure and the plasticity of supercoil domains in bacterial chromosomes.
Isolation of antibiotic resistance mutations in the rRNA by using an
in vitro
selection system
Authors: Cochella Luisa , Green Rachel
Published: March 2004

Genetic, biochemical, and structural data support an essential role for the ribosomal RNA in all steps of the translation process. Although
in vivo
genetic selection techniques have been used to identify mutations in the rRNAs that result in various miscoding phenotypes and resistance to known ribosome-targeted antibiotics, these are limited because the resulting mutant ribosomes must be only marginally disabled if they are able to support growth of the cell. Furthermore,
in vivo
, it is not possible to control the environment in precise ways that might allow for the isolation of certain types of rRNA variants. To overcome these limitations, we have developed an
in vitro
selection system for the isolation of functionally competent ribosomal particles from populations containing variant rRNAs. Here, we describe this system and present an example of its application to the selection of antibiotic resistance mutations. From a pool of 4,096 23S rRNA variants, a double mutant (A2058U/A2062G) was isolated after iteration of the selection process. This mutant was highly resistant to clindamycin in
in vitro
translation reactions and yet was not viable in
Escherichia coli
. These data establish that this system has the potential to identify mutations in the rRNA not readily accessed by comparable
in vivo
systems, thus allowing for more exhaustive ribosomal genetic screens.
Kinesin's second step
Authors: Klumpp Lisa M. , Hoenger Andreas , Gilbert Susan P.
Published: March 2004

We have identified dimeric kinesin mutants that become stalled on the microtubule after one ATP turnover, unable to bind and hydrolyze ATP at their second site. We have used these mutants to determine the regulatory signal that allows ATP to bind to the forward head, such that processive movement can continue. The results show that phosphate release occurs from the rearward head before detachment, and detachment triggers active-site accessibility for ATP binding at the forward head. This mechanism, in which the rearward head controls the behavior of the forward head, may be conserved among processive motors.
A single amino acid difference in the host APOBEC3G protein controls the primate species specificity of HIV type 1 virion infectivity factor
Authors: Bogerd Hal P. , Doehle Brian P. , Wiegand Heather L. , Cullen Bryan R.
Published: March 2004

The HIV type 1 (HIV-1) virion infectivity factor (Vif) protein blocks the action of the host defense factor APOBEC3G in human cells, thereby allowing release of infectious virions, but fails to inhibit similar APOBEC3G proteins present in some simian cells. Conversely, the Vif protein encoded by the African green monkey (agm) simian immunodeficiency virus (SIV) can block agm APOBEC3G function but fails to inhibit human APOBEC3G. This difference plays a key role in determining the primate species tropism of HIV-1 and SIV agm. Here, we demonstrate that a single APOBEC3G residue, which is an aspartic acid in human APOBEC3G and a lysine in agm APOBEC3G, controls the ability of the HIV-1 Vif protein to bind and inactivate these host defense factors. These data identify a critical charged residue that plays a key role in mediating the formation of the distinct Vif:APOBEC3G complexes formed in human and simian cells. Moreover, these results suggest that the biological barrier preventing the entry of additional SIV into the human population as zoonotic infections is potentially quite fragile.
Water in protein structure prediction
Authors: Papoian Garegin A. , Ulander Johan , Eastwood Michael P. , Luthey-Schulten Zaida , Wolynes Peter G.
Published: March 2004

Proteins have evolved to use water to help guide folding. A physically motivated, nonpairwise-additive model of water-mediated interactions added to a protein structure prediction Hamiltonian yields marked improvement in the quality of structure prediction for larger proteins. Free energy profile analysis suggests that long-range water-mediated potentials guide folding and smooth the underlying folding funnel. Analyzing simulation trajectories gives direct evidence that water-mediated interactions facilitate native-like packing of supersecondary structural elements. Long-range pairing of hydrophilic groups is an integral part of protein architecture. Specific water-mediated interactions are a universal feature of biomolecular recognition landscapes in both folding and binding.
Estrogen receptors ERα and ERβ in proliferation in the rodent mammary gland
Authors: Cheng Guojun , Weihua Zhang , Warner Margaret , Gustafsson Jan-Åke
Published: March 2004

Most evidence supports the view that ERα is responsible for estrogen (ovarian estradiol, E
2
)-induced proliferation in the epithelial cells of the mammary gland, but despite this, proliferating epithelial cells do not express ERα. We have examined this apparent paradox by studying the role of ERα and ERβ in E
2
-induced proliferation in mammary glands (measured by BrdUrd incorporation into DNA) in mice with intact ERβ (WT mice) and those in which the ERβ gene has been inactivated (ERβ
-/-
mice). On treatment of ERβ
-/-
mice with E
2
or ovariectomized WT mice with E
2
, tamoxifen, or a specific ERβ agonist (BAG), the number of BrdUrd-labeled cells in mammary glands increased from 3.4% in controls to 28-38% in the treated mice. This indicates that both ERα and ERβ can mediate E
2
-induced proliferation independently of each other. With specific antibodies, ERβ was found in both epithelial and stromal cells, whereas ERα was strictly epithelial. Within 4 h of a single dose of E
2
, ERα was lost from the nuclei of epithelial cells. In WT mice, ERα reappeared by 24 h, but in ERβ
-/-
mice, return to the nucleus was delayed by 24 h. At 4 h after E
2
, neither ERα nor progesterone receptor was detectable in BrdUrd-labeled nuclei but by 48 h after E
2
, 29% of the BrdUrd-labeled cells expressed ERα, and 21-38% expressed progesterone receptor. During 3 weeks of continuous E
2
treatment, ERβ remained in the nucleus, but there was no detectable ERα. With tamoxifen treatment, ERα remained in the nucleus, but ERβ was lost. From these results, we conclude that ERα receives the proliferation signal from E
2
, initiates DNA synthesis, and is then lost from cells. The subsequent steps in proliferation can proceed in the absence of either ERα or ERβ. ERβ facilitates the return of ERα to the nucleus and restores responsiveness to E
2
. By down-regulating ERβ, tamoxifen may prolong refractoriness to E
2
in mammary epithelium.