bluuebunny/crossref_metadata_2025 huggingface dataset:
Embryonic stem cells can form germ cells
in vitro
Authors: Toyooka Yayoi , Tsunekawa Naoki , Akasu Ryuko , Noce Toshiaki
Published: October 2003

Knock-in embryonic stem (ES) cells, in which
GFP
or
lacZ
was expressed from the endogenous mouse vasa homolog (
Mvh
), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during
in vitro
differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells
in vitro
.
In vitro
germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering.
Compensatory evolution of a precursor messenger RNA secondary structure in the
Drosophila melanogaster Adh
gene
Authors: Chen Ying , Stephan Wolfgang
Published: October 2003

Evidence for the evolutionary maintenance of a hairpin structure possibly involved in intron processing had been found in intron 1 of the alcohol dehydrogenase gene (
Adh
) in diverse
Drosophila
species. In this study, the putative hairpin structure was evaluated systematically in
Drosophila melanogaster
by elimination of either side of the stem using site-directed mutagenesis. The effects of these mutations and the compensatory double mutant on intron splicing efficiency and ADH protein production were assayed in
Drosophila melanogaster
Schneider L2 cells and germ-line transformed adult flies. Mutations that disrupt the putative hairpin structure right upstream of the intron branch point were found to cause a significant reduction in both splicing efficiency and ADH protein production. In contrast, the compensatory double mutant that restores the putative hairpin structure was indistinguishable from the WT in both splicing efficiency and ADH level. It was also observed by mutational analysis that a more stable secondary structure (with a longer stem) in this intron decreases both splicing efficiency and ADH protein production. Implications for RNA secondary structure and intron evolution are discussed.
Complete genome sequence and analysis of
Wolinella succinogenes
Authors: Baar Claudia , Eppinger Mark , Raddatz Guenter , Simon Jörg , Lanz Christa , Klimmek Oliver , Nandakumar Ramkumar , Gross Roland , Rosinus Andrea , Keller Heike , Jagtap Pratik , Linke Burkhard , Meyer Folker , Lederer Hermann , Schuster Stephan C.
Published: October 2003

To understand the origin and emergence of pathogenic bacteria, knowledge of the genetic inventory from their nonpathogenic relatives is a prerequisite. Therefore, the 2.11-megabase genome sequence of
Wolinella succinogenes
, which is closely related to the pathogenic bacteria
Helicobacter pylori
and
Campylobacter jejuni
, was determined. Despite being considered nonpathogenic to its bovine host,
W. succinogenes
holds an extensive repertoire of genes homologous to known bacterial virulence factors. Many of these genes have been acquired by lateral gene transfer, because part of the virulence plasmid pVir and an N-linked glycosylation gene cluster were found to be syntenic between
C. jejuni
and genomic islands of
W. succinogenes
. In contrast to other host-adapted bacteria,
W. succinogenes
does harbor the highest density of bacterial sensor kinases found in any bacterial genome to date, together with an elaborate signaling circuitry of the GGDEF family of proteins. Because the analysis of the
W. succinogenes
genome also revealed genes related to soil- and plant-associated bacteria such as the
nif
genes,
W. succinogenes
may represent a member of the epsilon proteobacteria with a life cycle outside its host.
A nonconserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved in degradation of Gap1 permease and stress-induced abnormal proteins
Authors: Hoshikawa Chikara , Shichiri Mika , Nakamori Shigeru , Takagi Hiroshi
Published: October 2003

A toxic
l
-proline analogue,
l
-azetidine-2-carboxylic acid (AZC), causes misfolding of the proteins into which it is incorporated competitively with
l
-proline, thereby inhibiting the growth of the cells. AZC enters budding yeast
Saccharomyces cerevisiae
cells primarily through the general amino acid permease Gap1, not through the proline-specific permease Put4. We isolated an AZC-hypersensitive mutant that cannot grow even at low concentrations of AZC because of the accumulation of intracellular AZC. By screening through a yeast genomic library, the mutant was found to carry an allele of
RSP5
encoding an E3 ubiquitin ligase. A single amino acid change replacing Ala (GCA) at position 401 with Glu (GAA) showed that Ala-401 in the third WW domain (a protein interaction module) is not conserved in the domain. The addition of

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to yeast cells growing on
l
-proline induced rapid ubiquitination, endocytosis, and vacuolar degradation of the plasma membrane protein Gap1. However, immunoblot and permease assays indicated that Gap1 in the
rsp5
mutant remained stable and active on the plasma membrane probably with no ubiquitination, leading to AZC accumulation and hypersensitivity. The
rsp5
mutants also showed hypersensitivity to various stresses (toxic amino acid analogues, high temperature in a rich medium, and oxidative treatments) and defects in spore growth. These results suggest that Rsp5 is involved in selective degradation of abnormal proteins and specific proteins for spore growth, in addition to nitrogen-regulated degradation of Gap1. Furthermore, Ala-401 of Rsp5 was considered to have an important role in the ubiquitination of targeted proteins.
EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells
Authors: Kleer Celina G. , Cao Qi , Varambally Sooryanarayana , Shen Ronglai , Ota Ichiro , Tomlins Scott A. , Ghosh Debashis , Sewalt Richard G. A. B. , Otte Arie P. , Hayes Daniel F. , Sabel Michael S. , Livant Donna , Weiss Stephen J. , Rubin Mark A. , Chinnaiyan Arul M.
Published: October 2003

The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. Here we investigate the functional role of EZH2 in cancer cell invasion and breast cancer progression. EZH2 transcript and protein were consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrated that EZH2 protein levels were strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion required an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation.
Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation
Authors: Matsuzaki Hitomi , Daitoku Hiroaki , Hatta Mitsutoki , Tanaka Keiji , Fukamizu Akiyoshi
Published: October 2003

Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. It has been shown that FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. Here, we investigate the fate of FKHR after cells are stimulated by insulin. We show that insulin treatment decreases endogenous FKHR proteins in HepG2 cells, which is inhibited by proteasome inhibitors. FKHR is ubiquitinated
in vivo
and
in vitro
, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and the mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination
in vivo
and
in vitro
. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation.
Sounds and silence: An optical topography study of language recognition at birth
Authors: Peña Marcela , Maki Atsushi , Kovac̆ić Damir , Dehaene-Lambertz Ghislaine , Koizumi Hideaki , Bouquet Furio , Mehler Jacques
Published: October 2003

Does the neonate's brain have left hemisphere (LH) dominance for speech? Twelve full-term neonates participated in an optical topography study designed to assess whether the neonate brain responds specifically to linguistic stimuli. Participants were tested with normal infant-directed speech, with the same utterances played in reverse and without auditory stimulation. We used a 24-channel optical topography device to assess changes in the concentration of total hemoglobin in response to auditory stimulation in 12 areas of the right hemisphere and 12 areas of the LH. We found that LH temporal areas showed significantly more activation when infants were exposed to normal speech than to backward speech or silence. We conclude that neonates are born with an LH superiority to process specific properties of speech.
CCAAT-binding factor regulates expression of the β
1
subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line
Authors: Sharina Iraida G. , Martin Emil , Thomas Anthony , Uray Karen L. , Murad Ferid
Published: October 2003

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of α and β subunits. We investigated human β
1
sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5′ upstream region of the β
1
sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the β
1
sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the β
1
sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of β
1
sGC by inhibiting transcription. In summary, our study represents an analysis of the human β
1
sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in β
1
sGC expression.
The
BCSC-1
locus at chromosome 11q23-q24 is a candidate tumor suppressor gene
Authors: Martin Eric S. , Cesari Rossano , Pentimalli Francesca , Yoder Kristine , Fishel Richard , Himelstein Andrew L. , Martin S. Eric , Godwin Andrew K. , Negrini Massimo , Croce Carlo M.
Published: October 2003

Frequent allelic loss at human chromosome 11q23-q24 occurs in a wide variety of cancers, suggesting that this region may harbor a tumor suppressor gene. By constructing a physical map of the LOH11CR2 minimal region of loss on 11q23-q24 associated with lung and breast carcinomas, we were able to clone a hereditary translocation, t(11;12)(q23;q24), in a patient with early-onset breast cancer and family history of cancer. The breakpoint was found within 6 kb of the
BCSC-1
candidate tumor suppressor gene located in the LOH11CR2 region whereas additional loss of heterozygosity (LOH) analysis in breast and ovarian tumors, including that of the patient with the t(11;12)(q23;q24), implicated the
BCSC-1
locus as the primary target of deletion. Northern analysis of the
BCSC-1
mRNA revealed a lack of expression in 33 of 41 (80%) tumor cell lines, and its ectopic expression led to the suppression of colony formation
in vitro
and tumorigenicity
in vivo
. These data suggest that
BCSC-1
may exert a tumor suppressor activity and is a likely target of the LOH observed on 11q23-q24 in cancer.
Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes
Authors: Klymchenko Andrey S. , Duportail Guy , Mély Yves , Demchenko Alexander P.
Published: October 2003

The principle of electrochromic modulation of excited-state intramolecular proton-transfer reaction was applied for the design of fluorescence probes with high two-color sensitivity to dipole potential, Ψ
d
, in phospholipid bilayers. We report on the effect of Ψ
d
variation on excitation and fluorescence spectra of two new 3-hydroxyflavone probes, which possess opposite orientations of the fluorescent moiety in the lipid bilayer. The dipole potential in the bilayer was modulated by the addition of 6-ketocholestanol or phloretin and by substitution of dimyristoyl phosphatidylcholine lipid with its ether analog 1,2-di-
o
-tetradecyl-
sn
-glycero-3-phosphocholine, and its value was estimated by the reference styryl dye 1-(3-sulfonatopropyl)-4-{β[2-(di-
n
-octylamino)-6-naphthyl]vinyl}pyridinium betaine. We demonstrate that after Ψ
d
changes, the probe orienting in the bilayer similarly to the reference dye shows similar shifts in the excitation spectra, whereas the probe with the opposite orientation shows the opposite shifts. The new observation is that the response of 3-hydroxyflavone probes to Ψ
d
in excitation spectra is accompanied by and quantitatively correlated with dramatic changes of relative intensities of the two well separated emission bands that belong to the initial normal and the product tautomer forms of the excited-state intramolecular proton-transfer reaction. This provides a strong response to Ψ
d
by change in emission color.
Negative selection imparts peptide specificity to the mature T cell repertoire
Authors: Huseby Eric S. , Crawford Frances , White Janice , Kappler John , Marrack Philippa
Published: October 2003

The T cell αβ receptor (TCR) recognizes foreign peptide antigens bound to proteins encoded in the MHC. The MHC portion of this complex contributes much to the footprint of the TCR on the ligand, yet T cells are usually very specific for individual foreign peptides. Here, we show that the development of peptide-specific T cells is not intrinsic to thymocytes that undergo thymic-positive selection but is an outcome of eliminating, through negative selection, thymocytes bearing TCRs with extensive peptide cross-reactivity. Hence, thymic-negative selection imposes peptide specificity on the mature T cell repertoire.
Antisense therapy targeting MDM2 oncogene in prostate cancer: Effects on proliferation, apoptosis, multiple gene expression, and chemotherapy
Authors: Zhang Zhuo , Li Mao , Wang Hui , Agrawal Sudhir , Zhang Ruiwen
Published: October 2003

This study was undertaken to investigate the role of mouse double minute 2 (MDM2) oncogene in prostate cancer growth and the potential of MDM2 as a target for prostate cancer therapy. An antisense anti-human–MDM2 mixed-backbone oligonucleotide was tested in human prostate cancer models with various p53 statuses, LNCaP (p53
wt/wt
), DU145 (p53
mt/mt
), and PC3 (p53
null
). In a dose- and time-dependent manner, it specifically inhibited MDM2 expression and modified expression of several genes, at both mRNA and protein levels. In LNCaP cells, p53, p21, Bax, and hypophosphorylated retinoblastoma tumor suppressor protein (pRb) levels increased, whereas Bcl2, pRb protein, and E2F transcription factor 1 (E2F1) levels decreased. In DU145 cells, p21 levels were elevated and E2F1 levels decreased, although mutant p53, Rb, and Bax levels remained unchanged. In PC3 cells, MDM2 inhibition resulted in elevated p21, Bax, and pRb levels and decreased ppRb and E2F1 levels. In all three cell lines, MDM2 inhibition reduced cell proliferation, induced apoptosis, and potentiated the effects of the chemotherapeutic agents 10-hydroxycamptothecin and paclitaxel. The anti-MDM2 oligonucleotide showed antitumor activity and increased therapeutic effectiveness of paclitaxel in both LNCaP and PC3 xenografts, causing changes in gene expression similar to those seen
in vitro
. In summary, this study demonstrates that MDM2 has a role in prostate cancer growth via p53-dependent and p53-independent mechanisms and that multiple genes are involved in the process. MDM2 inhibitors such as second-generation antisense oligonucleotides have a broad spectrum of antitumor activities in human cancers regardless of p53 status, providing novel approaches to therapy of human prostate cancer.
Genetically engineered mice with an additional class of cone photoreceptors: Implications for the evolution of color vision
Authors: Smallwood Philip M. , Ölveczky Bence P. , Williams Gary L. , Jacobs Gerald H. , Reese Benjamin E. , Meister Markus , Nathans Jeremy
Published: October 2003

Among eutherian mammals, only primates possess trichromatic color vision. In Old World primates, trichromacy was made possible by a visual pigment gene duplication. In most New World primates, trichromacy is based on polymorphic variation in a single X-linked gene that produces, by random X inactivation, a patchy mosaic of spectrally distinct cone photoreceptors in heterozygous females. In the present work, we have modeled the latter strategy in a nonprimate by replacing the X-linked mouse green pigment gene with one encoding the human red pigment. In the mouse retina, the human red pigment seems to function normally, and heterozygous female mice express the human red and mouse green pigments at levels that vary between animals. Multielectrode array recordings from heterozygous female retinas reveal significant variation in the chromatic sensitivities of retinal ganglion cells. The data are consistent with a model in which these retinal ganglion cells draw their inputs indiscriminately from a coarse-grained mosaic of red and green cones. These observations support the ideas that (
i
) chromatic signals could arise from stochastic variation in inputs drawn nonselectively from red and green cones and (
ii
) tissue mosaicism due to X chromosome inactivation could be one mechanism for driving the evolution of CNS diversity.
Cytosol-endoplasmic reticulum interplay by Sec61α translocon in polyglutamine-mediated neurotoxicity in
Drosophila
Authors: Kanuka Hirotaka , Kuranaga Erina , Hiratou Tetsuo , Igaki Tatsushi , Nelson Bryce , Okano Hideyuki , Miura Masayuki
Published: October 2003

Intracellular deposition of aggregated and ubiquitinated proteins is a prominent cytopathological feature of most neurodegenerative disorders frequently correlated with neural cell death. To elucidate mechanisms in neural cell death and degeneration, we characterized the
Drosophila
ortholog of Sec61α (DSec61α), a component of the translocon that is involved in both protein import and endoplasmic reticulum-associated degradation. Loss-of-function experiments for DSec61α revealed that the translocon contributes to expanded polyglutamine-mediated neuronal toxicity, likely resulting from proteasome inhibition and leading to accumulation of ubiquitinated proteins. Taken together, proteasome inhibition by expanded polyglutamine tracts may lead to the accumulation of toxic undegraded proteins normally transported by the Sec61α translocon.
Helix-stabilized cyclic peptides as selective inhibitors of steroid receptor–coactivator interactions
Authors: Leduc Anne-Marie , Trent John O. , Wittliff James L. , Bramlett Kelli S. , Briggs Stephen L. , Chirgadze Nikolay Y. , Wang Yong , Burris Thomas P. , Spatola Arno F.
Published: October 2003

The interaction between nuclear receptors and coactivators provides an arena for testing whether protein–protein interactions may be inhibited by small molecule drug candidates. We provide evidence that a short cyclic peptide, containing a copy of the LXXLL nuclear receptor box pentapeptide, binds tightly and selectively to estrogen receptor α. Furthermore, as shown by x-ray analysis, the disulfide-bridged nonapeptide, nonhelical in aqueous solutions, is able to adopt a quasihelical conformer while binding to the groove created by ligand attachment to estrogen receptor α. Ani, i+3 linked analog, H-Lys-cyclo(d-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln–NH2(peptidomimetic estrogen receptor modulator 1), binds with aKiof 25 nM, significantly better than ani, i+4 bridged cyclic amide, as predicted by molecular modeling design criteria. The induction of helical character, effective binding, and receptor selectivity exhibited by this peptide analog provide strong support for this strategy. The stabilization of minimalist surface motifs may prove useful for the control of other macromolecular assemblies, especially when an amphiphilic helix is crucial for the strong binding interaction between two proteins.
Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips
Authors: Zhong Xiao-bo , Reynolds Robert , Kidd Judith R. , Kidd Kenneth K. , Jenison Robert , Marlar Richard A. , Ward David C.
Published: October 2003

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30–40 min. Up to several hundred SNPs can be assayed on a 36-mm
2
chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.
The presence of p53 mutations in human osteosarcomas correlates with high levels of genomic instability
Authors: Overholtzer Michael , Rao Pulivarthi H. , Favis Reyna , Lu Xin-Yan , Elowitz Michael B. , Barany Francis , Ladanyi Marc , Gorlick Richard , Levine Arnold J.
Published: October 2003

Thep53gene is a critical tumor suppressor that is inactivated in a majority of cancers. The central role of p53 in response to stresses such as DNA damage, hypoxia, and oncogene activation underlies this high frequency of negative selection during tumorigenic transformation. Mutations inp53disrupt checkpoint responses to DNA damage and result in the potential for destabilization of the genome. Consistent with this,p53mutant cells have been shown to accumulate genomic alterations in cell culture, mouse models, and some human tumors. The relationship between p53 mutation and genomic instability in human osteosarcoma is addressed in this report. Similar to some other primary human tumors, the mutation ofp53correlates significantly with the presence of high levels of genomic instability in osteosarcomas. Surprisingly, osteosarcomas harboring an amplification of theHDM2oncogene, which inhibits the tumor-suppressive properties ofp53, do not display high levels of genomic instability. These results demonstrate that the inactivation of p53 in osteosarcomas directly by mutation versus indirectly byHDM2amplification may have different cellular consequences with respect to the stability of the genome.
Exon junction complexes mediate the enhancing effect of splicing on mRNA expression
Authors: Wiegand Heather L. , Lu Shihua , Cullen Bryan R.
Published: October 2003

Intron-containing genes are generally expressed more effectively in human cells than are intronless versions of the same gene. We have asked whether this effect is due directly to splicing or instead reflects the action of components of the exon junction complex (EJC) that is assembled at splice junctions after splicing is completed. Here, we show that intron removal does not enhance gene expression if EJC formation is blocked. Conversely, RNA tethering of the EJC components SRm160 or RNPS1 boosts the expression of intronless mRNAs but not of spliced mRNAs. Splicing and RNPS1 tethering are shown to enhance the same steps in mRNA biogenesis and function, including mRNA 3′ end processing and translation. Together, these data argue that the EJC is primarily responsible for the positive effect of splicing on gene expression.
Seasonality, density dependence, and population cycles in Hokkaido voles
Authors: Stenseth Nils Chr. , Viljugrein Hildegunn , Saitoh Takashi , Hansen Thomas F. , Kittilsen Marte O. , Bølviken Erik , Glöckner Fredrik
Published: October 2003

Voles and lemmings show extensive variation in population dynamics regulated across and within species. In an attempt to develop and test generic hypotheses explaining these differences, we studied 84 populations of the gray-sided vole (
Clethrionomys rufocanus
) in Hokkaido, Japan. We show that these populations are limited by a combination of density-independent factors (such as climate) and density-dependent processes (such as specialist predators). We show that density-dependent regulation primarily occurs in winter months, so that populations experiencing longer winters tend to have a stronger delayed density-dependence and, as a result, exhibit regular density cycles. Altogether, we demonstrate that seasonality plays a key role in determining whether a vole population is cyclic or not.
β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors
Authors: Azzi Mounia , Charest Pascale G. , Angers Stéphane , Rousseau Guy , Kohout Trudy , Bouvier Michel , Piñeyro Graciela
Published: October 2003

It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that β
2
AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1/2 thus behaving as dual efficacy ligands. ERK1/2 activation by dual efficacy ligands was not affected by ADP-ribosylation of Gαi and could be observed in S49-cyc
–
cells lacking Gαs indicating that, unlike the conventional agonist isoproterenol, these drugs induce ERK1/2 activation in a Gs/i-independent manner. In contrast, this activation was inhibited by a dominant negative mutant of β-arrestin and was abolished in mouse embryonic fibroblasts lacking β-arrestin 1 and 2. The role of β-arrestin was further confirmed by showing that transfection of β-arrestin 2 in these knockout cells restored ICI118551 promoted ERK1/2 activation. ICI118551 and propranolol also promoted β-arrestin recruitment to the receptor. Taken together, these observations suggest that β-arrestin recruitment is not an exclusive property of agonists, and that ligands classically classified as inverse agonists rely exclusively on β-arrestin for their positive signaling activity. This phenomenon is not unique to β
2
-adrenergic ligands because SR121463B, an inverse agonist on the V2 vasopressin receptor-stimulated adenylyl cyclase, recruited β-arrestin and stimulated ERK1/2. These results point to a multistate model of receptor activation in which ligand-specific conformations are capable of differentially activating distinct signaling partners.
Drosophila
screening model for metastasis: Semaphorin 5c is required for
l(2)gl
cancer phenotype
Authors: Woodhouse Elisa C. , Fisher Amy , Bandle Russell W. , Bryant-Greenwood Bianca , Charboneau Lula , Petricoin Emanuel F. , Liotta Lance A.
Published: October 2003

Cancer metastasis is a complex process involving many genes and pathways. This complexity hinders the identification of molecules functionally required for this process. We have developed and used a
Drosophila
screening system to identify genes that are functionally important for tumorigenicity and metastasis. Deletion of
Drosophila lethal giant larvae
(
l(2)gl
) leads to highly invasive and widely metastatic tumors on transplantation into adult flies. Random homozygous P element insertions were screened for the ability to modulate the
l(2)gl
phenotype. Analysis of metastasis patterns of the lines containing P element insertions and lacking wild-type
l(2)gl
expression identified three homozygous mutations that dramatically alter tumorigenesis and/or metastasis.
Semaphorin 5c
(
Sema 5c
) is required for tumorigenicity,
apontic
overexpression suppresses metastasis but not tumorigenicity, and
pointed
up-regulation accelerates lethality of
l(2)gl
tumors. Furthermore, class 5 semaphorins are shown to be expressed in cancer cells and localized to the membrane.
Drosophila
Sema-5c and the mammalian homologs are transmembrane proteins with extracellular thrombospondin type I (TspI) repeats. TspI repeats are known in some proteins to bind and activate transforming growth factor (TGF)-β ligand. Phospho-Mad and the downstream target gene
vestigial
were elevated in
l(2)gl
tumors, thus linking
Drosophila
neoplasia to the Dpp (TGF-β-like) signal pathway. The activation of the Dpp pathway in
l(2)gl
tumors occurred only in the presence of Sema-5c. This study demonstrates that the power of
Drosophila
genetics can be applied to screen, identify, and characterize molecules that are functionally required for invasion and metastasis.
Newborn infants can organize the auditory world
Authors: Winkler István , Kushnerenko Elena , Horváth János , Čeponienė Rita , Fellman Vineta , Huotilainen Minna , Näätänen Risto , Sussman Elyse
Published: October 2003

The perceptual world of neonates is usually regarded as not yet being fully organized in terms of objects in the same way as it is for adults. Using a recently developed method based on electric brain responses, we found that, similarly to adults, newborn infants segregate concurrent streams of sound, allowing them to organize the auditory input according to the existing sound source. The segregation of concurrent sound streams is a crucial step in the path leading to the identification of objects in the environment. Its presence in newborn infants shows that the basic abilities required for the development of conceptual objects are available already at the time of birth.
NMR structure of the forkhead-associated domain from the
Arabidopsis
receptor kinase-associated protein phosphatase
Authors: Lee Gui-in , Ding Zhaofeng , Walker John C. , Van Doren Steven R.
Published: October 2003

Forkhead-associated (FHA) domains are phosphoprotein-binding modules found in diverse signaling proteins that bind partners phosphorylated on threonine or serine. Kinase-associated protein phosphatase from
Arabidopsis
employs its FHA domain for negative regulation of receptor-like kinase signaling pathways, which are important in plant development. The solution structure of the free state of kinase-interacting FHA domain (KI-FHA) of kinase-associated protein phosphatase has been determined with high precision and accuracy using residual dipolar couplings. KI-FHA is a sandwich of a five-stranded mixed β-sheet with a six-stranded antiparallel β-sheet. Despite homology only in the recognition loops, this fold is shared with FHA domains from checkpoint proteins from yeast and humans, as well as with nonhomologous MH2 domains of Smad tumor suppressors. A shared pattern of hydrophobicity throughout FHA domains and Smad MH2 domains may stabilize the core of the β-sandwich. Evolutionary trace analysis of FHA domains suggests class-specific residues in the recognition loops that could tune their phosphoprotein-binding specificity. This surface agrees with that of KI-FHA in contact with a phosphothreonine peptide ligand. Evolutionary trace analysis also predicts an unexpected swath of class-specific residues on another face of FHA domains. Protein interactions with these faces may affect assembly of transmembrane signaling complexes in plants, and in other FHA domain-containing assemblies.
Blue light regulates an auxin-induced K
+
-channel gene in the maize coleoptile
Authors: Fuchs I. , Philippar K. , Ljung K. , Sandberg G. , Hedrich R.
Published: October 2003

Auxin redistribution along gravistimulated maize coleoptiles causes differential expression of the auxin-induced K
+
-channel gene
ZMK1
(
Zea mays
K
+
channel 1) and precedes the curvature response. To evaluate the role of
ZMK1
during phototropism, we here investigated blue light-stimulated coleoptiles. Four hours of blue light stimulation resulted in phototropic bending (23°). Rotation on a clinostat, at nominally “zero” gravity, and simultaneous stimulation with unidirectional blue light, however, resulted in up to 51° bending toward the light. Differential
ZMK1
transcription reached a maximum after 90 min of blue light stimulation under gravity, whereas
ZMK1
expression remained asymmetric for at least 180 min in photostimulated coleoptiles on a clinostat. We therefore conclude that the stronger phototropic bending under nominally “zero” gravity results from prolonged differential expression of
ZMK1
. Under both conditions, asymmetric expression of
ZMK1
could be superimposed on the lateral auxin gradient across the coleoptile tip, whereas the gene for the blue light receptor phototropin 1 (
PHOT1
), expressed in the tip only, was not differentially regulated in response to blue light. The activation of the two different receptors eliciting the photo- and gravitropic response of the coleoptile thus feeds into a common signaling pathway, resulting in auxin redistribution in the coleoptile tip and finally in differential transcription of
ZMK1
. In the process of signal integration, gravity transduction restricts the magnitude of the blue light-inducible
ZMK1
gradient. The spatial and temporal distribution of
ZMK1
transcripts and thus differential K
+
uptake in both flanks of the coleoptile seem to limit the stimulus-induced bending of this sensory organ.
Borrelia
oxidative stress response regulator, BosR: A distinctive Zn-dependent transcriptional activator
Authors: Boylan Julie A. , Posey James E. , Gherardini Frank C.
Published: October 2003

The ability of a pathogen to cause infection depends on successful colonization of the host, which, in turn, requires adaptation to various challenges presented by that host. For example, host immune cells use a variety of mechanisms to control infection by bacterial pathogens, including the production of bactericidal reactive oxygen species. Prokaryotic and eukaryotic cells have developed ways of protecting themselves against this oxidative damage; for instance,
Borrelia burgdorferi
alters the expression of oxidative-stress-related proteins, such as a Dps/Dpr homolog NapA (BB0690), in response to increasing levels of oxygen and reactive oxygen species. These stress-related genes appear to be regulated by a putative metal-dependent DNA-binding protein (BB0647) that has 50.7% similarity to the peroxide-specific stress response repressor of
Bacillus subtilis
, PerR. We overexpressed and purified this protein from
Escherichia coli
and designated it

B
orrelia

o
xidative
s
tress
r
egulator, BosR. BosR bound to a 50-nt region 180 bp upstream of the
napA
transcriptional start site and required DTT and Zn
2+
for optimal binding. Unlike the
Bacillus subtilis
PerR repressor, BosR did not require Fe
2+
and Mn
2+
for binding, and oxidizing agents, such as
t
-butyl peroxide, enhanced, not eliminated, BosR binding to the
napA
promoter region. Surprisingly, transcriptional fusion analysis indicated that BosR exerted a positive regulatory effect on
napA
that is inducible with
t
-butyl peroxide. On the basis of these data, we propose that, despite the similarity to PerR, BosR functions primarily as a transcriptional activator, not a repressor of oxidative stress response, in
B. burgdorferi
.
Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts using DNA microarray analysis
Authors: Shepard K. A. , Gerber A. P. , Jambhekar A. , Takizawa P. A. , Brown P. O. , Herschlag D. , DeRisi J. L. , Vale R. D.
Published: October 2003

Cytoplasmic mRNA localization provides a means of generating cell asymmetry and segregating protein activity. Previous studies have identified two mRNAs that localize to the bud tips of the yeast
Saccharomyces cerevisiae
. To identify additional localized mRNAs, we immunoprecipitated the RNA transport components She2p, She3p, and Myo4p and performed DNA microarray analysis of their associated RNAs. A secondary screen, using a GFP-tagged RNA reporter assay, identified 22 mRNAs that are localized to bud tips. These messages encode a wide variety of proteins, including several involved in stress responses and cell wall maintenance. Many of these proteins are asymmetrically localized to buds. However, asymmetric localization also occurs in the absence of RNA transport, suggesting the existence of redundant protein localization mechanisms. In contrast to findings in metazoans, the untranslated regions are dispensable for mRNA localization in yeast. This study reveals an unanticipated widespread use of RNA transport in budding yeast.
Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins
Authors: Giddings Kara S. , Johnson Arthur E. , Tweten Rodney K.
Published: October 2003

The cholesterol-dependent cytolysins (CDCs) constitute a large family of pore-forming toxins that function exclusively on cholesterol-containing membranes. A detailed analysis of the various stages in the cytolytic mechanism of three members of the CDC family revealed that significant depletion of cholesterol from the erythrocyte membrane stalls these toxins in the prepore complex. Therefore, the depletion of membrane cholesterol prevents the insertion of the transmembrane β-barrel and pore formation. These unprecedented findings provide a paradigm for the involvement of cholesterol in the CDC cytolytic mechanism and that of other pore-forming toxins whose activity is enhanced by the presence of membrane cholesterol.
Measurements of nitric oxide on the heme iron and β-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation
Authors: Xu Xiuli , Cho Man , Spencer Netanya Y. , Patel Neil , Huang Zhi , Shields Howard , King S. Bruce , Gladwin Mark T. , Hogg Neil , Kim-Shapiro Daniel B.
Published: October 2003

Nitric oxide has been proposed to be transported by hemoglobin as a third respiratory gas and to elicit vasodilation by an oxygen-linked (allosteric) mechanism. For hemoglobin to transport nitric oxide bioactivity it must capture nitric oxide as iron nitrosyl hemoglobin rather than destroy it by dioxygenation. Once bound to the heme iron, nitric oxide has been reported to migrate reversibly from the heme group of hemoglobin to the β-93 cysteinyl residue, in response to an oxygen saturation-dependent conformational change, to form an
S
-nitrosothiol. However, such a transfer requires redox chemistry with oxidation of the nitric oxide or β-93 cysteinyl residue. In this article, we examine the ability of nitric oxide to undergo this intramolecular transfer by cycling human hemoglobin between oxygenated and deoxygenated states. Under various conditions, we found no evidence for intramolecular transfer of nitric oxide from either cysteine to heme or heme to cysteine. In addition, we observed that contaminating nitrite can lead to formation of iron nitrosyl hemoglobin in deoxygenated hemoglobin preparations and a radical in oxygenated hemoglobin preparations. Using
15
N-labeled nitrite, we clearly demonstrate that nitrite chemistry could explain previously reported results that suggested apparent nitric oxide cycling from heme to thiol. Consistent with our results from these experiments conducted
in vitro
, we found no arterial/venous gradient of iron nitrosyl hemoglobin detectable by electron paramagnetic resonance spectroscopy. Our results do not support a role for allosterically controlled intramolecular transfer of nitric oxide in hemoglobin as a function of oxygen saturation.
HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in
Arabidopsis
Authors: Takahashi Akira , Casais Catarina , Ichimura Kazuya , Shirasu Ken
Published: October 2003

RAR1 and its interacting partner SGT1 play a central role in plant disease resistance triggered by a number of resistance (R) proteins. We identified cytosolic heat shock protein 90 (HSP90), a molecular chaperone, as another RAR1 interacting protein by yeast two-hybrid screening. RAR1 interacts with the N-terminal half of HSP90 that contains the ATPase domain. HSP90 also specifically interacts with SGT1 that contains a tetratricopeptide repeat motif and a domain with similarity to the cochaperone p23. In
Arabidopsis
, the HSP90 inhibitor geldanamycin reduces the hypersensitive response and abolishes resistance triggered by the R protein RPS2 against
Pseudomonas syringae pv. tomato
DC3000 (
avrRpt2
). One of four
Arabidopsis
cytosolic HSP90 isoforms, AtHSP90.1 is required for full RPS2 resistance and is rapidly induced upon pathogen challenge. We propose that RAR1 and SGT1 function closely with HSP90 in chaperoning roles that are essential for disease resistance.
Functional characterization of a portion of the Moloney murine leukemia virus
gag
gene by genetic footprinting
Authors: Auerbach Marcy R. , Shu Chang , Kaplan Artem , Singh Ila R.
Published: October 2003

Retroviral Gag proteins perform important functions in viral assembly, but are also involved in other steps in the viral life cycle. Conventional mutational analysis has yielded considerable information about domains essential for these functions, yet many regions of
gag
remain uncharacterized. We used genetic footprinting, a technique that permits the generation and simultaneous analysis of large numbers of mutations, to perform a near-saturation mutagenesis and functional analysis of 639 nucleotides in the
gag
region of Moloney murine leukemia virus. We report here the resulting functional map defined by eight footprints representing regions of Moloney murine leukemia virus
gag
, some previously uncharacterized, that are essential for replication. We found that significant portions of matrix and p12 proteins were tolerant of insertions, in contrast to the N-terminal half of capsid, which was not. We analyzed 30 mutants from our library by using conventional methods to validate the footprints. Six of these mutants were characterized in detail, identifying the precise stage at which their replication is blocked. In addition to providing the most comprehensive functional map of a retroviral
gag
gene, our study demonstrates the abundance of information that can be gleaned by genetic footprinting of viral sequences.
Observations of a “weekend effect” in diurnal temperature range
Authors: Forster Piers M. de F. , Solomon Susan
Published: October 2003

Using surface measurements of maximum and minimum temperatures from the Global Daily Climatological Network data set, we find evidence of a weekly cycle in diurnal temperature range (DTR) for many stations in the United States, Mexico, Japan, and China. The “weekend effect,” which we define as the average DTR for Saturday through Monday minus the average DTR for Wednesday through Friday, can be as large as 0.5 K, similar to the magnitude of observed long-term trends in DTR. This weekend effect has a distinct large-scale pattern that has changed only slightly over time, but its sign is not the same in all locations. The station procedures and the statistical robustness of both the individual station data and the patterns of DTR differences are thoroughly examined. We conclude that the weekend effect is a real short time scale and large spatial scale geophysical phenomenon, which is necessarily human in origin. We thus provide strong evidence of an anthropogenic link to DTR, an important climate indicator. Several possible anthropogenic mechanisms are discussed; we speculate that aerosol-cloud interactions are the most likely cause of this weekend effect, but we do not rule out others.
Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping
Authors: Lia Giuseppe , Bensimon David , Croquette Vincent , Allemand Jean-Francois , Dunlap David , Lewis Dale E. A. , Adhya Sankar , Finzi Laura
Published: October 2003

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the
gal
operon of
Escherichia coli
by forming a DNA loop of ≈40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for
gal
DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for
gal
repression observed both
in vitro
and
in vivo
. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.