bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal decomposition of surfactant coatings on Co and Ni nanocrystals
Authors: Pérez-Dieste V. , Castellini O. M. , Crain J. N. , Eriksson M. A. , Kirakosian A. , Lin J.-L. , McChesney J. L. , Himpsel F. J. , Black C. T. , Murray C. B.
Published: December 2003

The pathway for thermal decomposition of an oleic acid surfactant protecting Co and Ni nanocrystals is identified by probing the relevant molecular orbitals with x-ray absorption spectroscopy. The two steps observed previously in thermogravimetric measurements are identified with thermal desorption of entire molecules at ≈200 °C and dehydrogenation at ≈400 °C, which leaves a graphitic surface with alkane fragments underneath.
Time-resolved spectroscopy of recombination and relaxation dynamics in InN
Authors: Chen Fei , Cartwright A. N. , Lu Hai , Schaff William J.
Published: December 2003

Subpicosecond resolution differential transmission measurements of an InN epilayer have been employed to probe the carrier recombination dynamics and hot carrier relaxation processes in these materials at room temperature. We observed a fast initial hot carrier cooling followed by a slower recombination process with characteristic decay times of 300–400 ps. At short times after pulsed excitation, modeling of the observed relaxation suggests that the dominant energy relaxation process is longitudinal optical phonon scattering modified by a strong hot phonon effect. At longer times, a redshift of the peak energy in the differential transmission spectra was observed. This redshift is consistent with a reduction of the bandfilling effect that occurs as the photoexcited carriers recombine.
Vortex pinning by mesoscopic defects: A way to levitation at liquid oxygen temperature
Authors: Muralidhar M. , Sakai N. , Jirsa M. , Koshizuka N. , Murakami M.
Published: December 2003

The addition of long-term Zr2O3-ball milled Gd2BaCuO5 particles to a standard (Nd,Eu,Gd)Ba2Cu3Oy superconductor led to a spontaneous formation of extremely small (20–50 nm) Zr-rich (Nd,Eu,Gd)–Ba–Cu–O particles during the melt-growth process. This pinning medium, approaching the interesting physical limit of point-like pins from the side of large normal ones, dramatically enhanced supercurrents up to 90 K, both in low and intermediate fields. As a result, levitation experiment could be performed with liquid oxygen as a coolant. The particle size effect resemblance with fast neutron irradiation of RE–Ba–Cu–O single crystals is discussed.
Launching and decoupling surface plasmons via micro-gratings
Authors: Devaux Eloise , Ebbesen Thomas W. , Weeber Jean-Claude , Dereux Alain
Published: December 2003

Controlling separately the launching of surface plasmons and their recovery as freely propagating light is essential for the development of surface plasmon photonic circuits. With this target in mind, we have studied in the near-field the launching of surface plasmons in a well-defined direction by micro-arrays of subwavelength holes milled in a thick metal film. We show that surface plasmons can then be converted back to freely propagating light by means of another appropriately designed array. These results not only provide insight into the efficient decoupling of surface plasmons but also into their role in the enhanced transmission mechanism.
UV screening by phenolics in berries of grapevine (Vitis vinifera)
Authors: Kolb Christiane A. , Kopecký Jiri , Riederer Markus , Pfündel Erhard E.
Published: December 2003

The role of phenolics in UV-screening was investigated in berries of a white grape cultivar (Vitis vinifera L. cv. Bacchus). Fluorescence microscopy revealed accumulation of phenolics in the skin of berries and, by high performance liquid chromatography and mass spectrometry, flavonols and hydroxycinnamic acids were identified as the main groups of UV-absorbing phenolics. Relationships between natural radiation and the synthesis of phenolics were studied in plants that were cultivated in the absence of UV radiation in a greenhouse before outdoor exposure to three different light regimes: the entire solar spectrum, the solar spectrum minus UV-B radiation and only visible radiation. During six days of exposure, flavonol synthesis was significantly stimulated by natural UV, in particular UV-B, but concentrations of hydroxycinnamic acids decreased under all conditions. Direct comparison of fluorimetrically-determined skin absorbance with absorbance of extracted flavonols or hydroxycinnamic acids suggested that acclimation of UV screening depends almost exclusively on flavonol synthesis. While increased flavonol levels resulted in efficient UV-A shielding, UV-B shielding was incomplete, probably due to decreased levels of the UV-B-absorbing hydroxycinnamic acids during exposure.
Trans-stimulation of 13NH4+ efflux provides evidence for the cytosolic origin of tracer in the compartmental analysis of barley roots
Authors: Britto Dev T. , Kronzucker Herbert J.
Published: December 2003

The analysis of tracer efflux kinetics is fundamental to membrane transport studies, but requires the rigorous identification of subcellular tracer sources. We present a solution to this problem through the analysis of sharp increases in 13NH4+ efflux from roots of radiolabelled barley (Hordeum vulgare L.) seedlings, in response to a 100-fold increase in external [NH4+]. By comparing these trans-stimulation data with a mathematical model incorporating changes in subcellular NH4+ fluxes and pool sizes, we show that the cytosol of root cells is the origin of the tracer efflux. Our analysis provides new insight into the rapidly occurring events underlying compensatory flux regulation during transitions from one nutritional steady state to another, and confirms the validity of compartmental analysis by tracer efflux (CATE) in this important model system.
Control ofBcl-2expression by reactive oxygen species
Authors: Hildeman David A. , Mitchell Thomas , Aronow Bruce , Wojciechowski Sara , Kappler John , Marrack Philippa
Published: December 2003

Reactive oxygen species (ROS) mediate apoptosis in many different cell types. We have previously shown that the antioxidant Mn(III) tetrakis(5,10,15,20-benzoic acid)porphyrin (MnTBAP) decreased intracellular ROS and prevented the apoptosis of activated T cellsin vitro. To determine the mechanism(s) by which MnTBAP afforded such protection, we used Affymetrix (Santa Clara, CA) gene arrays to compare gene expression in T cells activated with staphylococcal enterotoxin Bin vivothen cultured with or without MnTBAP. This analysis showed that the antioxidant increased the expression of Bcl-2, an antiapoptotic molecule whose levels are normally decreased by T cell activation. Culture with MnTBAP revealed a tight inverse correlation between the levels of Bcl-2 and ROS within T cells.In vivo, production of ROS in activated T cells occurred before Bcl-2 down-regulation. Furthermore, MnTBAP's ability to prevent death required the expression of Bcl-2 in most T cells. Finally, neither ROS production nor the effects on Bcl-2 expression required Bim, the Bcl-2 antagonist that mediates the death of activated T cellsin vivo. Taken together, our results suggest that ROS sensitize T cells to apoptosis by decreasing expression of Bcl-2.
Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance
Authors: Singla-Pareek S. L. , Reddy M. K. , Sopory S. K.
Published: December 2003

The glyoxalase pathway involving glyoxalase I (gly I) and glyoxalase II (gly II) enzymes is required for glutathione-based detoxification of methylglyoxal. We had earlier indicated the potential of
gly I
as a probable candidate gene in conferring salinity tolerance. We report here that overexpression of
gly I
+
II
together confers improved salinity tolerance, thus offering another effective strategy for manipulating stress tolerance in crop plants. We have overexpressed the
gly II
gene either alone in untransformed plants or with gly I transgenic background. Both types of these transgenic plants stably expressed the foreign protein, and the enzyme activity was also higher. Compared with nontransformants, several independent gly II transgenic lines showed improved capability for tolerating exposure to high methylglyoxal and NaCl concentration and were able to grow, flower, and set normal viable seeds under continuous salinity stress conditions. Importantly, the double transgenic lines always showed a better response than either of the single gene-transformed lines and WT plants under salinity stress. Ionic measurements revealed higher accumulation of Na
+
and K
+
in old leaves and negligible accumulation of Na
+
in seeds of transgenic lines as compared with the WT plants. Comparison of various growth parameters and seed production demonstrated that there is hardly any yield penalty in the double transgenics under nonstress conditions and that these plants suffered only 5% loss in total productivity when grown in 200 mM NaCl. These findings establish the potential of manipulation of the glyoxalase pathway for increased salinity tolerance without affecting yield in crop plants.
HIV-1-suppressive factors are secreted by CD4+T cells during primary immune responses
Authors: Abdelwahab Sayed F. , Cocchi Fiorenza , Bagley Kenneth C. , Kamin-Lewis Roberta , Gallo Robert C. , DeVico Anthony , Lewis George K.
Published: December 2003

CD4+T cells are required for immunity against many viral infections, including HIV-1 where a positive correlation has been observed between strong recall responses and low HIV-1 viral loads. Some HIV-1-specific CD4+T cells are preferentially infected with HIV-1, whereas others escape infection by unknown mechanisms. One possibility is that some CD4+T cells are protected from infection by the secretion of soluble HIV-suppressive factors, although it is not known whether these factors are produced during primary antigen-specific responses. Here, we show that soluble suppressive factors are produced against CXCR4 and CCR5 isolates of HIV-1 during the primary immune response of human CD4+T cells. This activity requires antigenic stimulation of naïve CD4+T cells. One anti-CXCR4 factor is macrophage-derived chemokine (chemokine ligand 22, CCL22), and anti-CCR5 factors include macrophage inflammatory protein-1α (CCL3), macrophage inflammatory protein-1β (CCL4), and RANTES (regulated upon activation of normal T cells expressed and secreted) (CCL5). Intracellular staining confirms that CD3+CD4+T cells are the source of the prototype HIV-1-inhibiting chemokines CCL22 and CCL4. These results show that CD4+T cells secrete an evolving HIV-1-suppressive activity during the primary immune response and that this activity is comprised primarily of CC chemokines. The data also suggest that production of such factors should be considered in the design of vaccines against HIV-1 and as a mechanism whereby the host can control infections with this virus.
Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast
Authors: Storici Francesca , Durham Christopher L. , Gordenin Dmitry A. , Resnick Michael A.
Published: December 2003

The repair of chromosomal double-strand breaks (DSBs) can be accomplished through homologous recombination in most organisms. We report here that exogenous oligonucleotides can efficiently target for repair a single DSB induced in a chromosome of yeast. The efficiency of recombinational targeting leading to a desired DNA change can be as high as 20% of cells. The DSB was generated either by a regulatable I-
Sce
I endonuclease just before transformation or appeared spontaneously at the site of a long inverted repeat composed of human
Alu
sequences. The approach used features of our previously described
delitto perfetto
system for selecting transformants with integrative recombinant oligonucleotides. The DSB repair mediated by pairs of complementary integrative recombinant oligonucleotides was efficient for targeting to homologous sequences that were close to or distant from the DSB and in the presence of a competing homologous chromosome in diploid cells. We also demonstrate that a DSB can strongly stimulate recombination with single-stranded DNA, without strand bias. These findings expand current models of DSB repair. In addition, we establish a high-throughput system for rapid genome-wide modification with oligonucleotides.
Experimental test of predation's effect on divergent selection during character displacement in sticklebacks
Authors: Rundle Howard D. , Vamosi Steven M. , Schluter Dolph
Published: December 2003

Ecological character displacement is common in nature but the mechanisms causing divergence are not well understood. The contributions of ecological interactions other than competition have received little attention. We conducted a pond experiment to explore the contribution of both competition and predation to character divergence in threespine stickleback species. We estimated the strength of divergent selection on a morphologically intermediate target population between competition treatments under two alternate predation treatments. Divergent selection on the target population tended to be stronger in the predator-addition treatment than in the predator-reduction treatment, a difference that approached significance (P= 0.09). This trend occurred even though competition was strongest in the predator-reduction treatment. Overall, the strength of divergent selection was best predicted by stickleback mortality (P= 0.025) being strongest where mortality was highest. These results indicate that predation and other agents of mortality can enhance the rate of change in competition per unit of phenotypic divergence and, thereby, divergent selection, even as they lower the overall strength of competition. In this way, predation and other agents of mortality may facilitate, rather than hinder, character displacement.
Natural scene statistics as the universal basis of color context effects
Authors: Long Fuhui , Purves Dale
Published: December 2003

The color context effects referred to as color contrast, constancy, and assimilation underscore the fact that color percepts do not correspond to the spectral characteristics of the generative stimuli. Despite a variety of proposed theories, these phenomena have resisted explanation in a single principled framework. Using a hyperspectral image database of natural scenes, we here show that color contrast, constancy, and assimilation are all predicted by the statistical organization of spectral returns from natural visual environments.
Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations
Authors: Weninger Keith , Bowen Mark E. , Chu Steven , Brunger Axel T.
Published: December 2003

Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble
N
-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h
–1
. The existence of antiparallel configurations suggests a regulatory role of chaperones, such as
N
-ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly
in vivo
, and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments
in vitro
.
Random Boolean network models and the yeast transcriptional network
Authors: Kauffman Stuart , Peterson Carsten , Samuelsson Björn , Troein Carl
Published: December 2003

The recently measured yeast transcriptional network is analyzed in terms of simplified Boolean network models, with the aim of determining feasible rule structures, given the requirement of stable solutions of the generated Boolean networks. We find that, for ensembles of generated models, those with canalyzing Boolean rules are remarkably stable, whereas those with random Boolean rules are only marginally stable. Furthermore, substantial parts of the generated networks are frozen, in the sense that they reach the same state, regardless of initial state. Thus, our ensemble approach suggests that the yeast network shows highly ordered dynamics.
Structures and relative free energies of partially folded states of proteins
Authors: Vendruscolo Michele , Paci Emanuele , Karplus Martin , Dobson Christopher M.
Published: December 2003

The ability of proteins to fold to well defined compact structures is one of the most remarkable examples of the effect of natural selection on biological molecules. To understand their properties, including the stability, the mechanism of folding, and the possibilities of misfolding and association, it is necessary to know the protein free energy landscape. We use NMR data as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures populated by human α-lactalbumin in the presence of increasing concentrations of urea. The ensembles of structures that represent the partially folded states of the protein show that two structural cores, corresponding to portions of the α and β domains of the native protein, are preserved even when the native-like interactions that define their existence are substantially weakened. Analysis of the network of residual contacts reveals the presence of a complex interface region between the two structural cores and indicates that the development of specific interactions within this interface is the key step in achieving the native structure. The relative probabilities of the conformations determined from the NMR data are used to construct a coarse-grained free energy landscape for α-lactalbumin in the absence of urea. The form of the landscape, together with the existence of distinct cores, supports the concept that robustness and modularity are the properties that make possible the folding of complex proteins.
The population structure of
Mycobacterium bovis
in Great Britain: Clonal expansion
Authors: Smith Noel H. , Dale James , Inwald Jacqueline , Palmer Si , Gordon Stephen V. , Hewinson R. Glyn , Smith John Maynard
Published: December 2003

We have analyzed 11,500 isolates of
Mycobacterium bovis
(the cause of tuberculosis in cattle and other mammals) isolated in Great Britain (England, Wales and Scotland)] and characterized by spoligotype. Genetic exchange between cells is rare or absent in strains of the
Mycobacterium tuberculosis
complex so that, by using spoligotypes, it is possible to recognize “clones” with a recent common ancestor. The distribution of variable numbers of tandem repeats types in the most common clone in the data set is incompatible with random mutation and drift. The most plausible explanation is a series of “clonal expansions,” and this interpretation is supported by the geographical distribution of different genotypes. We suggest that the clonal expansion of a genotype is caused either by the spread of a favorable mutation, together with all other genes present in the ancestral cell in which the mutation occurred, or by the invasion of a novel geographical region by a limited number of genotypes. A similar pattern is observed in
M. tuberculosis
(the main cause of tuberculosis in humans). The significance of clonal expansion in other bacteria that have recombination is discussed.
Overcoming the hypoxic barrier to radiation therapy with anaerobic bacteria
Authors: Bettegowda Chetan , Dang Long H. , Abrams Ross , Huso David L. , Dillehay Larry , Cheong Ian , Agrawal Nishant , Borzillary Scott , McCaffery J. Michael , Watson E. Latice , Lin Kuo-Shyan , Bunz Fred , Baidoo Kwamena , Pomper Martin G. , Kinzler Kenneth W. , Vogelstein Bert , Zhou Shibin
Published: December 2003

The low level of oxygenation within tumors is a major cause of radiation treatment failures. We theorized that anaerobic bacteria that can selectively destroy the hypoxic regions of tumors would enhance the effects of radiation. To test this hypothesis, we used spores of
Clostridium novyi-NT
to treat transplanted tumors in mice. The bacteria were found to markedly improve the efficacy of radiotherapy in several of the mouse models tested. Enhancement was noted with external beam radiation derived from a Cs-137 source, systemic radioimmunotherapy with an I-131-conjugated monoclonal antibody, and a previously undescribed form of experimental brachytherapy using plaques loaded with I-125 seeds.
C. novyi-NT
spores added little toxicity to the radiotherapeutic regimens, and the combination resulted in long-term remissions in a significant fraction of animals.
An adenoviral vector cancer vaccine that delivers a tumor-associated antigen/CD40-ligand fusion protein to dendritic cells
Authors: Zhang Lixin , Tang Yucheng , Akbulut Hakan , Zelterman Daniel , Linton Phyllis-Jean , Deisseroth Albert B.
Published: December 2003

To develop a method to overcome the anergy that exists in tumor hosts to cancer, we have designed an adenoviral vector for thein vivoactivation and tumor antigen loading of dendritic cells. This adenoviral vector encodes a fusion protein composed of an amino-terminal tumor-associated antigen fragment fused to the CD40 ligand (CD40L). Subcutaneous injection of an adenoviral vector encoding a fusion protein of the human papillomavirus E7 foreign antigen linked to the CD40L generates CD8+T cell-dependent immunoresistance to the growth of the E7-positive syngeneic TC-1 cancer cells in C57BL/6 mice for up to 1 year. We also studied the s.c. injection of a vector carrying the gene for the human MUC-1 (hMUC-1) self-antigen fused to the CD40L. When this vector was injected into hMUC-1.Tg mice, which are transgenic for the hMUC-1 antigen, the growth of syngeneic hMUC-1-positive LL1/LL2hMUC-1 mouse cancer cells was suppressed in 100% of the injected animals. The hMUC-1.Tg mice are anergic to the hMUC-1 antigen before the injection of the vector. These experimental results show that it is possible to use vector injection to activate a long-lasting cellular immune response against self-antigens in anergic animals. The vector-mediatedin vivoactivation, and tumor-associated antigen loading of dendritic cells does not require additional cytokine boosting to induce the immune response against the tumor cells. This vector strategy may therefore be of use in the development of immunotherapy for the many carcinomas in which the hMUC-1 antigen is overexpressed.
Natural occurrence and synthesis of two new postspinel polymorphs of chromite
Authors: Chen Ming , Shu Jinfu , Mao Ho-kwang , Xie Xiande , Hemley Russell J.
Published: December 2003

A high-pressure polymorph of chromite, the first natural sample with the calcium ferrite structure, has been discovered in the shock veins of the Suizhou meteorite. Synchrotron x-ray diffraction analyses reveal an orthorhombic CaFe
2
O
4
-type (CF) structure. The unit-cell parameters are
a
= 8.954(7) Å,
b
= 2.986(2) Å,
c
= 9.891(7) Å,
V
= 264.5(4) Å
3
(
Z
= 4) with space group
Pnma
. The new phase has a density of 5.62 g/cm
3
, which is 9.4% denser than chromite-spinel. We performed laser-heated diamond anvil cell experiments to establish that chromite-spinel transforms to CF at 12.5 GPa and then to the recently discovered CaTi
2
O
4
-type (CT) structure above 20 GPa. With the ubiquitous presence of chromite, the CF and CT phases may be among the important index minerals for natural transition sequence and pressure and temperature conditions in mantle rocks, shock-metamorphosed terrestrial rocks, and meteorites.
Labeled cortical mantle distance maps of the cingulate quantify differences between dementia of the Alzheimer type and healthy aging
Authors: Miller M. I. , Hosakere M. , Barker A. R. , Priebe C. E. , Lee N. , Ratnanather J. T. , Wang L. , Gado M. , Morris J. C. , Csernansky J. G.
Published: December 2003

The cingulate gyri in 37 subjects with and without early dementia of the Alzheimer type (DAT) were studied by using MRI at 1.0 mm
3
isotropic resolution. Groups were segregated into young controls (
n
= 10), age-matched normal controls (
n
= 10), very mild DAT (
n
= 8), and mild DAT (
n
= 9). By using automated Bayesian segmentation of the cortex and gray matter/white matter (GM/WM) isosurface generation, tissue compartments were labeled into gray, white, and cerebrospinal fluid as a function of distance from the GM/WM isosurface. Cortical mantle distance maps are generated profiling the GM volume and cortical mantle distribution as a function of distance from the cortical surface. Probabilistic tests based on generalizations of Wilcoxon–Mann–Whitney tests were applied to quantify cortical mantle distribution changes with normal and abnormal aging. We find no significant change between young controls and healthy aging as measured by the GM volume and cortical mantle distribution as a function of distance in both anterior and posterior regions of the cingulate. Significant progression of GM loss is seen in the very mild DAT and mild DAT groups in all areas of the cingulate. Posterior regions show both GM volume loss as well as significant cortical mantle distribution decrease with the onset of mild DAT. The “shape of the cortical mantle” as measured by the cortical mantle distance profiles manifests a pronounced increase in variability with mild DAT.
Rapid Ca2+-dependent decrease of protein ubiquitination at synapses
Authors: Chen Hong , Polo Simona , Di Fiore Pier Paolo , De Camilli Pietro V.
Published: December 2003

Protein ubiquitination has been implicated in the regulation of axonal growth and synaptic plasticity as well as in the pathogenesis of neurodegenerative diseases. Here we show that depolarization-dependent Ca2+influx into synaptosomes produces a global, rapid (range of seconds), and reversible decrease of the ubiquitinated state of proteins, which correlates with the Ca2+-dependent dephosphorylation of several synaptic proteins. A similar general decrease in protein ubiquitination was observed in nonneuronal cells on Ca2+entry induced by ionomycin. Both in synaptosomes and in nonneuronal cells, this decrease was blocked by FK506 (a calcineurin antagonist). Proteins whose ubiquitinated state was decreased include epsin 1, a substrate for the deubiquitinating enzymefat facets/FAM, which we show here to be concentrated at synapses. These results reveal a fast regulated turnover of protein ubiquitination. In nerve terminals, protein ubiquitination may play a role both in the regulation of synaptic function, including vesicle traffic, and in the coordination of protein turnover with synaptic use.
Neural correlates of the emergence of consciousness of thirst
Authors: Egan Gary , Silk Tim , Zamarripa Frank , Williams John , Federico Paolo , Cunnington Ross , Carabott Leonie , Blair-West John , Shade Robert , McKinley Michael , Farrell Michael , Lancaster Jack , Jackson Graeme , Fox Peter , Denton Derek
Published: December 2003

Thirst was induced by rapid i.v. infusion of hypertonic saline (0.51 M at 13.4 ml/min). Ten humans were neuroimaged by positron-emission tomography (PET) and four by functional MRI (fMRI). PET images were made 25 min after beginning infusion, when the sensation of thirst began to enter the stream of consciousness. The fMRI images were made when the maximum rate of increase of thirst occurred. The PET results showed regional cerebral blood flow changes similar to those delineated when thirst was maximal. These loci involved the phylogenetically ancient areas of the brain. fMRI showed activation in the anterior wall of the third ventricle, an area that is key in the genesis of thirst but is not an area revealed by PET imaging. Thus, this region plays as major a role in thirst for humans as for animals. Strong activations in the brain with fMRI included the anterior cingulate, parahippocampal gyrus, inferior and middle frontal gyri, insula, and cerebellum. When the subjects drank water to satiation, thirst declined immediately to baseline. A precipitate decline in intensity of activation signal occurred in the anterior cingulate area (Brodmann area 32) putatively related to consciousness of thirst. The intensity of activation in the anterior wall of the third ventricle was essentially unchanged, which is consistent with the fact that a significant time (15–20 min) would be needed before plasma Na concentration changed as a result of water absorption from the gut.
Intracellular Ca
2+
and the phospholipid PIP
2
regulate the taste transduction ion channel TRPM5
Authors: Liu Dan , Liman Emily R.
Published: December 2003

The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase C β2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP
2
) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca
2+
from intracellular stores. The ion channel, TRPM5, is an essential component of this cascade; however, the mechanism by which it is activated is not known. Here we show that heterologously expressed TRPM5 forms a cation channel that is directly activated by micromolar concentrations of intracellular Ca
2+
(
K
1/2
= 21 μM). Sustained exposure to Ca
2+
desensitizes TRPM5 channels, but PIP
2
reverses desensitization, partially restoring channel activity. Whole-cell TRPM5 currents can be activated by intracellular Ca
2+
and show strong outward rectification because of voltage-sensitive gating of the channels. TRPM5 channels are nonselective among monovalent cations and not detectably permeable to divalent cations. We propose that the regulation of TRPM5 by Ca
2+
mediates sensory activation in the taste system.
Structure and functional significance of mechanically unfolded fibronectin type III
1
intermediates
Authors: Gao Mu , Craig David , Lequin Olivier , Campbell Iain D. , Vogel Viola , Schulten Klaus
Published: December 2003

Fibronectin (FN) forms fibrillar networks coupling cells to the extracellular matrix. The formation of FN fibrils, fibrillogenesis, is a tightly regulated process involving the exposure of cryptic binding sites in individual FN type III (FN-III) repeats presumably exposed by mechanical tension. The FN-III
1
module has been previously proposed to contain such cryptic sites that promote the assembly of extracellular matrix FN fibrils. We have combined NMR and steered molecular dynamics simulations to study the structure and mechanical unfolding pathway of FN-III
1
. This study finds that FN-III
1
consists of a β-sandwich structure that unfolds to a mechanically stable intermediate about four times the length of the native folded state. Considering previous experimental findings, our studies provide a structural model by which mechanical stretching of FN-III
1
may induce fibrillogenesis through this partially unfolded intermediate.
The base excision repair enzyme MED1 mediates DNA damage response to antitumor drugs and is associated with mismatch repair system integrity
Authors: Cortellino Salvatore , Turner David , Masciullo Valeria , Schepis Filippo , Albino Domenico , Daniel Rene , Skalka Anna Marie , Meropol Neal J. , Alberti Christophe , Larue Lionel , Bellacosa Alfonso
Published: December 2003

Cytotoxicity of methylating agents is caused mostly by methylation of the
O
6
position of guanine in DNA to form
O
6
–methylguanine (
O
6
–meG).
O
6
–meG can direct misincorporation of thymine during replication, generating
O
6
–meG:T mismatches. Recognition of these mispairs by the mismatch repair (MMR) system leads to cell cycle arrest and apoptosis. MMR also modulates sensitivity to other antitumor drugs. The base excision repair (BER) enzyme MED1 (also known as MBD4) interacts with the MMR protein MLH1. MED1 was found to exhibit thymine glycosylase activity on
O
6
–meG:T mismatches. To examine the biological significance of this activity, we generated mice with targeted inactivation of the
Med1
gene and prepared mouse embryonic fibroblasts (MEF) with different
Med1
genotype. Unlike wild-type and heterozygous cultures,
Med1
-/-
MEF failed to undergo G
2
-M cell cycle arrest and apoptosis upon treatment with the methylating agent
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine (MNNG). Similar results were obtained with platinum compounds' 5-fluorouracil and irinotecan. As is the case with MMR-defective cells, resistance of
Med1
-/-
MEF to MNNG was due to a tolerance mechanism because DNA damage accumulated but did not elicit checkpoint activation. Interestingly, steady state amounts of several MMR proteins are reduced in
Med1
-/-
MEF, in comparison with
Med1
+/+
and
Med1
+/-
MEF. We conclude that MED1 has an additional role in DNA damage response to antitumor agents and is associated with integrity of the MMR system. MED1 defects (much like MMR defects) may impair cell cycle arrest and apoptosis induced by DNA damage.
Thermodynamic treatment of oligonucleotide duplex–simplex equilibria
Authors: Owczarzy Richard , Dunietz Isard , Behlke Mark A. , Klotz Irving M. , Walder Joseph A.
Published: December 2003

Thermodynamic formulations have been devised to obtain Δ
G
° values directly from spectroscopic data at a fixed common temperature in nucleic acid duplex–simplex melting curves. In addition, the dependence of melting on salt concentration has been expressed in terms of a stepwise stoichiometric representation, which leads to a specific equation for the partition of the added sodium ions between the different oligonucleotide forms.
A pathway for the metabolism of vitamin D
3
: Unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1)
Authors: Guryev O. , Carvalho R. A. , Usanov S. , Gilep A. , Estabrook R. W.
Published: December 2003

Metabolites of vitamin D
3
(D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D
3
has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe
in vitro
experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D
3
and 20,22-dihydroxyvitamin D
3
) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their
in vivo
formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1
in vitro
suggests an important role.
Fragile site orthologs
FHIT/FRA3B
and
Fhit/Fra14A2
: Evolutionarily conserved but highly recombinogenic
Authors: Matsuyama Ayumi , Shiraishi Takeshi , Trapasso Francesco , Kuroki Tamotsu , Alder Hansjuerg , Mori Masaki , Huebner Kay , Croce Carlo M.
Published: December 2003

Common fragile sites are regions that show elevated susceptibility to DNA damage, leading to alterations that can contribute to cancer development.
FRA3B
, located at chromosome region 3p14.2, is the most frequently expressed human common fragile site, and allelic losses at
FRA3B
have been observed in many types of cancer. The
FHIT
gene, encompassing the
FRA3B
region, is a tumor-suppressor gene. To identify the features of
FHIT/FRA3B
that might contribute to fragility, sequences of the human
FHIT
and the flanking
PTPRG
gene were compared with those of murine
Fhit
and
Ptprg
. Human and mouse orthologous genes,
FHIT
and
Fhit
, are more highly conserved through evolution than
PTPRG/Ptprg
and yet contain more sequence elements that are exquisitely sensitive to genomic rearrangements, such as high-flexibility regions and long interspersed nuclear element 1s, suggesting that common fragile sites serve a function. The conserved AT-rich high-flexibility regions are the most characteristic of common fragile sites.
Thermodynamic stability of hydrogen clathrates
Authors: Patchkovskii Serguei , Tse John S.
Published: December 2003

The stability of the recently characterized type II hydrogen clathrate [Mao, W. L., Mao, H.-K., Goncharov, A. F., Struzhkin, V. V., Guo, Q.,et al. (2002)Science297, 2247–2249] with respect to hydrogen occupancy is examined with a statistical mechanical model in conjunction with first-principles quantum chemistry calculations. It is found that the stability of the clathrate is mainly caused by dispersive interactions between H2molecules and the water forming the cage walls. Theoretical analysis shows that both individual hydrogen molecules andnH2guest clusters undergo essentially free rotations inside the clathrate cages. Calculations at the experimental conditions – 2,000 bar (1 bar = 100 kPa) and 250 K confirm multiple occupancy of the clathrate cages with average occupations of 2.00 and 3.96 H2molecules perD-512(small) andH-51264(large) cage, respectively. The H2–H2O interactions also are responsible for the experimentally observed softening of the H—H stretching modes. The clathrate is found to be thermodynamically stable at 25 bar and 150 K.
Contribution of astrocytes to hippocampal long-term potentiation through release of
d
-serine
Authors: Yang Yunlei , Ge Wooping , Chen Yiren , Zhang Zhijun , Shen Wanhua , Wu Chienping , Poo Muming , Duan Shumin
Published: December 2003

Repetitive correlated activation of pre- and postsynaptic neurons induced long-term potentiation (LTP) of synaptic transmission among hippocampal neurons grown on a layer of astrocytes (mixed cultures) but not among neurons cultured in glial conditioned medium. Supplement of
d
-serine, an agonist for the glycine-binding site of
N
-methyl-
d
-aspartate (NMDA) receptors, enhanced NMDA receptor activation and enabled LTP induction in glial conditioned medium cultures. The induction of LTP in both mixed cultures and hippocampal slices was suppressed by NMDA receptor antagonists, glycine-binding-site blockers of NMDA receptors, or an enzyme that degrades endogenous
d
-serine. By providing extracellular
d
-serine that facilitates activation of NMDA receptors, astrocytes thus play a key role in long-term synaptic plasticity.
Structural basis of peptide–carbohydrate mimicry in an antibody-combining site
Authors: Vyas Nand K. , Vyas Meenakshi N. , Chervenak Mary C. , Bundle David R. , Pinto B. Mario , Quiocho Florante A.
Published: December 2003

The structure of a complex between the Fab fragment of the antibody (SYA/J6) specific for the cell surface O-antigen polysaccharide of the pathogenShigella flexneriY and an octapeptide (Met–Asp–Trp–Asn–Met–His–Ala–Ala), a functional mimic of the O-antigen, has been determined at 1.8-Å resolution. Comparison of the structure with that of the complex with the pentasaccharide antigen [→2)-α-l-Rha-(1→2)-α-l-Rha-(1→3)-α-l-Rha-(1→3)-β-d-GlcNAc-(1→2)-α-l-Rha-(1→] reveals the molecular recognition process by which a peptide mimics a carbohydrate in binding to an antibody. The binding modes of the two ligands differ considerably. Octapeptide binding complements the shape of the combining site groove much better than pentasaccharide binding. Moreover, the peptide makes a much greater number of contacts (126), which are mostly van der Waals interactions, with the Fab than the saccharide (74). An unusual feature is also the involvement of 12 water molecules in mediating hydrogen bonds between residues within the peptide or of the peptide and Fab. Despite better shape complementarity and greater number of contacts, the octapeptide binds with an affinity (KA= 2.5 × 105M-1, measured by calorimetry) only ≈2-fold tighter than the pentasaccharide. The structural results are relevant to the design of peptide mimetics with improved affinity for use as vaccines.
Frap
, FKBP12 rapamycin-associated protein, is a candidate gene for the plasmacytoma resistance locus
Pctr2
and can act as a tumor suppressor gene
Authors: Bliskovsky Valery , Ramsay Edward S. , Scott John , DuBois Wendy , Shi Wei , Zhang Shuling , Qian Xiaolan , Lowy Douglas R. , Mock Beverly A.
Published: December 2003

Susceptibility to mouse plasmacytomagenesis is a complex genetic trait controlled by several
Pctr
loci (
Pctr1, Pctr2
, etc). Congenic strain analysis narrowed the genetic interval surrounding the
Pctr2
locus, and genes identified in the interval were sequenced from susceptible BALB/c and resistant DBA/2 mice.
Frap
(FKBP12 rapamycin-associated protein, mTOR, RAFT) was the only gene differing in amino acid sequence between alleles that correlated with strain sensitivity to tumor development. The
in vitro
kinase activity of the BALB/c FRAP allele was lower than the DBA/2 allele; phosphorylation of p53 and PHAS1/4EBP1 (properties of heat and acid stability/eukaryotic initiation factor 4E-binding protein) and autophosphorylation of FRAP were less efficient with the BALB/c allele. FRAP also suppressed transformation of NIH 3T3 cells by ras, with DBA/2 FRAP being more efficient than BALB/c FRAP. Rapamycin, a specific inhibitor of FRAP, did not inhibit growth of plasmacytoma cell lines. These studies identify
Frap
as a candidate tumor suppressor gene, in contrast to many reports that have focused on its prooncogenic properties.
Frap
may be similar to
Tgfb
and
E2f
in exerting both positive and negative growth-regulatory signals, depending on the timing, pathway, or tumor system involved. The failure of rapamycin to inhibit plasma cell tumor growth suggests that FRAP antagonists may not be appropriate for the treatment of plasma cell tumors.
Pctr2
joins
Pctr1
in possessing alleles that modify susceptibility to plasmacytomagenesis by encoding differences in efficiency of function (efficiency alleles), rather than all-or-none, gain-of-function, or loss-of-function alleles. By analogy, human cancer may also result from the combined effects of several inefficient alleles.
Transmembrane phosphoprotein Cbp senses cell adhesion signaling mediated by Src family kinase in lipid rafts
Authors: Shima Takaki , Nada Shigeyuki , Okada Masato
Published: December 2003

Cbp, a C-terminal Src kinase (Csk)-binding protein, is a transmembrane phosphoprotein that has been implicated in the regulation of the Src family kinase (SFK) through recruiting Csk, a negative regulator of SFK, to a membrane microdomain of lipid rafts. To examine the contribution of Cbp to cell adhesion signaling mediated by SFK, we investigated the kinase responsible for phosphorylating Cbp and the mode of phosphorylation during the cell adhesion process. The results obtained by using mutant mice or cells that lack Csk and/or a member of SFK, Fyn, reveal that Cbp is phosphorylated predominantly by raft-localized Fyn
in vivo
. Upon cell adhesion onto fibronectin, Cbp becomes transiently phosphorylated (consistent with SFK activation) and recruits Csk to lipid rafts. These events are completed before the full activation of focal adhesion kinase, indicating that the transient activation and down-regulation of SFK in lipid rafts are earlier events in cell adhesion signaling. In Csk-deficient cells, continuous hyperactivation of SFK leads to continuous hyperphosphorylation of Cbp, accompanied by impaired cell spreading and migration. Silencing of Cbp by RNA interference also induced impaired cell spreading. These findings suggest that Cbp could serve as a sensor of SFK activity in early stages of cell adhesion signaling, and that Csk-mediated down-regulation of SFK is essential to allow dynamic cellular events involved in the regulation of cell spreading and migration.
Preferential megalin-mediated transcytosis of low-hormonogenic thyroglobulin: A control mechanism for thyroid hormone release
Authors: Lisi Simonetta , Pinchera Aldo , McCluskey Robert T. , Willnow Thomas E. , Refetoff Samuel , Marcocci Claudio , Vitti Paolo , Menconi Francesca , Grasso Lucia , Luchetti Fabiana , Collins A. Bernard , Marinò Michele
Published: December 2003

Hormone secretion by thyrocytes occurs by fluid phase uptake and lysosomal degradation of the prohormone thyroglobulin (Tg). However, some Tg internalized by megalin bypasses lysosomes and is transcytosed across cells and released into the bloodstream. Because the hormone content of Tg is variable, we investigated whether this affects transcytosis. We found that rat Tg with a low hormone content [low-hormonogenic rat Tg (low-horm-rTg)] is transcytosed by megalin across thyroid FRTL-5 cells to a greater extent than rat Tg with a high hormone content [hormonogenic rat Tg (horm-rTg)]. In immunoprecipitation experiments, the Tg sequence Arg-2489-Lys-2503 (required for binding to megalin and heparan sulfate proteoglycans) was found to be more exposed in low-horm-rTg, which accounted for its preferential transcytosis. Thus, removal of surface heparan sulfate proteoglycans from FRTL-5 cells or blocking of 2489–2503 reduced transcytosis of low-horm-rTg to a greater extent than that of horm-rTg. Preferential transcytosis of low-horm-rTg affected hormone release. Thus, the increase in hormone release from horm-rTg in FRTL-5 cells determined by megalin blocking (due to reduced transcytosis and enhanced Tg degradation) was rescued by low-horm-rTg, suggesting that megalin is required for effective hormone release. This finding was confirmed in a small number of megalin-deficient mice, which had serological features resembling mild hypothyroidism. Reduced hormone formation within Tgin vivo, due to treatment of rats with aminotriazole or of patients with Graves' disease with methimazole, resulted in increased Tg transcytosis via megalin, in confirmation of results with FRTL-5 cells. Our study points to a major role of megalin in thyroid homeostasis with possible implications in thyroid diseases.
Darwinian natural selection for orange bioluminescent color in a Jamaican click beetle
Authors: Stolz Uwe , Velez Sebastian , Wood Keith V. , Wood Monika , Feder Jeffrey L.
Published: December 2003

The Jamaican click beetlePyrophorus plagiophthalamus(Coleoptera: Elateridae) is unique among all bioluminescent organisms in displaying a striking light color polymorphism [Biggley, W. H., Lloyd, J. E. & Seliger, H. H.(1967)J. Gen. Physiol.50, 1681–1692]. Beetles on the island vary in the color of their ventral light organs from yellow–green to orange and their dorsal organs from green to yellow–green. The genetic basis for the color variation involves specific amino acid substitutions in the enzyme luciferase. Here, we show that dorsal and ventral light color inP. plagiophthalamusare under separate genetic control, we resolve the allelic basis for color variation, and, through analyses of luciferase sequence variation, we demonstrate that natural selection has produced a long-term adaptive trend for longer wavelength (more orange) ventral light on Jamaica. Our results constitute a novel example connecting the selective fixation of specific nucleotides in nature to their precisely determined phenotypic effects. We also present evidence suggesting that a recently derived ventral orange luciferase allele on the island has deterministically increased in frequency. Thus, the current luciferase polymorphism forP. plagiophthalamusappears to be mirroring the long-term anagenic trend on Jamaica, revealing a possible ongoing adaptive color transition in progress.