bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the parietal cortex in visual feature binding
Authors: Shafritz Keith M. , Gore John C. , Marois René
Published: September 2002

When multiple objects are simultaneously present in a scene, the visual system must properly integrate the features associated with each object. It has been proposed that this “binding problem” is solved by selective attention to the locations of the objects [Treisman, A.M. & Gelade, E. (1980)
Cogn. Psychol.
12, 97–136]. If spatial attention plays a role in feature integration, it should do so primarily when object location can serve as a binding cue. Using functional MRI (fMRI), we show that regions of the parietal cortex involved in spatial attention are more engaged in feature conjunction tasks than in single feature tasks when multiple objects are shown simultaneously at different locations but not when they are shown sequentially at the same location. These findings suggest that the spatial attention network of the parietal cortex is involved in feature binding but only when spatial information is available to resolve ambiguities about the relationships between object features.
The reduction potential of nitric oxide (NO) and its importance to NO biochemistry
Authors: Bartberger Michael D. , Liu Wei , Ford Eleonora , Miranda Katrina M. , Switzer Christopher , Fukuto Jon M. , Farmer Patrick J. , Wink David A. , Houk Kendall N.
Published: September 2002

A potential of about −0.8 (±0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion (
3
NO
−
) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to
1
NO
−
is highly unfavorable, with a predicted reduction potential of about −1.7 (±0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +0.39 V and −0.35 V for the NO/
3
NO
−
and NO/
1
NO
−
couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of
3
NO
−
has been reevaluated as 11.6 (±3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion,
1
NO
−
, is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O
2
under reductive conditions is discussed.
C1qRpdefines a new human stem cell population with hematopoietic and hepatic potential
Authors: Danet Guénahel H. , Luongo Jennifer L. , Butler Gary , Lu Min Min , Tenner Andrea J. , Simon M. Celeste , Bonnet Dominique A.
Published: September 2002

The characterization of two distinct classes of hematopoietic stem cells based on CD34 expression and the ability of human bone marrow (BM) cells to differentiate into nonhematopoietic cells introduced new levels of complexity within the stem cell compartment. Here we report the identification and purification of a rare human stem cell population with hematopoietic and hepatic potential based on the expression of a receptor for the complement molecule C1q (C1qRp). We show that C1qRpis a positive marker of all BM-repopulating stem cells because it is expressed on both CD34−and CD34+stem cells from umbilical cord blood and adult BM. In addition, we show that highly purified lineage-negative CD45+CD38−CD34+or−C1qR\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{p}^{+}\end{equation*}\end{document}cells not only have BM-repopulating capacity but also can differentiate into human hepatocytesin vivo. The identification of human hepatocytes in mouse livers indicates that the NOD/SCID (nonobese diabetic/severe combined immunodeficient) mouse model can be a valuable tool to study the differentiation potential of adult human stem cells. These findings may have important scientific and clinical implications in the field of human stem cell biology and transplantation.
Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop
Authors: Roumiantsev Sergei , Shah Neil P. , Gorre Mercedes E. , Nicoll John , Brasher Bradley B. , Sawyers Charles L. , Van Etten Richard A.
Published: September 2002

The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) were previously described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Here we identify mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrated intermediate resistance to STI-571
in vitro
and
in vivo
when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 was influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 was increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulated c-Abl and conferred intrinsic but not absolute resistance to STI-571 that was independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management.
Ephrin-A5 restricts topographically specific arborization in the chick retinotectal projection
in vivo
Authors: Sakurai Takashi , Wong Eric , Drescher Uwe , Tanaka Hideaki , Jay Daniel G.
Published: September 2002

The retinotectal map is the best characterized model system to study how axons respond to guidance cues during the formation of the nervous system. Recent studies have shown that the critical event in forming this map is topographic-specific axon branching. To elucidate the
in vivo
role of the repulsive cue ephrin-A5 in this event, we used chromophore-assisted laser inactivation (CALI) to generate acute loss of ephrin-A5 function in localized areas of the posterior tectum of chick embryos
in ovo
and analyzed the resulting changes of retinal projections during initial outgrowth (E11) and when retinal axons arborize in the deep layers in the tectum (E12). We confirmed that ephrin-A5 functions to restrict initial axon outgrowth at E11. At E12, CALI of ephrin-A5 did not affect the extent of axon outgrowth on the tectal surface but instead caused ectopic arborization posterior to the topographically correct site in deeper layers of the tectum. This shows that ephrin-A5 restricts arborization during this critical process for developing the retinotopic map. CALI provides an approach to inactivate
in vivo
function in higher vertebrates with high temporal and spatial specificity that may have wide application.
A class B scavenger receptor mediates the cellular uptake of carotenoids in
Drosophila
Authors: Kiefer Cornelia , Sumser Emerich , Wernet Mathias F. , von Lintig Johannes
Published: September 2002

Carotenoids are currently being intensely investigated regarding their potential to lower the risk of chronic disease and vitamin A deficiency. Invertebrate models in which vitamin A deficiency is not lethal allow the isolation of blind but viable mutants affected in the pathway leading from dietary carotenoids to vitamin A. Using a mutant in one of these model systems,
Drosophila
, the vitamin A-forming enzyme has recently been molecularly identified. We now show that the molecular basis for the blindness of a different
Drosophila
mutant,
ninaD
, is a defect in the cellular uptake of carotenoids. The
ninaD
gene encodes a class B scavenger receptor essential for the formation of the visual chromophore. A loss of this function results in a carotenoid-free and thus vitamin A-deficient phenotype. Our investigations provide molecular insight into how carotenoids may be distributed into cells of target tissues in animals and indicate a crucial role of class B scavenger receptors rendering dietary carotenoids available for subsequent cell metabolism, as needed for their various physiological functions.
Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness
Authors: Jessani Nadim , Liu Yongsheng , Humphrey Mark , Cravatt Benjamin F.
Published: September 2002

By primarily measuring changes in transcript and protein abundance, conventional genomics and proteomics methods may fail to detect significant posttranslational events that regulate protein activity and, ultimately, cell behavior. To address these limitations, activity-based proteomic technologies that measure dynamics in protein function on a global scale would be of particular value. Here, we describe the application of a chemical proteomics strategy to quantitatively compare enzyme activities across a panel of human breast and melanoma cancer cell lines. A global analysis of the activity, subcellular distribution, and glycosylation state for the serine hydrolase superfamily resulted in the identification of a cluster of proteases, lipases, and esterases that distinguished cancer lines based on tissue of origin. Strikingly, nearly all of these enzyme activities were down-regulated in the most invasive cancer lines examined, which instead up-regulated a distinct set of secreted and membrane-associated enzyme activities. These invasiveness-associated enzymes included urokinase, a secreted serine protease with a recognized role in tumor progression, and a membrane-associated hydrolase KIAA1363, for which no previous link to cancer had been made. Collectively, these results suggest that invasive cancer cells share discrete proteomic signatures that are more reflective of their biological phenotype than cellular heritage, highlighting that a common set of enzymes may support the progression of tumors from a variety of origins and thus represent attractive targets for the diagnosis and treatment of cancer.
Chimeric human CstF-77/
Drosophila
Suppressor of forked proteins rescue
suppressor of forked
mutant lethality and mRNA 3′ end processing in
Drosophila
Authors: Benoit Béatrice , Juge François , Iral Florence , Audibert Agnès , Simonelig Martine
Published: September 2002

The Suppressor of forked [Su(f)] protein is the
Drosophila
homologue of CstF-77, a subunit of human cleavage stimulation factor (CstF) that is required for the first step of the mRNA 3′ end processing reaction
in vitro
. We have addressed directly the role of
su(f)
in the mRNA 3′ end processing reaction
in vivo
. We show that
su(f)
is required for the cleavage of pre-mRNA during mRNA 3′ end formation. Analysis of the functional complementation between Su(f) and CstF-77 shows that most of the
Drosophila
protein (85%) can be exchanged for the human protein to produce chimeric CstF-77/Su(f) proteins that rescue lethality and cleavage defect during mRNA 3′ end formation in
su(f)
mutants. Interestingly, we show that a domain in human CstF-77 is limiting for the rescue and that this domain is not able to reproduce protein interactions with the CstF subunits of
Drosophila
. We also show that chimeric CstF-77/Su(f) proteins that rescue lethality of
su(f)
mutants cannot restore utilization of a regulated poly(A) site in
Drosophila
. Taken together, these results demonstrate that CstF-77 and Su(f) have the same function in mRNA 3′ end formation
in vivo
, but that these two proteins are not interchangeable for regulation of poly(A) site utilization.
Tetracyclines affect prion infectivity
Authors: Forloni Gianluigi , Iussich Selina , Awan Tazeen , Colombo Laura , Angeretti Nadia , Girola Laura , Bertani Ilaria , Poli Giorgio , Caramelli Maria , Grazia Bruzzone Maria , Farina Laura , Limido Lucia , Rossi Giacomina , Giaccone Giorgio , Ironside James W. , Bugiani Orso , Salmona Mario , Tagliavini Fabrizio
Published: September 2002

Prion diseases are transmissible neurodegenerative disorders of humans and animals for which no effective treatment is available. Conformationally altered, protease-resistant forms of the prion protein (PrP) termed PrP
Sc
are critical for disease transmissibility and pathogenesis, thus representing a primary target for therapeutic strategies. Based on previous findings that tetracyclines revert abnormal physicochemical properties and abolish neurotoxicity of PrP peptides
in vitro
, we tested the ability of these compounds to interact with PrP
Sc
from patients with the new variant of Creutzfeldt–Jakob disease (vCJD) and cattle with bovine spongiform encephalopathy (BSE). The incubation with tetracycline hydrochloride or doxycycline hyclate at concentrations ranging from 10 μM to 1 mM resulted in a dose-dependent decrease in protease resistance of PrP
Sc
. This finding prompted us to investigate whether tetracyclines affect prion infectivity by using an animal model of disease. Syrian hamsters were injected intracerebrally with 263K scrapie-infected brain homogenate that was coincubated with 1 mM tetracycline hydrochloride, 1 mM doxycycline hyclate, or vehicle solution before inoculation. Hamsters injected with tetracycline-treated inoculum showed a significant delay in the onset of clinical signs of disease and prolonged survival time. These effects were paralleled by a delay in the appearance of magnetic-resonance abnormalities in the thalamus, neuropathological changes, and PrP
Sc
accumulation. When tetracycline was preincubated with highly diluted scrapie-infected inoculum, one third of hamsters did not develop disease. Our data suggest that these well characterized antibiotics reduce prion infectivity through a direct interaction with PrP
Sc
and are potentially useful for inactivation of BSE- or vCJD-contaminated products and prevention strategies.
A −1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA
Authors: Barry Jennifer K. , Miller W. Allen
Published: September 2002

Programmed −1 ribosomal frameshifting is necessary for translation of the polymerase genes of many viruses. In addition to the consensus elements in the mRNA around the frameshift site, we found previously that frameshifting on
Barley yellow dwarf virus
RNA requires viral sequence located four kilobases downstream. By using dual luciferase reporter constructs, we now show that a predicted loop in the far downstream frameshift element must base pair to a bulge in a bulged stem loop adjacent to the frameshift site. Introduction of either two or six base mismatches in either the bulge or the far downstream loop abolished frameshifting, whereas mutations in both sites that restored base pairing reestablished frameshifting. Likewise, disruption of this base pairing abolished viral RNA replication in plant cells, and restoration of base pairing completely reestablished virus replication. We propose a model in which
Barley yellow dwarf virus
uses this and another long-distance base-pairing event required for cap-independent translation to allow the replicase copying from the 3′ end to shut off translation of upstream ORFs and free the RNA of ribosomes to allow unimpeded replication. This would be a means of solving the “problem,” common to positive strand RNA viruses, of competition between ribosomes and replicase for the same RNA template.
The metapopulation genetic algorithm: An efficient solution for the problem of large phylogeny estimation
Authors: Lemmon Alan R. , Milinkovitch Michel C.
Published: September 2002

Large phylogeny estimation is a combinatorial optimization problem that no future computer will ever be able to solve exactly in practical computing time. The difficulty of the problem is amplified by the need to use complex evolutionary models and large taxon samplings. Hence, many heuristic approaches have been developed, with varying degrees of success. Here, we report on a heuristic approach, the metapopulation genetic algorithm, involving several populations of trees that are forced to cooperate in the search for the optimal tree. Within each population, trees are subjected to evaluation, selection, and mutation events, which are directed by using inter-population consensus information. The method proves to be both very accurate and vastly faster than existing heuristics, such that data sets comprised of hundreds of taxa can be analyzed in practical computing times under complex models of maximum-likelihood evolution. Branch support values produced by the metapopulation genetic algorithm might closely approximate the posterior probabilities of the corresponding branches.
Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism
Authors: Baglin Trevor P. , Carrell Robin W. , Church Frank C. , Esmon Charles T. , Huntington James A.
Published: September 2002

The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the serpin family, which use a unique β-sheet expansion mechanism to trap and destroy their targets. Since the discovery that serpins were a family of serine protease inhibitors there has been controversy as to the role of conformational change in their mechanism. It now is clear that protease inhibition depends entirely on rapid serpin β-sheet expansion after proteolytic attack. The regulatory advantage afforded by the conformational mobility of serpins is demonstrated here by the structures of native and S195A thrombin-complexed heparin cofactor II (HCII). HCII inhibits thrombin, the final protease of the coagulation cascade, in a glycosaminoglycan-dependent manner that involves the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. The native structure of HCII resembles that of native antithrombin and suggests an alternative mechanism of allosteric activation, whereas the structure of the S195A thrombin–HCII complex defines the molecular basis of allostery. Together, these structures reveal a multistep allosteric mechanism that relies on sequential contraction and expansion of the central β-sheet of HCII.
Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting
Authors: Shenolikar S. , Voltz J. W. , Minkoff C. M. , Wade J. B. , Weinman E. J.
Published: September 2002

Na
+
/H
+
exchanger regulatory factor (NHERF)-1 and NHERF-2, two structurally related protein adapters containing tandem PSD-95/Discs large/ZO-1 (PDZ) domains, were identified as essential factors for protein kinase A-mediated inhibition of the sodium-hydrogen exchanger, NHE3. NHERF-1 and NHERF-2 also bound other cellular targets including the sodium-phosphate cotransporter type IIa encoded by the NPT2 gene. Targeted disruption of the mouse NHERF-1 gene eliminated NHERF-1 expression in kidney and other tissues of the mutant mice without altering NHERF-2 levels in these tissues. NHERF-1 (+/−) and (−/−) male mice maintained normal blood electrolytes but showed increased urinary excretion of phosphate when compared with wild-type (+/+) animals. Although the overall levels of renal NHERF-1 targets, NHE3 and Npt2, were unchanged in the mutant mice, immunocytochemistry showed that the Npt2 protein was aberrantly localized at internal sites in the renal proximal tubule cells. The mislocalization of Npt2 paralleled a reduction in the transporter protein in renal brush–border membranes isolated from the mutant mice. In contrast, NHE3 was appropriately localized at the apical surface of proximal tubules in both wild-type and mutant mice. These data suggested that NHERF-1 played a unique role in the apical targeting and/or trafficking of Npt2 in the mammalian kidney, a function not shared by NHERF-2 or other renal PDZ proteins. Phosphate wasting seen in the NHERF-1(−/−) null mice provided a new experimental system for defining the role of PDZ adapters in the hormonal control of ion transport and renal disease.
The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy
Authors: Xie Qing , Bu Weishu , Bhatia Smita , Hare Joan , Somasundaram Thayumanasamy , Azzi Arezki , Chapman Michael S.
Published: September 2002

The structure of the adeno-associated virus (AAV-2) has been determined to 3-Å resolution by x-ray crystallography. AAV is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. As in the distantly related autonomous parvoviruses, the capsid protein has a β-barrel fold, but long loops between the β-strands share little structural homology with other parvoviruses, leading to unique surface features. Most prominent are groups of threefold-related peaks, each an intimate association of loops from two neighboring subunits. Mutations affecting cell entry and receptor binding are clustered near the positively charged side of each peak, implicating the region in attachment to the cellular receptor, heparan sulfate proteoglycan. Amino acids involved in antibody binding are in the same general vicinity. The structure will guide rational engineering of vector capsids to tailor cellular targeting and to avoid immediate neutralization by an immune system sensitized by prior exposure to AAV.
The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation
Authors: Shimada Jun , Shakhnovich Eugene I.
Published: September 2002

Protein G is folded with an all-atom Monte Carlo simulation by using a Gō potential. When folding is monitored by using burial of the lone tryptophan in protein G as the reaction coordinate, the ensemble kinetics is single exponential. Other experimental observations, such as the burst phase and mutational data, are also reproduced. However, more detailed analysis reveals that folding occurs over three distinct, three-state pathways. We show that, because of this tryptophan's asymmetric location in the tertiary fold, its burial (
i
) does not detect certain intermediates and (
ii
) may not correspond to the folding event. This finding demonstrates that ensemble averaging can disguise the presence of multiple pathways and intermediates when a non-ideal reaction coordinate is used. Finally, all observed folding pathways eventually converge to a common rate-limiting step, which is the formation of a specific nucleus involving hydrophobic core residues. These residues are conserved in the ubiquitin superfamily and in a phage display experiment, suggesting that fold topology is a strong determinant of the transition state.
A human embryonic hemoglobin inhibits Hb S polymerization
in vitro
and restores a normal phenotype to mouse models of sickle cell disease
Authors: He Zhenning , Russell J. Eric
Published: September 2002

The principle that developmentally silenced globin genes can be reactivated in adults with defects in β-globin gene expression has been well established both
in vitro
and
in vivo
. In practice, levels of developmental stage-discordant fetal γ globin that can be achieved by using currently approved therapies are generally insufficient to fully resolve typical clincopathological features of sickle cell disease. The therapeutic potential of another developmentally silenced globin—embryonic ɛ globin—has been difficult to evaluate in the absence of a convenient expression system or an appropriate experimental model. The current work analyzes the antisickling properties of an ɛ-globin-containing heterotetramer (Hb Gower-2) both
in vitro
as well as
in vivo
in a well-established mouse model of sickle cell anemia. These animals, expressing 100% human Hb S, display a chronic hemolytic anemia with compensatory marrow and extramedullary erythropoiesis, abundant circulating sickled erythrocytes, and chronic tissue damage evidenced by parallel histopathological and functional deficits. By comparison, related mice that coexpress Hb S as well as Hb Gower-2 exhibit normal physiological, morphological, histological, and functional attributes. Subsequent
in vitro
analyses substantiate results from whole-animal studies, indicating that the polymerization of deoxygenated Hb S can be significantly slowed by relatively small quantities of Hb Gower-2. Together, the
in vivo
and
in vitro
analyses suggest that reactivation of ɛ-globin gene expression would be therapeutically beneficial to adults with sickle phenotypes, and provide a rationale for detailed investigations into the molecular basis for its developmental silencing.
Clusters of transcription-coupled repair in the human genome
Authors: Surrallés Jordi , Ramírez María J. , Marcos Ricard , Natarajan Adayapalam T. , Mullenders Leon H. F.
Published: September 2002

A specialized nucleotide excision repair pathway known as transcription-coupled repair (TCR) counteracts the toxic effects of DNA damage in transcriptionally active genes. The clustering of active genes into gene-rich chromosomal domains predicts that the sites of TCR are unevenly distributed through the genome. To elucidate the genomic organization and chromosomal localization of TCR, we isolated DNA fragments encompassing TCR-mediated repair sites from UV-C irradiated xeroderma pigmentosum group C cells, which can only repair the transcribed strand of active genes. This DNA was used as a molecular probe to visualize TCR in normal metaphase spreads by reverse fluorescence
in situ
hybridization. Whereas DNA repair sites in normal human cells are evenly distributed through the genome, TCR is highly localized at specific chromosomal domains. Particularly, clusters of TCR sites were identified at early-replicating gene-rich bands and telomeric regions of several chromosomes. High gene-density chromosomes such as chromosome 19 and the GC-rich domains of several chromosomes (T bands) are preferential locations of TCR. Our results demonstrate that the intragenomic localization of TCR resembles the uneven distribution of the human transcriptome, CpG islands, and hyperacetylated histones, enforcing the basic link between DNA repair, transcription, and nuclear organization in a complex genome.
The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in
Arabidopsis
Authors: Gagne Jennifer M. , Downes Brian P. , Shiu Shin-Han , Durski Adam M. , Vierstra Richard D.
Published: September 2002

The covalent attachment of ubiquitin is an important determinant for selective protein degradation by the 26S proteasome in plants and animals. The specificity of ubiquitination is often controlled by ubiquitin-protein ligases (or E3s), which facilitate the transfer of ubiquitin to appropriate targets. One ligase type, the SCF E3s are composed of four proteins, cullin1/Cdc53, Rbx1/Roc1/Hrt1, Skp1, and an F-box protein. The F-box protein, which identifies the targets, binds to the Skp1 component of the complex through a degenerate N-terminal ≈60-aa motif called the F-box. Using published F-boxes as queries, we have identified 694 potential F-box genes in
Arabidopsis
, making this gene superfamily one of the largest currently known in plants. Most of the encoded proteins contain interaction domains C-terminal to the F-box that presumably participate in substrate recognition. The F-box proteins can be classified via a phylogenetic approach into five major families, which can be further organized into multiple subfamilies. Sequence diversity within the subfamilies suggests that many F-box proteins have distinct functions and/or substrates. Representatives of all of the major families interact in yeast two-hybrid experiments with members of the
Arabidopsis
Skp family supporting their classification as F-box proteins. For some, a limited preference for Skps was observed, suggesting that a hierarchical organization of SCF complexes exists defined by distinct Skp/F-box protein pairs. Collectively, the data shows that
Arabidopsis
has exploited the SCF complex and the ubiquitin/26S proteasome pathway as a major route for cellular regulation and that a diverse array of SCF targets is likely present in plants.
Graded contribution of the Gβγ binding domains to GIRK channel activation
Authors: Sadja Rona , Alagem Noga , Reuveny Eitan
Published: September 2002

G protein coupled inwardly rectifying K
+
channels (GIRK/Kir3.x) are mainly activated by a direct interaction with Gβγ subunits, released upon the activation of inhibitory neurotransmitter receptors. Although Gβγ binding domains on all four subunits have been found, the relative contribution of each of these binding sites to channel gating has not yet been defined. It is also not known whether GIRK channels open once all Gβγ sites are occupied, or whether gating is a graded process. We used a tandem tetrameric approach to enable the selective elimination of specific Gβγ binding domains in the tetrameric context. Here, we show that tandem tetramers are fully operational. Tetramers with only one wild-type channel subunit showed receptor-independent high constitutive activity. The presence of two or three wild-type subunits reconstituted receptor activation gradually. Furthermore, a tetramer with no GIRK1 Gβγ binding domain displayed slower kinetics of activation. The slowdown in activation was found to be independent of regulator of G protein signaling or receptor coupling, but this slowdown could be reversed once only one Gβγ binding domain of GIRK1 was added. These results suggest that partial activation can occur under low Gβγ occupancy and that full activation can be accomplished by the interaction with three Gβγ binding subunits.
Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21
CIP-1
-mediated G
1
cell cycle arrest through CCAAT/enhancer-binding protein-α
Authors: Wu Frederick Y. , Tang Qi-Qun , Chen Honglin , ApRhys Colette , Farrell Christopher , Chen Jianmeng , Fujimuro Masahiro , Lane M. Daniel , Hayward Gary S.
Published: September 2002

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G
1
cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12–24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPα and p21
CIP-1
proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPα
in vitro
and stimulates C/EBPα-induced expression from both the C/EBPα and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPα and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G
1
to S cell cycle progression in the same cells. All of these effects were abolished in cells that lack C/EBPα or by deletion of the basic/leucine zipper region in RAP that interacts with C/EBPα. Therefore, C/EBPα is essential for the p21-mediated inhibition of G
1
to S-phase progression by RAP in KSHV-infected host cells.
Increased peripheral expansion of naive CD4+ T cellsin vivoafter IL-2 treatment of patients with HIV infection
Authors: Natarajan Ven , Lempicki Richard A. , Sereti Irini , Badralmaa Yunden , Adelsberger Joseph W. , Metcalf Julia A. , Prieto Darue A. , Stevens Randy , Baseler Michael W. , Kovacs Joseph A. , Lane H. Clifford
Published: September 2002

Intermittent interleukin-2 (IL-2) therapy has been shown to increase the number of CD4+ T cells, preferentially cells with a naive phenotype, in patients with HIV infection. For this report we investigated the mechanism underlying this expansion by studying the relative roles of peripheral expansion and thymic output. In a cohort of six patients receiving IL-2 over a period of 1 year, the mean number of naive CD4+ T cells increased from 139 to 387 cells per μl while levels of T cell receptor rearrangement excision circles (TRECs) declined from 47,946 to 26,510 copies per 106naive T cells, thus making it unlikely that the CD4+ T cell count increases were secondary to increase in thymic output. To examine directly the impact of IL-2 on peripheral expansion, peripheral blood mature, naive CD4+ T cells were labeledex vivowith 5-bromodeoxyuridine as well as stained directly for Ki67. These studies revealed a 7-fold increase in the percentage of 5-bromodeoxyuridine-positive cells and a 20–40-fold increase in Ki67 staining in the naive CD4+ T cell pool in the setting of IL-2 administration. This degree of increase in mature CD4+ T cell turnover induced by IL-2 does not compromise the future replicative potential of these cells, because longitudinal measurements of telomere length went from 6,981 to 7,153 bp after 1 year of IL-2 therapy. These data strongly suggest that much of the increase in CD4+ cells associated with IL-2 treatment is caused by peripheral expansion of existing naive CD4+ T cells rather than increased thymic output and that these increases occur without compromising the potential of these cells for further cell division.
Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects
Authors: Zhu Xiaorong , Zhou An , Dey Arunangsu , Norrbom Christina , Carroll Raymond , Zhang Chunling , Laurent Virginie , Lindberg Iris , Ugleholdt Randi , Holst Jens J. , Steiner Donald F.
Published: September 2002

The subtilisin-like proprotein convertases PC1/3 (SPC3) and PC2 (SPC2) are believed to be the major endoproteolytic processing enzymes of the regulated secretory pathway. They are expressed together or separately in neuroendocrine cells throughout the brain and dispersed endocrine system in both vertebrates and invertebrates. Disruption of the gene-encoding mouse PC1/3 has now been accomplished and results in a syndrome of severe postnatal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2. Mice lacking PC1/3 are normal at birth, but fail to grow normally and are about 60% of normal size at 10 weeks. They lack mature GHRH, have low pituitary growth hormone (GH) and hepatic insulin-like growth factor-1 mRNA levels and resemble phenotypically the “little” mouse (Gaylinn, B. D., Dealmeida, V. I., Lyons, C. E., Jr., Wu, K. C., Mayo, K. E. & Thorner, M. O. (1999)
Endocrinology
140, 5066–5074) that has a mutant GHRH receptor. Despite a severe defect in pituitary proopiomelanocortin processing to mature adrenocorticotropic hormone, blood corticosterone levels are essentially normal. There is marked hyperproinsulinemia but without impairment of glucose tolerance. In contrast, PC2-null mice lack mature glucagon and are chronically hypoglycemic (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H. & Steiner, D. (1997)
Proc. Natl. Acad. Sci. USA
94, 6646–6651). The PC1/3-null mice differ from a human subject reported with compound heterozygosity for defects in this gene, who was of normal stature but markedly obese from early life. The PC1/3-null mice are not obese. The basis for these phenotypic differences is an interesting topic for further study. These findings prove the importance of PC1/3 as a key neuroendocrine convertase.
Identification of oncogenes collaborating with p27
Kip1
loss by insertional mutagenesis and high-throughput insertion site analysis
Authors: Hwang Harry C. , Martins Carla P. , Bronkhorst Yvon , Randel Erin , Berns Anton , Fero Matthew , Clurman Bruce E.
Published: September 2002

The p27
Kip1
protein is a cyclin-dependent kinase inhibitor that blocks cell division in response to antimitogenic cues. p27 expression is reduced in many human cancers, and p27 functions as a tumor suppressor that exhibits haploinsufficiency in mice. Despite the well characterized role of p27 as a cyclin-dependent kinase inhibitor, its mechanism of tumor suppression is unknown. We used Moloney murine leukemia virus to induce lymphomas in p27+/+ and p27−/− mice and observed that lymphomagenesis was accelerated in the p27−/− animals. To identify candidate oncogenes that collaborate with p27 loss, we used a high-throughput strategy to sequence 277 viral insertion sites derived from two distinct sets of p27−/− lymphomas and determined their chromosomal location by comparison with the Celera and public (Ensembl) mouse genome databases. This analysis identified a remarkable number of putative protooncogenes in these lymphomas, which included loci that were novel as well as those that were overrepresented in p27−/− tumors. We found that
Myc
activations occurred more frequently in p27−/− lymphomas than in p27+/+ tumors. We also characterized insertions within two novel loci: (
i
) the
Jun
dimerization protein 2 gene (
Jundp2
), and (
ii
) an X-linked locus termed
Xpcl1
. Each of the loci that we found to be frequently involved in p27−/− lymphomas represents a candidate oncogene collaborating with p27 loss. This study illustrates the power of high-throughput insertion site analysis in cancer gene discovery.
Sex differences in the neural basis of emotional memories
Authors: Canli Turhan , Desmond John E. , Zhao Zuo , Gabrieli John D. E.
Published: September 2002

Psychological studies have found better memory in women than men for emotional events, but the neural basis for this difference is unknown. We used event-related functional MRI to assess whether sex differences in memory for emotional stimuli is associated with activation of different neural systems in men and women. Brain activation in 12 men and 12 women was recorded while they rated their experience of emotional arousal in response to neutral and emotionally negative pictures. In a recognition memory test 3 weeks after scanning, highly emotional pictures were remembered best, and remembered better by women than by men. Men and women activated different neural circuits to encode stimuli effectively into memory even when the analysis was restricted to pictures rated equally arousing by both groups. Men activated significantly more structures than women in a network that included the right amygdala, whereas women activated significantly fewer structures in a network that included the left amygdala. Women had significantly more brain regions where activation correlated with both ongoing evaluation of emotional experience and with subsequent memory for the most emotionally arousing pictures. Greater overlap in brain regions sensitive to current emotion and contributing to subsequent memory may be a neural mechanism for emotions to enhance memory more powerfully in women than in men.
Reconsidering the null hypothesis: Is maternal rank associated with birth sex ratios in primate groups?
Authors: Brown Gillian R. , Silk Joan B.
Published: September 2002

Trivers and Willard hypothesized that vertebrates adaptively vary the sex ratio of their offspring in response to the mother's physical condition [Trivers, R. L. & Willard, D. (1973)
Science
179, 90–92]. This hypothesis has produced considerable debate within evolutionary biology. Here we use meta-analysis techniques to evaluate claims that nonhuman primate females facultatively adjust the sex ratio of their progeny in relation to their own dominance rank in a uniform way. The magnitude of the difference in birth sex ratios of high- and low-ranking females declines as sample sizes increase, and the mean difference in birth sex ratios of high- and low-ranking females is zero. These results suggest that the observed effects could be the product of stochastic variation in small samples. These findings indicate that presently we cannot reject the null hypothesis that maternal dominance rank is unrelated to birth sex ratios.
Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease
Authors: Keene C. Dirk , Rodrigues Cecilia M. P. , Eich Tacjana , Chhabra Manik S. , Steer Clifford J. , Low Walter C.
Published: September 2002

Huntington's disease (HD) is an untreatable neurological disorder caused by selective and progressive degeneration of the caudate nucleus and putamen of the basal ganglia. Although the etiology of HD pathology is not fully understood, the observed loss of neuronal cells is thought to occur primarily through apoptosis. Furthermore, there is evidence in HD that cell death is mediated through mitochondrial pathways, and mitochondrial deficits are commonly associated with HD. We have previously reported that treatment with tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, prevented neuropathology and associated behavioral deficits in the 3-nitropropionic acid rat model of HD. We therefore examined whether TUDCA would also be neuroprotective in a genetic mouse model of HD. Our results showed that systemically administered TUDCA led to a significant reduction in striatal neuropathology of the R6/2 transgenic HD mouse. Specifically, R6/2 mice began receiving TUDCA at 6 weeks of age and exhibited reduced striatal atrophy, decreased striatal apoptosis, as well as fewer and smaller size ubiquitinated neuronal intranuclear huntingtin inclusions. Moreover, locomotor and sensorimotor deficits were significantly improved in the TUDCA-treated mice. In conclusion, TUDCA is a nontoxic, endogenously produced hydrophilic bile acid that is neuroprotective in a transgenic mouse model of HD and, therefore, may provide a novel and effective treatment in patients with HD.
Structure of the voltage-gated L-type Ca
2+
channel by electron cryomicroscopy
Authors: Serysheva I. I. , Ludtke S. J. , Baker M. R. , Chiu W. , Hamilton S. L.
Published: September 2002

Voltage-dependent L-type Ca
2+
channels play important functional roles in many excitable cells. We present a three-dimensional structure of an L-type Ca
2+
channel. Electron cryomicroscopy in conjunction with single-particle processing was used to determine a 30-Å resolution structure of the channel protein. The asymmetrical channel structure consists of two major regions: a heart-shaped region connected at its widest end with a handle-shaped region. A molecular model is proposed for the arrangement of this skeletal muscle L-type Ca
2+
channel structure with respect to the sarcoplasmic reticulum Ca
2+
-release channel, the physical partner of the L-type channel for signal transduction during the excitation-contraction coupling in muscle.
Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish
Authors: Lee Percy , Goishi Katsutoshi , Davidson Alan J. , Mannix Robert , Zon Leonard , Klagsbrun Michael
Published: September 2002

Neuropilin-1 (NRP1) is a cell-surface receptor for both vascular endothelial growth factor
165
(VEGF
165
) and class 3 semaphorins that is expressed by neurons and endothelial cells. NRP1 is required for normal developmental angiogenesis in mice. The zebrafish is an excellent system for analyzing vascular development. Zebrafish intersegmental vessels correspond to mammalian capillary sprouts, whereas the axial vessels correspond to larger blood vessels, such as arteries. The zebrafish NRP1 gene (z
nrp1
) was isolated and when overexpressed in cells, zNRP1 protein was a functional receptor for human VEGF
165
. Whole-mount
in situ
hybridization showed that transcripts for
znrp1
during embryonic and early larval development were detected mainly in neuronal and vascular tissues. Morpholino-mediated knockdown of zNRP1 in embryos resulted in vascular defects, most notably impaired circulation in the intersegmental vessels. Circulation via trunk axial vessels was not affected. Embryos treated with VEGF receptor-2 kinase inhibitor had a similar intersegmental vessel defect suggesting that knockdown of zNRP1 reduces VEGF activity. To determine whether NRP1 and VEGF activities were interdependent
in vivo
, zNRP1 and VEGF morpholinos were coinjected into embryos at concentrations that individually did not significantly inhibit blood vessel development. The result was a potent inhibition of blood cell circulation via both intersegmental and axial vessels demonstrating that VEGF and NRP1 act synergistically to promote a functional circulatory system. These results provide the first physiological demonstration that NRP1 regulates angiogenesis through a VEGF-dependent pathway.
A model for the simultaneous analysis of reflectance spectra and basis factors of Munsell color samples under D65 illumination in three-dimensional Euclidean space
Authors: Romney A. Kimball , Indow Tarow
Published: September 2002

In this paper we present the results of an analysis of the physically measured surface reflectance spectra of 360 matte Munsell chromatic color chips plus 10 flat achromatic vectors corresponding to Munsell value levels 10 (white) to 1 (near black) for a total sample size of 370. Each of the 370 spectra was multiplied by the spectral radiant power distribution of D65 light so that the final results represent the spectra of reflected light from Munsell color chips under D65 illumination. We simultaneously model the structure of the color chips and the spectra in a common three-dimensional Euclidean space, oriented to yield the most interpretable structure with respect of the Munsell color structure. In this orientation, axis 1 roughly corresponds to the mean power of the spectral reflectance (approximate Munsell value), axis 2 goes from Munsell red to blue-green, and axis 3 goes from Munsell green-yellow to purple. Basis factors for the spectra are also plotted against wavelength and Munsell hue. These plots have implications for theories of opponent processes. By plotting the chips and spectra in the same space we obtain virtually exact correspondences between the various Munsell hues and spectral values in nanometers for comparison to those obtained by previous researchers. Mathematical derivations are provided to validate the common Euclidean model.