bluuebunny/crossref_metadata_2025 huggingface dataset:
Internalization and recycling of 5-HT
2A
receptors activated by serotonin and protein kinase C-mediated mechanisms
Authors: Bhattacharyya Samarjit , Puri Sapna , Miledi Ricardo , Panicker Mitradas M.
Published: October 2002

Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT
2
receptor subtypes increase IP
3
levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT
2A
-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT
2A
receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT
2A
receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT
2A
receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT
2A
receptors are recycled.
Karyopherin β2B participates in mRNA export from the nucleus
Authors: Shamsher Monee K. , Ploski Jonathan , Radu Aurelian
Published: October 2002

Transport of macromolecules between the cell nucleus and cytoplasm occurs through the nuclear pores and is mediated by soluble carriers known as karyopherins (Kaps), transportins, importins, or exportins. We report that Kap β2B (transportin-2) forms complexes with the mRNA export factor TAP in the presence of RanGTP, as shown by coimmunoprecipitation from HeLa cells. The interaction strictly depends on the presence of RanGTP. In digitonin-permeabilized cells, Kap β2B mediates TAP-GFP export from the nuclei in the presence of RanGTP. A TAP mutant that does not coimmunoprecipitate with Kap β2B is also not exported by Kap β2B. In the permeabilized cells assay, TAP is also exported independently of Kap β2B by direct interaction with nucleoporins, in agreement with previous reports. The export rate is, however, significantly lower than the Kap β2B-mediated pathway. Both Kap β2B and TAP are present and enriched in the poly(A)
+
RNA complexes isolated from HeLa cell nuclear lysates. Poly(A)
+
RNA strongly accumulates in the nuclei of HeLa cells treated with Kap β2B short interfering RNA, indicating that Kap β2B is involved in the export of at least a large proportion of the mRNA species. The export of β-actin and GAPDH mRNA is also inhibited, whereas 28S RNA is not affected. The data support the conclusion that Kap β2B participates directly in the export of a large proportion of cellular mRNAs, and TAP connects Kap β2B to the mRNAs to be exported.
Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide
Authors: Donaldson Logan W. , Gish Gerald , Pawson Tony , Kay Lewis E. , Forman-Kay Julie D.
Published: October 2002

On phosphorylation of Y221 by Abelson (Abl) kinase, the Crk-II adapter protein undergoes an intramolecular reorganization initiated by the binding of its own Src homology 2 (SH2) domain to the pY221 site. Conformational changes induced by phosphotyrosine recognition promote the binding of the Src homology 3 (SH3) domain of the Abl tyrosine kinase to a proline-rich loop located between the βD and βE strands of the SH2 domain (DE loop). We have determined the NMR solution structure of the ternary complex of the Abl SH3 domain with the Crk SH2 domain bound to a Crk pY221 phosphopeptide. The SH2 domain bridges two ligands that bind at distinct sites. The interaction between the Abl SH3 domain and the Crk SH2 domain is localized to a canonical eight-residue site within the DE loop. From
15
N relaxation experiments, the DE loop of the SH2 domain in the complex displays a significant degree of conformational freedom. The structural and dynamic data therefore indicate that these SH2 and SH3 domains do not assume a unique orientation with respect to one another; rather, they appear to be only tethered via the DE loop. Thus, SH2 domain–SH3 domain interactions do not require additional tertiary contacts or restriction of domain orientation when a recognition motif is presented in a mobile loop. This complex between the Abl SH3 domain, Crk SH2 domain, and Crk phosphopeptide is an example of the extremely modular nature of regulatory proteins that provides a rich repertoire of mechanisms for control of biological function.
RB activation defect in tumor cell lines
Authors: Broceño Cristina , Wilkie Scott , Mittnacht Sibylle
Published: October 2002

Activation of the retinoblastoma (RB) protein through dephosphorylation arises in cells upon exit from M phase and in response to environmental stresses, including DNA damage. We provide here for the first time evidence that these responses are co-ordinately affected in a subset of tumor derived cell lines. We find that RB dephosphorylation is not apparent in these cells during progression into G
1
. Importantly these cells also do not respond with RB activation after DNA damage during S phase. Moreover and as a consequence they display phenotypes classically associated with RB
−
cells, showing accelerated apoptosis after DNA damage and DNA re-replication after spindle-checkpoint activation. A large body of literature provides evidence that controls governing inactivation of RB are lost in tumors. The results presented here indicate that the reverse reaction, namely the activation of RB from an inactive precursor, may also be compromised. Our findings indicate that this type of defect may be coupled with hypersensitivity to DNA damage and an increase in genomic instability in response to spindle-checkpoint activation thus bearing potentially important medical implications.
Bottlenecks for metabolic engineering of isoflavone glycoconjugates inArabidopsis
Authors: Liu Chang-Jun , Blount Jack W. , Steele Christopher L. , Dixon Richard A.
Published: October 2002

In view of their perceived chemopreventive activities against hormone-dependent cancers, cardiovascular disease, and postmenopausal ailments, there is considerable interest in engineering plants to contain isoflavone phytoestrogens. However, attempts to date have only resulted in low levels of isoflavone accumulation in non-legumes. Introducing soybean isoflavone synthase (IFS) intoArabidopsis thalianaleads to accumulation of low levels of genistein glycosides. Leaves of wild-typeA. thalianacontain high levels of similar conjugates of the flavonols quercetin and kaempferol, which could be increased by threefold on introduction of an alfalfa chalcone isomerase transgene. Levels of genistein were not increased by expressing both IFS and alfalfa chalcone isomerase, but levels of flavonol conjugates were reduced to a greater extent than could be accounted for by flux into isoflavone. Introduction of IFS into thett6/tt3double mutant blocked in flavonol, and anthocyanin synthesis resulted in high levels of genistein. The bottleneck for constitutive isoflavone production inArabidopsisis, therefore, competition for flavanone between IFS and endogenous flavonol synthesis, and the flavonol pathway is reciprocally but disproportionately affected by IFS.
Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres
Authors: Suslov Oleg N. , Kukekov Valery G. , Ignatova Tatyana N. , Steindler Dennis A.
Published: October 2002

Neural stem cells (NSCs)in vitroare able to generate clonal structures, “neurospheres,” that exhibit intra-clonal neural cell-lineage diversity; i.e., they contain, in addition to NSCs, neuronal and glial progenitors in different states of differentiation. The present study focuses on a subset of neurospheres derived from fresh clinical specimens of human brain by using anin vitrosystem that relies on particular growth factors, serum, and anchorage withdrawal. Thirty individual and exemplary cDNA libraries from these neurosphere clones were clustered and rearranged within a panel after characterization of differentially expressed transcripts. The molecular phenotypes that were obtained indicate that clonogenic NSCs in ourin vitrosystem are heterogeneous, with subsets reflecting distinct neural developmental commitments. This approach is useful for the sorting and expansion of NSCs and facilitates the discovery of genes involved in cell proliferation, communication, fate control, and differentiation.
Isolation and characterization of lymphocyte-like cells from a lamprey
Authors: Mayer Werner E. , Uinuk-ool Tatiana , Tichy Herbert , Gartland Lanier A. , Klein Jan , Cooper Max D.
Published: October 2002

Lymphocyte-like cells in the intestine of the sea lamprey,Petromyzon marinus, were isolated by flow cytometry under light-scatter conditions used for the purification of mouse intestinal lymphocytes. The purified lamprey cells were morphologically indistinguishable from mammalian lymphocytes. A cDNA library was prepared from the lamprey lymphocyte-like cells, and more than 8,000 randomly selected clones were sequenced. Homology searches comparing these ESTs with sequences deposited in the databases led to the identification of numerous genes homologous to those predominantly or characteristically expressed in mammalian lymphocytes, which included genes controlling lymphopoiesis, intracellular signaling, proliferation, migration, and involvement of lymphocytes in innate immune responses. Genes closely related to those that in gnathostomes control antigen processing and transport of antigenic peptides could be ascertained, although no sequences with significant similarity to MHC, T cell receptor, or Ig genes were found. The data suggest that the evolution of lymphocytes in the lamprey has reached a stage poised for the emergence of adaptive immunity.
The genome sequence of
Bifidobacterium longum
reflects its adaptation to the human gastrointestinal tract
Authors: Schell Mark A. , Karmirantzou Maria , Snel Berend , Vilanova David , Berger Bernard , Pessi Gabriella , Zwahlen Marie-Camille , Desiere Frank , Bork Peer , Delley Michele , Pridmore R. David , Arigoni Fabrizio
Published: October 2002

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gastrointestinal tract (GIT) and vagina. Although not numerically dominant in the complex intestinal microflora, they are considered as key commensals that promote a healthy GIT. We determined the 2.26-Mb genome sequence of an infant-derived strain of
Bifidobacterium longum
, and identified 1,730 possible coding sequences organized in a 60%–GC circular chromosome. Bioinformatic analysis revealed several physiological traits that could partially explain the successful adaptation of this bacteria to the colon. An unexpectedly large number of the predicted proteins appeared to be specialized for catabolism of a variety of oligosaccharides, some possibly released by rare or novel glycosyl hydrolases acting on “nondigestible” plant polymers or host-derived glycoproteins and glycoconjugates. This ability to scavenge from a large variety of nutrients likely contributes to the competitiveness and persistence of bifidobacteria in the colon. Many genes for oligosaccharide metabolism were found in self-regulated modules that appear to have arisen in part from gene duplication or horizontal acquisition. Complete pathways for all amino acids, nucleotides, and some key vitamins were identified; however, routes for Asp and Cys were atypical. More importantly, genome analysis provided insights into the reciprocal interactions of bifidobacteria with their hosts. We identified polypeptides that showed homology to most major proteins needed for production of glycoprotein-binding fimbriae, structures that could possibly be important for adhesion and persistence in the GIT. We also found a eukaryotic-type serine protease inhibitor (serpin) possibly involved in the reported immunomodulatory activity of bifidobacteria.
Lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes
Authors: Uinuk-ool Tatiana , Mayer Werner E. , Sato Akie , Dongak Roman , Cooper Max D. , Klein Jan
Published: October 2002

To shed light on the origin of adaptive immunity, a cDNA library was prepared from purified lymphocyte-like cells of a jawless vertebrate, the sea lamprey (Petromyzon marinus). Randomly selected cDNA clones were sequenced, and their homologies to proteins in the databases were determined. Of the sequences homologous to proteins involved in immune responses, five were selected for further characterization. Their encoding genes corresponded to loci that in jawed vertebrates are essential for activities of lymphocytes. These activities include regulation of T and B cell stimulation and proliferation (CD45); stabilization of molecular complexes involved in lymphocyte activation, adhesion, migration, and differentiation (CD9/CD81); adaptor functions in signaling leading to the activation of B lymphocytes (BCAP) and T lymphocytes (CAST); and amino acid transport associated with cell activation (CD98). The presence of these genes in the lamprey genome and their expression in lymphocyte-like cells support the notion that these cells perform many of the functions of gnathostome lymphocytes. It reopens the question of the stage jawless fishes reached in the evolution of their immune system.
Inositol pyrophosphates regulate endocytic trafficking
Authors: Saiardi Adolfo , Sciambi Catherine , McCaffery J. Michael , Wendland Beverly , Snyder Solomon H.
Published: October 2002

The high energy potential and rapid turnover of the recently discovered inositol pyrophosphates, such as diphosphoinositol-pentakisphosphate and bis-diphosphoinositol-tetrakisphosphate, suggest a dynamic cellular role, but no specific functions have yet been established. Using several yeast mutants with defects in inositol phosphate metabolism, we identify dramatic membrane defects selectively associated with deficient formation of inositol pyrophosphates. We show that this phenotype reflects specific abnormalities in endocytic pathways and not other components of membrane trafficking. Thus, inositol pyrophosphates are major regulators of endocytosis.
Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development
Authors: Chen Lan , Segal Daniel , Hukriede Neil A. , Podtelejnikov Alexandre V. , Bayarsaihan Dashzeveg , Kennison James A. , Ogryzko Vasily V. , Dawid Igor B. , Westphal Heiner
Published: October 2002

The LIM-domain-binding protein Ldb1 is a key factor in the assembly of transcriptional complexes involving LIM-homeodomain proteins and other transcription factors that regulate animal development. We identified Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) as components of Ldb1-associated nuclear complexes in HeLa cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. This association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from
Drosophila
to humans. Whereas the vertebrate
Ssdp
gene family has several closely related members, the
Drosophila Ssdp
gene is unique. In
Xenopus
, Ssdp encoded by
Drosophila Ssdp
or mouse
Ssdp1
mRNA enhances axis induction by
Ldb1
in conjunction with the LIM-homeobox gene
Xlim1
. Furthermore, we were able to demonstrate an interaction between
Ssdp
and
Chip
(the fly homolog of
Ldb1
) in
Drosophila
wing development. These findings indicate functional conservation of Ssdp as a cofactor of Ldb1 during invertebrate and vertebrate development.
Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees
Authors: Bukh Jens , Pietschmann Thomas , Lohmann Volker , Krieger Nicole , Faulk Kristina , Engle Ronald E. , Govindarajan Sugantha , Shapiro Max , St. Claire Marisa , Bartenschlager Ralf
Published: October 2002

The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced
in vivo
infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating
in vivo
.
Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells
Authors: Johnson James D. , Misler Stanley
Published: October 2002

Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca2+signals is uncharacterized. We examined Ca2+-dependent insulin signaling in human beta cells. Two hundred nanomolar insulin elevated [Ca2+]cto 284 ± 27 nM above baseline in ≈30% of Fura-4F-loaded cells. Insulin evoked multiple Ca2+signal waveforms, 60% of which included oscillations. Although the amplitude of Ca2+signals was dose-dependent between 0.002 and 2,000 nM, the percentage of cells responding was highest at 0.2 nM insulin, suggesting the interaction of stimulatory and inhibitory pathways. Ca2+-free solutions did not affect the initiation of insulin-stimulated Ca2+signals, but abolished the second phase of plateaus/oscillations. Likewise, inositol 1,4,5-trisphosphate (IP3) receptor antagonists xestospongin C and caffeine selectively blocked the second phase, but not the initiation of insulin signaling. Thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ) blocked insulin signaling, implicating sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA)-containing Ca2+stores. Insulin-stimulated Ca2+signals were insensitive to ryanodine. Injection of the CD38-derived Ca2+mobilizing metabolite, nicotinic acid-adenine dinucleotide phosphate (NAADP), at nanomolar concentrations, evoked oscillatory Ca2+signals that could be initiated in the presence of ryanodine, xestospongin C, and Ca2+-free solutions. Desensitizing concentrations of NAADP abolished insulin-stimulated Ca2+signals. Insulin-stimulated Ca2+signals led to a Ca2+-dependent increase in cellular insulin contents, but not secretion. These data reveal the complexity of insulin signal transduction and function in human beta cells and demonstrate functional NAADP-sensitive Ca2+stores in a human primary cultured cell type.
Quantitative noise analysis for gene expression microarray experiments
Authors: Tu Y. , Stolovitzky G. , Klein U.
Published: October 2002

A major challenge in DNA microarray analysis is to effectively dissociate actual gene expression values from experimental noise. We report here a detailed noise analysis for oligonuleotide-based microarray experiments involving reverse transcription, generation of labeled cRNA (target) through
in vitro
transcription, and hybridization of the target to the probe immobilized on the substrate. By designing sets of replicate experiments that bifurcate at different steps of the assay, we are able to separate the noise caused by sample preparation and the hybridization processes. We quantitatively characterize the strength of these different sources of noise and their respective dependence on the gene expression level. We find that the sample preparation noise is small, implying that the amplification process during the sample preparation is relatively accurate. The hybridization noise is found to have very strong dependence on the expression level, with different characteristics for the low and high expression values. The hybridization noise characteristics at the high expression regime are mostly Poisson-like, whereas its characteristics for the small expression levels are more complex, probably due to cross-hybridization. A method to evaluate the significance of gene expression fold changes based on noise characteristics is proposed.
Fractionating the neural substrate of cognitive control processes
Authors: Dreher Jean-Claude , Berman Karen Faith
Published: October 2002

Psychological and neurobiological theories of cognitive control must account for flexible, seemless transitions among cognitive operations. When subjects switch between tasks, they must both inhibit the previous task and re-engage in a different task. Inhibition of the disengaged task remains active for a period of time and has to be overcome when re-engaging in the same task. Here we used a task-switching paradigm that allows distinction of two control processes: overcoming the inhibition of a previously performed task when re-engaging it and restarting a sequence of tasks after a period of interruption. Behaviorally, these processes were reflected in the facts that: (
i
) switching to a recently performed task, that is thus unlikely to have fully recovered from inhibition, takes longer than switching to a task less recently performed and (
ii
) re-engaging in a sequence of tasks after a period of interruption transiently increases response time. Using event-related functional MRI, we found that these two behavioral effects were accompanied by a double dissociation: the right lateral prefrontal cortex was more activated when switching to a task recently performed compared to a task less recently performed, while the anterior cingulate cortex was recruited when a sequence of tasks was initiated. These results provide insights into the functional organization of the frontal lobe in humans and its role in distinct processes involved in cognitive control.
Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells
Authors: Kato-Stankiewicz Juran , Hakimi Irina , Zhi Gang , Zhang Jie , Serebriiskii Ilya , Guo Lea , Edamatsu Hironori , Koide Hiroshi , Menon Sanjay , Eckl Robert , Sakamuri Sukumar , Lu Yingchun , Chen Quin-Zene , Agarwal Seema , Baumbach William R. , Golemis Erica A. , Tamanoi Fuyuhiko , Khazak Vladimir
Published: October 2002

The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced serum-induced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site. They also inhibited Ras-induced Raf-1 activation in human embryonic kidney 293 cells, Raf-1 and mitogen-activated protein kinase kinase 1 activities in HT1080 fibrosarcoma cells, and epidermal growth factor-induced Raf-1 activation in A549 lung carcinoma cells. The MCP compounds caused reversion of
ras
-transformed phenotypes including morphology,
in vitro
invasiveness, and anchorage-independent growth of HT1080 cells. Decreased level of matrix metalloproteinases was also observed. Further characterization showed that MCP compounds restore actin stress fibers and cause flat reversion in NIH 3T3 cells transformed with H-Ras (V12) but not in NIH 3T3 cells transformed with constitutively active Raf-1 (RafΔN). Finally, we show that MCP compounds inhibit anchorage-independent growth of A549 and PANC-1 cells harboring K-
ras
mutation. Furthermore, MCP110 caused G
1
enrichment of A549 cells with the decrease of cyclin D level. These results highlight potent and specific effects of MCP compounds on cancer cells with intrinsic Ras activation.
Genome fragment ofWolbachiaendosymbiont transferred to X chromosome of host insect
Authors: Kondo Natsuko , Nikoh Naruo , Ijichi Nobuyuki , Shimada Masakazu , Fukatsu Takema
Published: October 2002

The adzuki bean beetle,Callosobruchus chinensis, is triple-infected with distinct lineages ofWolbachiaendosymbiont, wBruCon, wBruOri, and wBruAus, which were identified by theirwsp(Wolbachiasurface protein) gene sequences. Whereas wBruCon and wBruOri caused cytoplasmic incompatibility of the host insect, wBruAus did not. Although wBruCon and wBruOri were easily eliminated by antibiotic treatments, wBruAus persisted over five treated generations and could not be eliminated. The inheritance pattern of wBruAus was, surprisingly, explained by sex-linked inheritance in male-heterozygotic organisms, which agreed with the karyotype ofC. chinensis(2n= 20, XY). Quantitative PCR analysis demonstrated that females contain around twice as muchwsptiter as males, which is concordant with an X chromosome linkage. Specific PCR and Southern blot analyses indicated that the wBruAus-bearing strain ofC. chinensiscontains only a fraction of theWolbachiagene repertoire. Several genome fragments of wBruAus were isolated using an inverse PCR technique. The fragments exhibited a bacterial genome structure containing a number of ORFs typical of the α-proteobacteria, although some of the ORFs contained disruptive mutations. In the flanking region offtsZgene, a non-long terminal repeat (non-LTR) retrotransposon sequence, which is typical of insects but not found from bacteria, was present. These results strongly suggest that wBruAus has no microbial entity but is a genome fragment ofWolbachiaendosymbiont transferred to the X chromosome of the host insect.
Modularity, individuality, and evo-devo in butterfly wings
Authors: Beldade Patrícia , Koops Kees , Brakefield Paul M.
Published: October 2002

Modularity in animal development is thought to have facilitated morphological diversification, but independent change of those traits integrated within a module might be restricted. Correlations among traits describe potential developmental constraints on evolution. These have often been postulated to explain patterns of morphological variation and have been examined theoretically but seldom analyzed experimentally. Here, we use artificial selection to explore the modular organization of butterfly wing patterns and the extent to which their evolution is constrained by the genetic correlations among repeated pattern elements. We show that, in
Bicyclus anynana
butterflies, despite the evidence that all eyespots are developmentally coupled, the response to selection for increased size of one individual eyespot can proceed in a manner largely independent from selection imposed on another eyespot. We argue that among-eyespot correlations are unlikely to have constrained the evolutionary diversification of butterfly wing patterns but might be important when only limited time is available for adaptive evolution to occur. The ease with which we have been able to produce independent responses to artificial selection on different eyespots may be linked to a legacy of natural selection favoring individuality. Our results are discussed within the context of the evolution of modularity and individuality of serially repeated morphological traits.
φ value analysis of heterogeneity in pathways of allosteric transitions: Evidence for parallel pathways of ATP-induced conformational changes in a GroEL ring
Authors: Horovitz Amnon , Amir Amnon , Danziger Oded , Kafri Galit
Published: October 2002

What are the mechanisms of ligand-induced allosteric transitions in proteins? A powerful method to characterize pathways and transition states of reactions is φ value analysis. A φ value is the ratio between the changes on a perturbation (e.g., mutation) in the activation and equilibrium free energies of a reaction. Here, φ value analysis is used to characterize the ATP-induced allosteric transitions of GroEL by using changes in ATP concentration as perturbations. GroEL consists of two stacked back-to-back heptameric rings that bind ATP with positive cooperativity within rings and negative cooperativity between rings. Evidence is presented for the existence of parallel pathways for the allosteric transition of each ring. In both allosteric transitions, there is an abrupt ATP-dependent switch from a pathway with ATP-binding sites in the transition state that are very similar to those of the initial T state (φ = 0) to a pathway with a φ value of ≈0.3. The φ value procedure outlined here should be useful in mapping the energy landscape of allosteric transitions of other proteins.
Crystal structure of the DNA-binding domain from Ndt80, a transcriptional activator required for meiosis in yeast
Authors: Montano Sherwin P. , Coté Marie L. , Fingerman Ian , Pierce Michael , Vershon Andrew K. , Georgiadis Millie M.
Published: October 2002

Ndt80 is a transcriptional activator required for meiosis in the yeast
Saccharomyces cerevisiae
. Here, we report the crystal structure at 2.3 Å resolution of the DNA-binding domain of Ndt80 experimentally phased by using the anomalous and isomorphous signal from a single ordered Se atom per molecule of 272-aa residues. The structure reveals a single ≈32-kDa domain with a distinct fold comprising a β-sandwich core elaborated with seven additional β-sheets and three short α-helices. Inspired by the structure, we have performed a mutational analysis and defined a DNA-binding motif in this domain. The DNA-binding domain of Ndt80 is homologous to a number of proteins from higher eukaryotes, and the residues that we have shown are required for DNA binding by Ndt80 are highly conserved among this group of proteins. These results suggest that Ndt80 is the defining member of a previously uncharacterized family of transcription factors, including the human protein (C11orf9), which has been shown to be highly expressed in invasive or metastatic tumor cells.
Control of lactotrop proliferation by dopamine: Essential role of signaling through D2 receptors and ERKs
Authors: Iaccarino Ciro , Samad Tarek A. , Mathis Carole , Kercret Henri , Picetti Roberto , Borrelli Emiliana
Published: October 2002

Dopamine is thought to exert a negative control on lactotrop cell proliferation and prolactin production. Indeed, mice lacking the D2 receptor develop pituitary tumors of lactotrop origin. Because lactotrops express two isoforms of D2R, D2L, and D2S, in a specific ratio, we decided to explore the physiological importance of their relative abundance
in vivo
. Thus, we generated transgenic animals overexpressing either D2L or D2S in lactotrops. Increased expression of D2S, but not of D2L, leads to mitogen-activated protein kinase (MAPK) induction, which results in pituitary hypoplasia. On the other hand, levels of phosphorylated MAPKs are drastically reduced in pituitary tumors generated by the absence of D2-dependent signaling. These results underline a critical role of D2-mediated MAPK activation in lactotrop proliferation. Furthermore, whereas D2S overexpression results to a drastic reduction of prolactin, D2L overexpression elevates it. Our findings underscore a different role of the two D2R isoforms in the pituitary gland physiology.
A test of evolutionary theories of aging
Authors: Hughes Kimberly A. , Alipaz Julie A. , Drnevich Jenny M. , Reynolds Rose M.
Published: October 2002

Senescence is a nearly universal feature of multicellular organisms, and understanding why it occurs is a long-standing problem in biology. The two leading theories posit that aging is due to (i) pleiotropic genes with beneficial early-life effects but deleterious late-life effects (“antagonistic pleiotropy”) or (ii) mutations with purely deleterious late-life effects (“mutation accumulation”). Previous attempts to distinguish these theories have been inconclusive because of a lack of unambiguous, contrasting predictions. We conducted experiments withDrosophilabased on recent population-genetic models that yield contrasting predictions. Genetic variation and inbreeding effects increased dramatically with age, as predicted by the mutation theory. This increase occurs because genes with deleterious effects with a late age of onset are unopposed by natural selection. Our findings provide the strongest support yet for the mutation theory.
Bradyoxetin, a unique chemical signal involved in symbiotic gene regulation
Authors: Loh John , Carlson Russell W. , York William S. , Stacey Gary
Published: October 2002

Bradyrhizobium japonicum
is a symbiotic bacterium that nodulates soybean. Critical for the infection and establishment of this symbiosis are the bacterial nodulation genes (
nod
,
nol
,
noe
), which are induced in the presence of plant produced isoflavones. Transcription of the nodulation genes is also controlled in a population density-dependent fashion. Expression of the
nod
genes is maximal at low population densities, and decreases significantly at higher culture densities. Population density control of the nodulation genes involves NolA and NodD2, both of which function in tandem to repress
nod
gene expression. An extracellular secreted factor (CDF) is known to mediate this repression. Here, we report that CDF is a novel signaling molecule, designated bradyoxetin, different from other Gram-negative quorum signals. The proposed structure of bradyoxetin is 2-{4-[[4-(3-aminooxetan-2-yl)phenyl](imino)methyl]phenyl}oxetan-3-ylamine. Interestingly, expression of bradyoxetin is iron-regulated, and is maximally produced under iron-starved conditions. Consistent with this, expression of the nodulation genes occurred in an iron-dependent fashion. Addition of iron to
B. japonicum
cultures at high optical densities resulted in decreased bradyoxetin production, and a concomitant reduction in
nolA
expression. A corresponding increase in
nodY
–
lacZ
expression was observed with iron treatment.
Coupling of c-Src to large conductance voltage- and Ca
2+
-activated K
+
channels as a new mechanism of agonist-induced vasoconstriction
Authors: Alioua Abderrahmane , Mahajan Aman , Nishimaru Kazuhide , Zarei Masoud M. , Stefani Enrico , Toro Ligia
Published: October 2002

The voltage-dependent and Ca
2+
-activated K
+
channel (MaxiK, BK) and the cellular proto-oncogene pp60
c-Src
(c-Src) are abundant proteins in vascular smooth muscle. The role of MaxiK channels as a vasorelaxing force is well established, but their role in vasoconstriction is unclear. Because Src participates in regulating vasoconstriction, we investigated whether c-Src inhibits MaxiK as a mechanism for agonist-induced vasoconstriction. Functional experiments in human and rat show that inhibitors of Src (Lavendustin A, PP2) but not inactive compounds (Lavendustin B, PP3) induce a pronounced relaxation of coronary or aortic smooth muscle precontracted with 5-hydroxytriptamine, phenylephrine, or Angiotensin II. Iberiotoxin, a MaxiK blocker, antagonizes the relaxation induced by Lavendustin A or PP2, indicating that c-Src inhibits the Iberiotoxin-sensitive component, likely MaxiK channels. In agreement, coronary muscle MaxiK currents were enhanced by Lavendustin A. To investigate the molecular mechanism of c-Src action on MaxiK channels, we transiently expressed its α subunit, hSlo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSlo was right-shifted by ≈16 mV. hSlo inhibition by c-Src is due to channel direct phosphorylation because: (
i
) excised patches exposed to protein tyrosine phosphatase (CD45) resulted in a partial reversal of the inhibitory effect by ≈10 mV, and (
ii
) immunoprecipitated hSlo channels were recognized by an anti-phosphotyrosine Ab. Furthermore, coexpression of hSlo and c-Src demonstrate a striking colocalization in HEK293T cells. We propose that MaxiK channels via direct c-Src-dependent phosphorylation play a significant role supporting vasoconstriction after activation of G protein-coupled receptors by vasoactive substances and neurotransmitters.
Structure of HIV-2 reverse transcriptase at 2.35-Å resolution and the mechanism of resistance to non-nucleoside inhibitors
Authors: Ren J. , Bird L. E. , Chamberlain P. P. , Stewart-Jones G. B. , Stuart D. I. , Stammers D. K.
Published: October 2002

The HIV-2 serotype of HIV is a cause of disease in parts of the West African population, and there is evidence for its spread to Europe and Asia. HIV-2 reverse transcriptase (RT) demonstrates an intrinsic resistance to non-nucleoside RT inhibitors (NNRTIs), one of two classes of anti-AIDS drugs that target the viral RT. We report the crystal structure of HIV-2 RT to 2.35 Å resolution, which reveals molecular details of the resistance to NNRTIs. HIV-2 RT has a similar overall fold to HIV-1 RT but has structural differences within the “NNRTI pocket” at both conserved and nonconserved residues. The structure points to the role of sequence differences that can give rise to unfavorable inhibitor contacts or destabilization of part of the binding pocket at positions 101, 106, 138, 181, 188, and 190. We also present evidence that the conformation of Ile-181 compared with the HIV-1 Tyr-181 could be a significant contributory factor to this inherent drug resistance of HIV-2 to NNRTIs. The availability of a refined structure of HIV-2 RT will provide a stimulus for the structure-based design of novel non-nucleoside inhibitors that could be used against HIV-2 infection.
Overexpression of motor protein KIF17 enhances spatial and working memory in transgenic mice
Authors: Wong Richard Wing-Chuen , Setou Mitsutoshi , Teng Junlin , Takei Yosuke , Hirokawa Nobutaka
Published: October 2002

The kinesin superfamily proteins (KIFs) play essential roles in receptor transportation along the microtubules. KIF17 transports the
N
-methyl-
d
-aspartate receptor NR2B subunit
in vitro
, but its role
in vivo
is unknown. To clarify this role, we generated transgenic mice overexpressing KIF17 tagged with GFP. The KIF17 transgenic mice exhibited enhanced learning and memory in a series of behavioral tasks, up-regulated NR2B expression with the potential involvement of a transcriptional factor, the cAMP-dependent response element-binding protein, and increased phosphorylation of the cAMP-dependent response element-binding protein. Our results suggest that the motor protein KIF17 contributes to neuronal events required for learning and memory by trafficking fundamental
N
-methyl-
d
-aspartate-type glutamate receptors.
Experimental evolution of learning ability in fruit flies
Authors: Mery Frederic , Kawecki Tadeusz J.
Published: October 2002

The presence of genetic variation for learning ability in animals opens the way for experiments asking how and under what ecological circumstances improved learning ability should evolve. Here we report experimental evolution of learning ability in
Drosophila melanogaster
. We exposed experimental populations for 51 generations to conditions that we expected to favor associative learning with regard to oviposition substrate choice. Flies that learned to associate a chemical cue (quinine) with a particular substrate, and still avoided this substrate several hours after the cue had been removed, were expected to contribute more alleles to the next generation. From about generation 15 on, the experimental populations showed marked ability to avoid oviposition substrates that several hours earlier had contained the chemical cue. The improved response to conditioning was also expressed when the flies were faced with a choice of novel media. We demonstrate that these behavioral changes are caused by the evolution of both a higher learning rate and a better memory.
The Cretaceous superchron geodynamo: Observations near the tangent cylinder
Authors: Tarduno John A. , Cottrell Rory D. , Smirnov Alexei V.
Published: October 2002

If relationships exist between the frequency of geomagnetic reversals and the morphology, secular variation, and intensity of Earth's magnetic field, they should be best expressed during superchrons, intervals tens of millions of years long lacking reversals. Here we report paleomagnetic and paleointensity data from lavas of the Cretaceous Normal Polarity Superchron that formed at high latitudes near the tangent cylinder that surrounds the solid inner core. The time-averaged field recorded by these lavas is remarkably strong and stable. When combined with global results available from lower latitudes, these data define a time-averaged field that is overwhelmingly dominated by the axial dipole (octupole components are insignificant). These observations suggest that the basic features of the geomagnetic field are intrinsically related. Superchrons may reflect times when the nature of core–mantle boundary heat flux allows the geodynamo to operate at peak efficiency.
Structure of the GCN5 histone acetyltransferase bound to a bisubstrate inhibitor
Authors: Poux Arienne N. , Cebrat Marek , Kim Cheol M. , Cole Philip A. , Marmorstein Ronen
Published: October 2002

Histone acetyltransferases (HATs) use acetyl CoA to acetylate target lysine residues within histones and other transcription factors, such as the p53 tumor suppressor, to promote gene activation. HAT enzymes fall into subfamilies with divergence in sequence and substrate preference. Several HAT proteins have been implicated in human cancer. We have previously reported on the preparation of peptide-CoA conjugate inhibitors with distinct specificities for the p300/CBP [cAMP response element binding protein (CREB)-binding protein] or GCN5 HAT subfamilies. Here we report on the crystal structure of the GCN5 HAT bound to a peptide-CoA conjugate containing CoA covalently attached through an isopropionyl linker to Lys-14 of a 20-aa N-terminal fragment of histone H3. Surprisingly, the structure reveals that the H3 portion of the inhibitor is bound outside of the binding site for the histone substrate and that only five of the 20 aa residues of the inhibitor are ordered. Rearrangements within the C-terminal region of the GCN5 protein appear to mediate this peptide displacement. Mutational and enzymatic data support the hypothesis that the observed structure corresponds to a late catalytic intermediate. The structure also provides a structural scaffold for the design of HAT-specific inhibitors that may have therapeutic applications for the treatment of HAT-mediated cancers.
Biological activity of the
Helicobacter pylori
virulence factor CagA is determined by variation in the tyrosine phosphorylation sites
Authors: Higashi Hideaki , Tsutsumi Ryouhei , Fujita Akiko , Yamazaki Shiho , Asaka Masahiro , Azuma Takeshi , Hatakeyama Masanori
Published: October 2002

Helicobacter pylori
is a causative agent of gastritis and peptic ulcer.
cagA
+
H. pylori
strains are more virulent than
cagA
−
strains and are associated with gastric carcinoma. The
cagA
gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western
H. pylori
isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by
H. pylori
strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian
H. pylori
isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.
Two different inward rectifier K
+
channels are effectors for transmitter-induced slow excitation in brain neurons
Authors: Bajic D. , Koike M. , Albsoul-Younes A. M. , Nakajima S. , Nakajima Y.
Published: October 2002

Substance P (SP) excites large neurons of the nucleus basalis (NB) by inhibiting an inward rectifier K
+
channel (Kir). The properties of the Kir in NB (KirNB) in comparison with the G protein-coupled Kir (GIRK) were investigated. Single-channel recordings with the cell-attached mode showed constitutively active KirNB channels, which were inhibited by SP. When the recording method was changed from the on-cell to the inside-out mode, the channel activity of KirNB remained intact with its constitutive activity unaltered. Application of Gβ
1γ2
to inside-out patches induced activity of a second type of Kir (GIRK). Application of Gβ
1γ2
, however, did not change the KirNB activity. Sequestering Gβ
1γ2
with Gα
i2
abolished the GIRK activity, whereas the KirNB activity was not affected. The mean open time of KirNB channels (1.1 ms) was almost the same as that of GIRKs. The unitary conductance of KirNB was 23 pS (155 mM [K
+
]
o
), whereas that of the GIRK was larger (32–39 pS). The results indicate that KirNB is different from GIRKs and from any of the classical Kirs (IRKs). Whole-cell current recordings revealed that application of muscarine to NB neurons induced a GIRK current, and this GIRK current was also inhibited by SP. Thus, SP inhibits both KirNB and GIRKs. We conclude that the excitatory transmitter SP has two types of Kirs as its effectors: the constitutively active, Gβγ-independent KirNB channel and the Gβγ-dependent GIRK.