bluuebunny/crossref_metadata_2025 huggingface dataset:
SecA-dependent quality control of intracellular protein localization
Authors: Eser Markus , Ehrmann Michael
Published: November 2003

Complex secretion machineries mediate protein translocation across cellular membranes. These machines typically recognize their substrates via signal sequences, which are required for proper targeting to the translocon. We report that during posttranslational secretion the widely conserved targeting factor SecA performs a quality-control function that is based on a general chaperone activity. This quality-control mechanism involves assisted folding of signal sequenceless proteins, thereby excluding them from the secretion process. These results suggest that SecA channels proteins into one of two key pathways, posttranslational secretion or folding in the cytoplasm. Implications of this finding for intracellular protein localization are discussed.
Posttranscriptional control of the competence factor βFTZ-F1 by juvenile hormone in the mosquito
Aedes aegypti
Authors: Zhu Jinsong , Chen Li , Raikhel Alexander S.
Published: November 2003

In anautogenous mosquitoes, vitellogenesis, which includes production of yolk protein precursors, requires blood feeding. Consequently, mosquitoes transmit many diseases. Understanding the molecular mechanisms of vitellogenesis regulation will contribute significantly to vector control strategies. Newly emerged
Aedes aegypti
females require 3 days before becoming competent to activate vitellogenesis in response to a blood-meal-initiated, elevated titer of 20-hydroxyecdysone (20E). An orphan nuclear receptor gene β
FTZ-F1
is transcribed in the fat body of newly emerged mosquito females; however, the βFTZ-F1 protein is only found 3 days later. Dramatically increased titer of the juvenile hormone III (JH III) is essential for the acquisition of 20E competence.
In vitro
fat body culture experiments have shown that βFTZ-F1 protein appears after exposure to JH III. Injection of double-stranded RNA complementary to β
FTZ-F1
into newly emerged females attenuated expression of the early genes
EcR-B, E74B
, and
E75A
and the target
YPP
gene
Vg
, in response to a blood meal. Thus, βFTZ-F1 is indeed the factor defining the acquisition of competence to 20E in the mosquito fat body. Moreover, this is achieved through JH III-mediated posttranscriptional control of β
FTZ-F1
.
Isorhodopsin rather than rhodopsin mediates rod function in RPE65 knock-out mice
Authors: Fan Jie , Rohrer Baerbel , Moiseyev Gennadiy , Ma Jian-xing , Crouch Rosalie K.
Published: November 2003

The chromophore of visual pigments is 11-
cis
-retinal and, thus, in its absence, opsin is not photosensitive and no visual function exists. However, in the RPE65 knockout (
Rpe65
-/-
) mouse, where synthesis of 11-
cis
-retinal does not occur, a minimal visual response from rod photoreceptors is obtained. We have examined if an alternative pathway exists for
cis
-retinoid generation in the absence of RPE65. Cyclic-light-reared, 2-month-old
Rpe65
-/-
mice were placed in complete darkness. No exogenous retinoids were administered. After 4 weeks, enhanced a- and b-wave amplitudes were obtained, increasing >10-fold for the a-wave and >3-fold for the b-wave as compared with cyclic-light-reared
Rpe65
-/-
mice. Visual-pigment levels increased to ≈10 pmol per retina, compared with no measurable pigment for cyclic-light-reared
Rpe65
-/-
mice. The λ
max
of the isolated pigment was 487 nm, characteristic for isorhodopsin. Retinoid extractions confirmed the presence of 9-
cis
-retinal and the absence of 11-
cis
-retinal. Once the
Rpe65
-/-
mice were returned to cyclic light, within 48 h the electroretinogram function returned to levels found in
Rpe65
-/-
mice maintained in cyclic light. This dark-mediated pathway is also operational in older animals, because 13-month-old
Rpe65
-/-
mice kept in prolonged darkness (12 weeks) had increased isorhodopsin levels and electroretinogram a- and b-wave amplitudes. These studies demonstrate that a pathway exists in the eye for the generation of 9-
cis
-retinal that is independent of RPE65 and light.
A panoramic view of gene expression in the human kidney
Authors: Chabardès-Garonne Danielle , Méjean Arnaud , Aude Jean-Christophe , Cheval Lydie , Di Stefano Antonio , Gaillard Marie-Claude , Imbert-Teboul Martine , Wittner Monika , Balian Chanth , Anthouard Véronique , Robert Catherine , Ségurens Béatrice , Wincker Patrick , Weissenbach Jean , Doucet Alain , Elalouf Jean-Marc
Published: November 2003

To gain a molecular understanding of kidney functions, we established a high-resolution map of gene expression patterns in the human kidney. The glomerulus and seven different nephron segments were isolated by microdissection from fresh tissue specimens, and their transcriptome was characterized by using the serial analysis of gene expression (SAGE) method. More than 400,000 mRNA SAGE tags were sequenced, making it possible to detect in each structure transcripts present at 18 copies per cell with a 95% confidence level. Expression of genes responsible for nephron transport and permeability properties was evidenced through transcripts for 119 solute carriers, 84 channels, 43 ion-transport ATPases, and 12 claudins. Searching for differences between the transcriptomes, we found 998 transcripts greatly varying in abundance from one nephron portion to another. Clustering analysis of these transcripts evidenced different extents of similarity between the nephron portions. Approximately 75% of the differentially distributed transcripts corresponded to cDNAs of known or unknown function that are accurately mapped in the human genome. This systematic large-scale analysis of individual structures of a complex human tissue reveals sets of genes underlying the function of well-defined nephron portions. It also provides quantitative expression data for a variety of genes mutated in hereditary diseases and helps in sorting candidate genes for renal diseases that affect specific portions of the human nephron.
PA-457: A potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing
Authors: Li F. , Goila-Gaur R. , Salzwedel K. , Kilgore N. R. , Reddick M. , Matallana C. , Castillo A. , Zoumplis D. , Martin D. E. , Orenstein J. M. , Allaway G. P. , Freed E. O. , Wild C. T.
Published: November 2003

New HIV therapies are urgently needed to address the growing problem of drug resistance. In this article, we characterize the anti-HIV drug candidate 3-
O
-(3′,3′-dimethylsuccinyl) betulinic acid (PA-457). We show that PA-457 potently inhibits replication of both WT and drug-resistant HIV-1 isolates and demonstrate that the compound acts by disrupting a late step in Gag processing involving conversion of the capsid precursor (p25) to mature capsid protein (p24). We find that virions from PA-457-treated cultures are noninfectious and exhibit an aberrant particle morphology characterized by a spherical, acentric core and a crescent-shaped, electron-dense shell lying just inside the viral membrane. To identify the determinants of compound activity we selected for PA-457-resistant virus
in vitro
. Consistent with the effect on Gag processing, we found that mutations conferring resistance to PA-457 map to the p25 to p24 cleavage site. PA-457 represents a unique class of anti-HIV compounds termed maturation inhibitors that exploit a previously unidentified viral target, providing additional opportunities for HIV drug discovery.
Multipoint linkage-disequilibrium mapping narrows location interval and identifies mutation heterogeneity
Authors: Morris Andrew P. , Whittaker John C. , Xu Chun-Fang , Hosking Louise K. , Balding David J.
Published: November 2003

Single-nucleotide polymorphism (SNP) genotypes were recently examined in an 890-kb region flanking the human gene
CYP2D6
. Single-marker and haplotype-based analyses identified, with genomewide significance (
P
< 10
-7
), a 403-kb interval displaying strong linkage disequilibrium (LD) with predicted poor-metabolizer phenotype. However, the width of this interval makes the location of causal variants difficult: for example, the interval contains seven known or predicted genes in addition to
CYP2D6
. We have developed the Bayesian fine-mapping software
coldmap
, which, applied to these genotype data, yields a 95% location interval covering only 185 kb and establishes genomewide significance for a causal locus within the region. Strikingly, our interval correctly excludes four SNPs, which individually display association with genomewide significance, including the SNP showing strongest LD (
P
< 10
-34
). In addition,
coldmap
distinguishes homozygous cases for the major
CYP2D6
mutation from those bearing minor mutations. We further investigate a selection of SNP subsets and find that previously reported methods lead to a 38% savings in SNPs at the cost of an increase of <20% in the width of the location interval.
Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in
Bacillus subtilis
Authors: Nakano Shunji , Küster-Schöck Elke , Grossman Alan D. , Zuber Peter
Published: November 2003

The Spx protein of
Bacillus subtilis
represses activator-stimulated transcription by interacting with the C-terminal domain of RNA polymerase (RNAP) α subunit. Its concentration increases in cells lacking the ATP-dependent protease, ClpXP, resulting in severe effects on growth and developmental processes. Microarray analysis was undertaken to identify genes that are induced or repressed when Spx interacts with RNAP. The induced genes included those encoding products known to function in maintaining thiol homeostasis. Two genes, thioredoxin (
trxA
) and thioredoxin reductase (
trxB
), are transcriptionally induced under conditions of thiol-specific oxidative (disulfide) stress by a mechanism involving Spx-RNAP interaction. Disulfide stress also results in an increase in Spx-dependent transcriptional repression. The increase in Spx activity in cells encountering disulfide stress is due in part to a posttranscriptional mechanism of
spx
control resulting in an increase in Spx concentration. An
spx
null mutant and a strain bearing an allele of
rpoA
that prevents Spx-RNAP interaction show hypersensitivity to disulfide stress. From these results, it is proposed that Spx is an activator that mobilizes the operations necessary to reverse the effects of oxidative damage, but it also serves as a negative regulator that causes the postponement of developmental programs and energy-consuming growth-related functions while the cell copes with the period of stress.
Conscious and subliminal conflicts in normal subjects and patients with schizophrenia: The role of the anterior cingulate
Authors: Dehaene Stanislas , Artiges Eric , Naccache Lionel , Martelli Catherine , Viard Armelle , Schürhoff Franck , Recasens Christophe , Martinot Marie Laure Paillère , Leboyer Marion , Martinot Jean-Luc
Published: November 2003

The human anterior cingulate cortex (ACC), which is active during conflict-monitoring tasks, is thought to participate with prefrontal cortices in a distributed network for conscious self-regulation. This hypothesis predicts that conflict-related ACC activation should occur only when the conflicting stimuli are consciously perceived. To dissociate conflict from consciousness, we measured the behavioral and brain imaging correlates of a motor conflict induced by task-irrelevant subliminal or conscious primes. The same task was studied in normal subjects and in patients with schizophrenia in whom the ACC and prefrontal cortex are thought to be dysfunctional. Conscious, but not subliminal, conflict affected anterior cingulate activity in normal subjects. Furthermore, patients with schizophrenia, who exhibited a hypoactivation of the ACC and other frontal, temporal, hippocampal, and striatal sites, showed impaired conscious priming but normal subliminal priming. Those findings suggest that subliminal conflicts are resolved without ACC contribution and that the ACC participates in a distributed conscious control network that is altered in schizophrenia.
An experimental platform for studying growth and invasiveness of tumor cells within teratomas derived from human embryonic stem cells
Authors: Tzukerman Maty , Rosenberg Tzur , Ravel Yael , Reiter Irena , Coleman Raymond , Skorecki Karl
Published: November 2003

There is currently no available experimental system wherein human cancer cells can be grown in the context of a mixed population of normal differentiated human cells for testing biological aspects of cancer cell growth (e.g., tumor cell invasion and angiogenesis) or response to anti-cancer therapies. When implanted into immunocompromised mice, human embryonic stem cells develop teratomas containing complex structures comprising differentiated cell types representing the major germ line-derived lineages. We sought to determine whether human cancer cells would grow within such teratomas and display properties associated with malignancy, such as invasiveness and recruitment of blood vessels. HEY ovarian cancer cells stably expressing an H2A-GFP fusion protein (HEY-GFP) injected into mature teratomas developed into tumors, which allowed tracking of tumor cell invasion and recruitment of human teratoma-derived blood vessels. This provides a straightforward and powerful approach to studying the biological properties of cancer cells within the microenvironment of normal differentiated human cells.
Autoimmune cardiomyopathy and heart block develop spontaneously in HLA-DQ8 transgenic IAβ knockout NOD mice
Authors: Elliott John F. , Liu Junliang , Yuan Zuan-Ning , Bautista-Lopez Norma , Wallbank Sarah L. , Suzuki Kunimasa , Rayner David , Nation Patrick , Robertson Murray A. , Liu Gang , Kavanagh Katherine M.
Published: November 2003

A line of nonobese diabetic (NOD) mice expressing the human diabetes-associated HLA-DQ8 transgene in the absence of mouse IAβfailed to show spontaneous insulitis or diabetes, but rather developed dilated cardiomyopathy, leading to early death from heart failure. Pathology in these animals results from an organ- and cell-specific autoimmune response against normal cardiomyoctes in the atrial and ventricular walls, as well as against very similar myocytes present in the outermost muscle layer surrounding the pulmonary veins. Progression of the autoimmune process could be followed by serial ECG measurements; irradiation of young animals significantly delayed disease progression, and this effect could be reversed by adoptive transfer of splenocytes taken from older animals with complete heart block. Disease progression could also be blocked by cyclosporin A treatment, but was accelerated by injection of complete Fruend's adjuvant. The constellation of findings of spontaneously arising destructive focal lymphocytic infiltrates within the myocardium, rising titers of circulating anticardiac autoantibodies, dilation of the cardiac chambers, and gradual progression to end-stage heart failure bears a striking resemblance to what is seen in humans with idiopathic dilated cardiomyopathy, a serious and often life-threatening medical condition. This transgenic strain provides a highly relevant animal model for human autoimmune myocarditis and postinflammatory dilated cardiomyopathy.
A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs
Authors: Li Yong , Altman Sidney
Published: November 2003

The
rnpA
mutation, A49, in
Escherichia coli
reduces the level of RNase P at 43°C because of a temperature-sensitive mutation in C5 protein, the protein subunit of the enzyme. Microarray analysis reveals the expression of several noncoding intergenic regions that are increased at 43°C compared with 30°C. These regions are substrates for RNase P, and they are cleaved less efficiently than, for example, tRNA precursors. An analysis of the
tna, secG, rbs
, and
his
operons, all of which contain RNase P cleavage sites, indicates that RNase P affects gene expression for regions downstream of its cleavage sites.
NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells
Authors: Schneider Benoît , Mutel Vincent , Pietri Mathéa , Ermonval Myriam , Mouillet-Richard Sophie , Kellermann Odile
Published: November 2003

Putative functions of the cellular prion protein, PrP
C
, include resistance to oxidative stress, copper uptake, cell adhesion, and cell signaling. Here, we report NADPH oxidase-dependent reactive oxygen species (ROS) production and extracellular regulated kinases 1/2 (ERK1/2) phosphorylation on PrP
C
stimulation in the 1C11 neuroectodermal precursor, in its neuronal differentiated progenies, and in GT1-7 neurohypothalamic and BW5147 lymphoid cells. In neuroprogenitor, hypothalamic, and lymphoid cells, ERK1/2 activation is fully controlled by the NADPH oxidase-dependent ROS production. In 1C11-derived bioaminergic cells, ROS signaling and ERK1/2 phosphorylation are both controlled by Fyn kinase activation, introducing some specificity in PrP
C
transduction associated with this neuronal context. These data argue for an ubiquitous function of PrP
C
in cell-redox homeostasis through ROS production.
RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito
Aedes aegypti
Authors: Attardo Geoffrey M. , Higgs Stephen , Klingler Kimberley A. , Vanlandingham Dana L. , Raikhel Alexander S.
Published: November 2003

Blood feeding tightly regulates the reproductive cycle in anautogenous mosquitoes. Vitellogenesis (the synthesis of yolk protein precursors) is a key event in the mosquito reproductive cycle and is activated in response to a blood meal. Before blood feeding,
Aedes aegypti
is in a state of reproductive arrest during which the yolk protein precursor genes (
YPP
s) are repressed. The regulatory region of the major
YPP
gene
vitellogenin
(
Vg
) has multiple GATA-binding sites required for the high expression level of this gene. However, a GATA factor (
AaGATAr
) likely acts as a repressor, preventing activation of this gene before a blood meal. Here we report
in vivo
data confirming the role of
AaGATAr
as a repressor of the
Vg
gene at the state of previtellogenic arrest. Using an RNA interference (RNAi)-mediated technique in conjunction with the Sindbis viral expression system, we show that knockdown of the
AaGATAr
gene results in an increased basal level of expression of the
Vg
gene and an elevated response to the steroid hormone 20-hydroxyecdysone in mosquitoes in a state of arrest. These experiments have revealed a component in the molecular mechanism by which anautogeny is maintained in
A. aegypti
.
Calcium-dependent molecular spring elements in the giant protein titin
Authors: Labeit Dietmar , Watanabe Kaori , Witt Christian , Fujita Hideaki , Wu Yiming , Lahmers Sunshine , Funck Theodor , Labeit Siegfried , Granzier Henk
Published: November 2003

Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: ≈28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calcium-dependent molecular spring that adapts to the physiological state of the cell.
A comparative study of motor-protein motions by using a simple elastic-network model
Authors: Zheng Wenjun , Doniach Sebastian
Published: November 2003

In this work, we report on a study of the structure-function relationships for three families of motor proteins, including kinesins, myosins, and F1-ATPases, by using a version of the simple elastic-network model of large-scale protein motions originally proposed by Tirion [Tirion, M. (1996)
Phys. Rev. Lett.
77, 1905–1908]. We find a surprising dichotomy between kinesins and the other motor proteins (myosins and F1-ATPase). For the latter, there exist one or two dominant lowest-frequency modes (one for myosin, two for F1-ATPase) obtained from normal-mode analysis of the elastic-network model, which overlap remarkably well with the measured conformational changes derived from pairs of solved crystal structures in different states. Furthermore, we find that the computed global conformational changes induced by the measured deformation of the nucleotide-binding pocket also overlap well with the measured conformational changes, which is consistent with the “nucleotide-binding-induced power-stroke” scenario. In contrast, for kinesins, this simplicity breaks down. Multiple modes are needed to generate the measured conformational changes, and the computed displacements induced by deforming the nucleotide-binding pocket also overlap poorly with the measured conformational changes, and are insufficient to explain the large-scale motion of the relay helix and the linker region. This finding may suggest the presence of two different mechanisms for myosins and kinesins, despite their strong evolutionary ties and structural similarities.
Loss-of-function mutations in the human
GLI2
gene are associated with pituitary anomalies and holoprosencephaly-like features
Authors: Roessler Erich , Du Yang-Zhu , Mullor Jose L. , Casas Esther , Allen William P. , Gillessen-Kaesbach Gabriele , Roeder Elizabeth R. , Ming Jeffrey E. , Altaba Ariel Ruiz i , Muenke Maximilian
Published: November 2003

Diminished Sonic Hedgehog (Shh) signaling is associated with the most common forebrain defect in humans, holoprosencephaly (HPE), which includes cyclopia, a phenotype also seen in mice and other vertebrates with defective Shh signaling. The secreted protein Shh acts as a crucial factor that patterns the ventral forebrain and is required for the division of the primordial eye field and brain into two discrete halves. Gli2 is one of three vertebrate transcription factors implicated as obligatory mediators of Shh signal transduction. Here, we show that loss-of-function mutations in the human
GLI2
gene are associated with a distinctive phenotype (within the HPE spectrum) whose primary features include defective anterior pituitary formation and pan-hypopituitarism, with or without overt forebrain cleavage abnormalities, and HPE-like midfacial hypoplasia. We also demonstrate that these mutations lack GLI2 activity. We report on a functional association between
GLI2
and human disease and highlight the role of GLI2 in human head development.
Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors
Authors: Sperger Jamie M. , Chen Xin , Draper Jonathan S. , Antosiewicz Jessica E. , Chon Chris H. , Jones Sunita B. , Brooks James D. , Andrews Peter W. , Brown Patrick O. , Thomson James A.
Published: November 2003

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.
Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease
Authors: Neher Saskia B. , Sauer Robert T. , Baker Tania A.
Published: November 2003

The
Escherichia coli
UmuD′ protein is a component of DNA polymerase V, an error-prone polymerase that carries out translesion synthesis on damaged DNA templates. The intracellular concentration of UmuD′ is strictly controlled by regulated transcription, by posttranslational processing of UmuD to UmuD′, and by ClpXP degradation. UmuD′ is a substrate for the ClpXP protease but must form a heterodimer with its unabbreviated precursor, UmuD, for efficient degradation to occur. Here, we show that UmuD functions as a UmuD′ delivery protein for ClpXP. UmuD can also deliver a UmuD partner for degradation. UmuD resembles SspB, a well-characterized substrate-delivery protein for ClpX, in that both proteins use related peptide motifs to bind to the N-terminal domain of ClpX, thereby tethering substrate complexes to ClpXP. The combined use of a weak substrate recognition signal and a delivery factor that tethers the substrate to the protease allows regulated proteolysis of UmuD/D′ in the cell. Dual recognition strategies of this type may be a relatively common feature of intracellular protein turnover.
Revealing competitive Förster-type resonance energy-transfer pathways in single bichromophoric molecules
Authors: Hofkens Johan , Cotlet Mircea , Vosch Tom , Tinnefeld Philip , Weston Kenneth D. , Ego Christophe , Grimsdale Andrew , Müllen Klaus , Beljonne David , Brédas Jean Luc , Jordens Sven , Schweitzer Gerd , Sauer Markus , De Schryver Frans
Published: November 2003

We demonstrate measurements of the efficiency of competing Förster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.
The T cell receptor β enhancer promotes access and pairing of Dβ and Jβ gene segments during V(D)J recombination
Authors: Ryu Chun Jeih , Haines Brian B. , Draganov Dobrin D. , Kang Yun Hee , Whitehurst Charles E. , Schmidt Tara , Hong Hyo Jeong , Chen Jianzhu
Published: November 2003

The precise function of cis elements in regulating V(D)J recombination is still controversial. Here, we determined the effect of inactivation of the TCRβ enhancer (Eβ) on cleavage and rearrangement of Dβ1, Dβ2, Jβ1, and Jβ2 gene segments in CD4-CD8-[double-negative (DN)] and CD4+CD8+[double-positive (DP)] thymocytes. In Eβ-deficient mice, (i) Dβ1 rearrangements were more severely impaired than Dβ2 rearrangements; (ii) most of the Dβ and Jβ cleavages and rearrangements occurred in DP, rather than in DN, thymocytes; and (iii) most of the 3′ Dβ1 cleavages were coupled to 5′ Dβ2 cleavages instead of to Jβ cleavages, resulting in nonstandard Dβ1-Dβ2-Jβ2 joints. These findings suggest that the Eβ regulates TCRβ rearrangement by promoting accessibility of Dβ and Jβ gene segments in DN thymocytes and proper pairing between Dβ1 and Jβ gene segments for cleavage and joining in DP thymocytes.
Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam
Authors: Cox Paul Alan , Banack Sandra Anne , Murch Susan J.
Published: November 2003

We here report biomagnification (the increasing accumulation of bioactive, often deleterious molecules through higher trophic levels of a food chain) of the neurotoxic nonprotein amino acid β-methylamino-
l
-alanine (BMAA) in the Guam ecosystem. Free-living cyanobacteria produce 0.3 μg/g BMAA, but produce 2-37 μg/g as symbionts in the coralloid roots of cycad trees. BMAA is concentrated in the developing reproductive tissues of the cycad
Cycas micronesica
, averaging 9 μg/g in the fleshy seed sarcotesta and a mean of 1,161 μg/g BMAA in the outermost seed layer. Flying foxes (
Pteropus mariannus
), which forage on the seeds, accumulate a mean of 3,556 μg/g BMAA. Flying foxes are a prized food item of the indigenous Chamorro people who boil them in coconut cream and eat them whole. Chamorros who die of amyotrophic lateral sclerosis/parkinsonism-dementia complex (AL-SPDC), a neurodegenerative disease with symptoms similar to amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease, have an average of 6.6 μg/g BMAA in their brain tissues. The biomagnification of BMAA through the Guam ecosystem fits a classic triangle of increasing concentrations of toxic compounds up the food chain. This may explain why the incidence of ALS-PDC among the Chamorro was 50-100 times the incidence of amyotrophic lateral sclerosis elsewhere. Biomagnification of cyanobacterial BMAA may not be unique to Guam; our discovery of BMAA in the brain tissue from Alzheimer's patients from Canada suggests alternative ecological pathways for the bioaccumulation of BMAA in aquatic or terrestrial ecosystems.
Interaction of sensory responses with spontaneous depolarization in layer 2/3 barrel cortex
Authors: Petersen Carl C. H. , Hahn Thomas T. G. , Mehta Mayank , Grinvald Amiram , Sakmann Bert
Published: November 2003

The rodent primary somatosensory cortex is spontaneously active in the form of locally synchronous membrane depolarizations (UP states) separated by quiescent hyperpolarized periods (DOWN states) both under anesthesia and during quiet wakefulness.
In vivo
whole-cell recordings and tetrode unit recordings were combined with voltage-sensitive dye imaging to analyze the relationship of the activity of individual pyramidal neurons in layer 2/3 to the ensemble spatiotemporal dynamics of the spontaneous depolarizations. These were either brief and localized to an area of a barrel column or occurred as propagating waves dependent on local glutamatergic synaptic transmission in layer 2/3. Spontaneous activity inhibited the sensory responses evoked by whisker deflection, accounting almost entirely for the large trial-to-trial variability of sensory-evoked postsynaptic potentials and action potentials. Subthreshold sensory synaptic responses evoked while a cortical area was spontaneously depolarized were smaller, briefer and spatially more confined. Surprisingly, whisker deflections evoked fewer action potentials during the spontaneous depolarizations despite neurons being closer to threshold. The ongoing spontaneous activity thus regulates the amplitude and the time-dependent spread of the sensory response in layer 2/3 barrel cortex.
Saccharomyces cerevisiae
phenotypes can be predicted by using constraint-based analysis of a genome-scale reconstructed metabolic network
Authors: Famili Iman , Förster Jochen , Nielsen Jens , Palsson Bernhard O.
Published: November 2003

Full genome sequences of prokaryotic organisms have led to reconstruction of genome-scale metabolic networks and
in silico
computation of their integrated functions. The first genome-scale metabolic reconstruction for a eukaryotic cell,
Saccharomyces cerevisiae
, consisting of 1,175 metabolic reactions and 733 metabolites, has appeared. A constraint-based
in silico
analysis procedure was used to compute properties of the
S. cerevisiae
metabolic network. The computed number of ATP molecules produced per pair of electrons donated to the electron transport system (ETS) and energy-maintenance requirements were quantitatively in agreement with experimental results. Computed whole-cell functions of growth and metabolic by-product secretion in aerobic and anaerobic culture were consistent with experimental data, and thus mRNA expression profiles during metabolic shifts were computed. The computed consequences of gene knockouts on growth phenotypes were consistent with experimental observations. Thus, constraint-based analysis of a genome-scale metabolic network for the eukaryotic
S. cerevisiae
allows for computation of its integrated functions, producing
in silico
results that were consistent with observed phenotypic functions for ≈70–80% of the conditions considered.
Aquaporin-4 square array assembly: Opposing actions of M1 and M23 isoforms
Authors: Furman C. Sue , Gorelick-Feldman Daniel A. , Davidson Kimberly G. V. , Yasumura Thomas , Neely John D. , Agre Peter , Rash John E.
Published: November 2003

Osmotic homeostasis in the brain involves movement of water through aquaporin-4 (AQP4) membrane channels. Perivascular astrocyte end-feet contain distinctive orthogonal lattices (square arrays) assembled from 4- to 6-nm intramembrane particles (IMPs) corresponding to individual AQP4 tetramers. Two isoforms of AQP4 result from translation initiation at methionine residues M1 and M23, but no functional differences are known. In this study, Chinese hamster ovary cells were transfected with M1, M23, or M1+M23 isoforms, and AQP4 expression was confirmed by immunoblotting, immunocytochemistry, and immunogold labeling. Square array organization was examined by freeze-fracture electron microscopy. In astrocyte end-feet, >90% of 4- to 6-nm IMPs were found in square arrays, with 65% in arrays of 13-30 IMPs. In cells transfected with M23, 95% of 4- to 6-nm IMPs were in large assemblies (rafts), 85% of which contained >100 IMPs. However, in M1 cells, >95% of 4- to 6-nm IMPs were present as singlets, with <5% in incipient arrays of 2-12 IMPs. In M1+M23 cells, 4- to 6-nm IMPs were in arrays of intermediate sizes, resembling square arrays in astrocytes. Structural cross-bridges of 1 × 2 nm linked >90% of IMPs in M23 arrays (≈1,000 cross-bridges per μm
2
) but were rarely seen in M1 cells. These studies show that M23 and M1 isoforms have opposing effects on intramembrane organization of AQP4: M23 forms large square arrays with abundant cross-bridges; M1 restricts square array assembly.
A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors
Authors: Rubin Joshua B. , Kung Andrew L. , Klein Robyn S. , Chan Jennifer A. , Sun YanPing , Schmidt Karl , Kieran Mark W. , Luster Andrew D. , Segal Rosalind A.
Published: November 2003

The vast majority of brain tumors in adults exhibit glial characteristics. Brain tumors in children are diverse: Many have neuronal characteristics, whereas others have glial features. Here we show that activation of the Giprotein-coupled receptor CXCR4 is critical for the growth of both malignant neuronal and glial tumors. Systemic administration of CXCR4 antagonist AMD 3100 inhibits growth of intracranial glioblastoma and medulloblastoma xenografts by increasing apoptosis and decreasing the proliferation of tumor cells. This reflects the ability of AMD 3100 to reduce the activation of extracellular signal-regulated kinases 1 and 2 and Akt, all of which are pathways downstream of CXCR4 that promote survival, proliferation, and migration. These studies (i) demonstrate that CXCR4 is critical to the progression of diverse brain malignances and (ii) provide a scientific rationale for clinical evaluation of AMD 3100 in treating both adults and children with malignant brain tumors.
Developmental gene regulatory network architecture across 500 million years of echinoderm evolution
Authors: Hinman Veronica F. , Nguyen Albert T. , Cameron R. Andrew , Davidson Eric H.
Published: November 2003

Evolutionary change in morphological features must depend on architectural reorganization of developmental gene regulatory networks (GRNs), just as true conservation of morphological features must imply retention of ancestral developmental GRN features. Key elements of the provisional GRN for embryonic endomesoderm development in the sea urchin are here compared with those operating in embryos of a distantly related echinoderm, a starfish. These animals diverged from their common ancestor 520-480 million years ago. Their endomesodermal fate maps are similar, except that sea urchins generate a skeletogenic cell lineage that produces a prominent skeleton lacking entirely in starfish larvae. A relevant set of regulatory genes was isolated from the starfish
Asterina miniata
, their expression patterns determined, and effects on the other genes of perturbing the expression of each were demonstrated. A three-gene feedback loop that is a fundamental feature of the sea urchin GRN for endoderm specification is found in almost identical form in the starfish: a detailed element of GRN architecture has been retained since the Cambrian Period in both echinoderm lineages. The significance of this retention is highlighted by the observation of numerous specific differences in the GRN connections as well. A regulatory gene used to drive skeletogenesis in the sea urchin is used entirely differently in the starfish, where it responds to endomesodermal inputs that do not affect it in the sea urchin embryo. Evolutionary changes in the GRNs since divergence are limited sharply to certain cis-regulatory elements, whereas others have persisted unaltered.
Randomly inserted and targeted
Hox/
reporter fusions transcriptionally silenced in
Polycomb
mutants
Authors: de Graaff Wim , Tomotsune Daihachiro , Oosterveen Tony , Takihara Yoshihiro , Koseki Haruhiko , Deschamps Jacqueline
Published: November 2003

Polycomb-group (Pc-G) proteins ensure late maintenance of transcriptional repression outside the expression domain of target genes in flies and vertebrates. They act in complexes, presumably by modulating chromatin structure. In
Drosophila
, they have been found to be associated with transcriptionally inactive loci but seem to be present in association with actively transcribed promoters as well, a feature which is not yet understood. In the mouse, mutations in several
Pc
-
G
genes result in an often subtle, local derepression of only a subset of the
Hox
genes rostral to their expression domains. We report here that
Hox
/reporter fusion genes, either randomly integrated as transgenes or as insertions within endogenous loci, are transcriptionally silenced in two mouse
Pc
-
G
-null mutants,
Mel18
and
rae28
. Transcriptional silencing of
Hox
/reporter transgenes in
Pc
-
G
mutants was accompanied by increased DNA methylation in the promoter region. Gene silencing was observed at early developmental stages, long before Pc-G and trithorax-group proteins exert their function in maintenance of the
Hox
patterns. Although all five
Hox
genes tested as
Hox
/reporter fusions were silenced in the
Pc
-
G
mutants, transcription of the endogenous loci was mildly decreased in a subset of these
Hox
genes, and
Hoxb1
was the most strongly affected. We discuss the possibilities that the observed negative effect of
Pc
-
G
mutations on
Hox
and
Hox
/reporter expression may reflect a positive involvement of the Pc-G epigenetic repressors in initial
Hox
gene transcription and that this requirement is exacerbated by the reporter insertion.
The anthropoid status of a primate from the late middle Eocene Pondaung Formation (Central Myanmar): Tarsal evidence
Authors: Marivaux Laurent , Chaimanee Yaowalak , Ducrocq Stéphane , Marandat Bernard , Sudre Jean , Soe Aung Naing , Tun Soe Thura , Htoon Wanna , Jaeger Jean-Jacques
Published: November 2003

Primate dental and postcranial remains from the Eocene Pondaung Formation (Myanmar) have been the subject of considerable confusion since their initial discoveries, and their anthropoid status has been widely debated. We report here a well preserved primate talus discovered in the Segyauk locality near Mogaung that displays derived anatomical features typical of haplorhines, notably anthropoids, and lacks strepsirhine synapomorphies. Linear discriminant and parsimony analyses indicate that the talus from Myanmar is more similar structurally to those of living and extinct anthropoids than to those of adapiforms, and its overall osteological characteristics further point to arboreal quadrupedalism. Regressions of talar dimensions versus body mass in living primates indicate that this foot bone might have belonged toAmphipithecus. This evidence supports hypotheses favoring anthropoid affinities for the large-bodied primates from Pondaung and runs contrary to the hypothesis thatPondaungiaandAmphipithecusare strepsirhine adapiforms.
An endogenous Suppressor of Hairy-wing insulator separates regulatory domains in
Drosophila
Authors: Parnell Timothy J. , Viering Michaela M. , Skjesol Astrid , Helou Cecilia , Kuhn Emily J. , Geyer Pamela K.
Published: November 2003

Insulators define independent domains of gene function throughout the genome. The
Drosophila gypsy
insulator was isolated from the
gypsy
retrotransposon as a region that contains a cluster of binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. To study the effects of the
gypsy
insulator on gene expression within a single genomic domain, targeted gene replacement was used to exchange the endogenous
yellow
gene, located at cytological location 1A, with a set of
gypsy
-modified
yellow
genes. Replaced
yellow
genes carried a
gypsy
insulator positioned between the
yellow
promoter and either the upstream or the downstream tissue-specific enhancers. Whereas the
gypsy
insulator blocked the function of the upstream enhancers at the endogenous location, the downstream enhancers were not blocked. Investigation of the 1A region revealed two clustered Su(Hw)-binding sites downstream of the
yellow
gene, named 1A-2, that bind Su(Hw)
in vivo
and possess enhancer blocking function. We propose that interaction between 1A-2 and the
gypsy
insulator permits activation of
yellow
expression by enhancers in the neighboring
achaete
-
scute
complex, causing an apparent absence of the block of the downstream
yellow
enhancers. Based on these data, we suggest that 1A-2 is an endogenous Su(Hw) insulator that separates regulatory domains within the
Drosophila
genome.
Escherichia coli nucleoside diphosphate kinase is a uracil-processing DNA repair nuclease
Authors: Postel Edith H. , Abramczyk Bozena M.
Published: November 2003

Escherichia coli
nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23/NDK family.
E. coli
cells disrupted in the
ndk
gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that e
ndk
-
cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (
i
) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; (
ii
) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (
iii
) DNA repair phosphodiesterase that removes 3′-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.
Subadult experience influences adult mate choice in an arthropod: Exposed female wolf spiders prefer males of a familiar phenotype
Authors: Hebets Eileen A.
Published: November 2003

Current sexual selection theory proposes several potential mechanisms driving the evolution of female mating preferences, few of which involve social interactions. Although vertebrate examples of socially influenced mating preferences do exist, the invertebrate examples are virtually nonexistent. Here I demonstrate that the mating preferences of female wolf spiders can be acquired through exposure as subadults to unrelated, sexually active adult males. I first conducted exposure trials during which subadult females of the wolf spider
Schizocosa uetzi
were allowed to interact with mature males of an experimentally manipulated phenotype (either black or brown forelegs). After maturation, these previously exposed females were paired with a male of either a familiar or unfamiliar manipulated phenotype for mate-choice trials. Subadult females that were exposed to directed courtship by mature males of a particular morphological phenotype were subsequently more likely to mate with a male of a familiar phenotype as adults. Furthermore, females that were exposed as subadults were more likely, as adults, to cannibalize a courting male with an unfamiliar phenotype. Unexposed females did not distinguish between phenotypes in either mate choice or cannibalism frequency. These results suggest a previously uncharacterized mechanism influencing the origin of female mating preferences and ultimately the evolution of male traits: subadult experience. This study also stresses the potential importance of learning and memory on adult mate choice in an arthropod.
Generation of hair cells by stepwise differentiation of embryonic stem cells
Authors: Li Huawei , Roblin Graham , Liu Hong , Heller Stefan
Published: November 2003

The increase in life expectancy is accompanied by the growing burden of chronic diseases. Hearing loss is perhaps the most prevalent of all chronic diseases. In addition to age-related hearing loss, a substantial number of cases of audiological impairment are either congenital in nature or acquired during childhood. The permanence of hearing loss is mainly due to the inability of the cochlear sensory epithelium to replace lost mechanoreceptor cells, or hair cells. Generation of hair cells from a renewable source of progenitors that can be transplanted into damaged inner ears is a principal requirement for potential cell replacement therapy in this organ. Here, we present an experimental protocol that enables us to routinely create inner ear progenitors from murine embryonic stem cells
in vitro
. These progenitors express a comprehensive set of marker genes that define the developing inner ear, in particular the organ's developing sensory patches. We further demonstrate that cells that express markers characteristic of hair cells differentiate from embryonic stem cell-derived progenitors. Finally, we show that these progenitors integrate into the developing inner ear at sites of epithelial injury and that integrated cells start expressing hair cell markers and display hair bundles when situated in cochlear or vestibular sensory epithelia
in vivo
.