bluuebunny/crossref_metadata_2025 huggingface dataset:
Vacuolar Na
+
/H
+
antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca
2+
- and pH-dependent manner
Authors: Yamaguchi Toshio , Aharon Gilad S. , Sottosanto Jordan B. , Blumwald Eduardo
Published: October 2005

The selective movement of ions between intracellular compartments is fundamental for eukaryotes.
Arabidopsis thaliana
Na
+
/H
+
exchanger 1 (AtNHX1), the most abundant vacuolar Na
+
/H
+
antiporter in
A. thaliana
, has important roles affecting the maintenance of cellular pH, ion homeostasis, and the regulation of protein trafficking. Previously, we have shown that the AtNHX1 C-terminal hydrophilic region localized in the vacuolar lumen plays an important role in regulating the antiporter's activity. Here, we have identified
A. thaliana
calmodulin-like protein 15 (AtCaM15), which interacts with the AtNHX1 C terminus. When expressed in yeast, AtCaM15 is localized in the vacuolar lumen. The transient expression of AtCaM15 in
Arabidopsis
leaf protoplasts showed that AtCaM15 is present in the central vacuole. The binding of AtCaM15 to AtNHX1 was Ca
2+
- and pH-dependent and decreased with increasing pH values. Our results also show that the binding of AtCaM15 to AtNHX1 modified the Na
+
/K
+
selectivity of the antiporter, decreasing its Na
+
/H
+
exchange activity. Taken together, the presence of a vacuolar calmodulin-like protein acting on the vacuolar-localized AtNHX1 C terminus in a Ca
2+
- pH-dependent manner suggests the presence of signaling entities acting within the vacuole.
The error-free component of the
RAD6/RAD18
DNA damage tolerance pathway of budding yeast employs sister-strand recombination
Authors: Zhang Hengshan , Lawrence Christopher W.
Published: October 2005

Evidence for an error-free DNA damage tolerance process in eukaryotes (also called postreplication repair) has existed for more than two decades, but its underlying mechanism, although known to be different from that in prokaryotes, has remained elusive. We have investigated this mechanism in
Saccharomyces cerevisiae
, in which it is the major component of the
RAD6/RAD18
pathway, by transforming an isogenic set of
rad1
Δ excision-defective strains with plasmids that carry a single thymine-thymine pyrimidine (6-4) pyrimidinone photoadduct in each strand at staggered positions 28 base pairs apart. C-C mismatches placed opposite each of the T-T photoproducts permit unambiguous detection of the events that can lead to the completion of replication: sister-strand recombination or translesion replication on one or the other strand. Despite the severe block to replication that these lesions impose, we find that more than half of the plasmids were fully replicated in a
rad1
Δ strain and that >90% of them achieved this end by recombination between partially replicated sister strands within the interlesion region. Approximately 60-70% of these events depended on the error-free component of the
RAD6/RAD18
pathway, with the remaining events depended on
RAD52
; these two processes account for almost all of the recombination, which depended neither on DNA polymerase ζ nor on mismatch repair. We conclude that the error-free component of the
RAD6/RAD18
pathway completes replication by a mechanism employing recombination between partially replicated sister strands, possibly by means of transient template strand switching or copy choice.
In vitro
and
in vivo
two-photon luminescence imaging of single gold nanorods
Authors: Wang Haifeng , Huff Terry B. , Zweifel Daniel A. , He Wei , Low Philip S. , Wei Alexander , Cheng Ji-Xin
Published: October 2005

Gold nanorods excited at 830 nm on a far-field laser-scanning microscope produced strong two-photon luminescence (TPL) intensities, with a cos
4
dependence on the incident polarization. The TPL excitation spectrum can be superimposed onto the longitudinal plasmon band, indicating a plasmon-enhanced two-photon absorption cross section. The TPL signal from a single nanorod is 58 times that of the two-photon fluorescence signal from a single rhodamine molecule. The application of gold nanorods as TPL imaging agents is demonstrated by
in vivo
imaging of single nanorods flowing in mouse ear blood vessels.
Prostaglandin E receptor EP1 controls impulsive behavior under stress
Authors: Matsuoka Yoko , Furuyashiki Tomoyuki , Yamada Kiyofumi , Nagai Taku , Bito Haruhiko , Tanaka Yasuhiro , Kitaoka Shiho , Ushikubi Fumitaka , Nabeshima Toshitaka , Narumiya Shuh
Published: October 2005

Animals under stress take adaptive actions that may lead to various types of behavioral disinhibition. Such behavioral disinhibition, when expressed excessively and impulsively, can result in harm in individuals and cause a problem in our society. We now show that, under social or environmental stress, mice deficient in prostaglandin E receptor subtype EP1 (
Ptger1
-/-
) manifest behavioral disinhibition, including impulsive aggression with defective social interaction, impaired cliff avoidance, and an exaggerated acoustic startle response. This phenotype was reproduced in wild-type mice by administration of an EP1-selective antagonist, whereas administration of an EP1-selective agonist suppressed electric-shock-induced impulsive aggression. Dopamine turnover in the frontal cortex and striatum was increased in
Ptger1
-/-
mice, and administration of dopaminergic antagonists corrected their behavioral phenotype. These results suggest that prostaglandin E
2
acts through EP1 to control impulsive behavior under stress, a finding potentially exploitable for development of drugs that attenuate impulsive behavior in humans.
Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice
Authors: Takayanagi Yuki , Yoshida Masahide , Bielsky Isadora F. , Ross Heather E. , Kawamata Masaki , Onaka Tatsushi , Yanagisawa Teruyuki , Kimura Tadashi , Matzuk Martin M. , Young Larry J. , Nishimori Katsuhiko
Published: October 2005

The oxytocin receptor (OXTR) and its ligand, oxytocin (OXT), regulate reproductive physiology (i.e., parturition and lactation) and sociosexual behaviors. To define the essential functions of OXTR, we generated mice with a null mutation in the
Oxtr
gene (
Oxtr
-/-
) and compared them with OXT-deficient (
Oxt
-/-
) mice.
Oxtr
-/-
mice were viable and had no obvious deficits in fertility or reproductive behavior.
Oxtr
-/-
dams exhibited normal parturition but demonstrated defects in lactation and maternal nurturing. Infant
Oxtr
-/-
males emitted fewer ultrasonic vocalizations than wild-type littermates in response to social isolation. Adult
Oxtr
-/-
males also showed deficits in social discrimination and elevated aggressive behavior. Ligand
Oxt
-/-
males from
Oxt
-/-
dams, but not from
Oxt
+/-
dams, showed similar high levels of aggression. These data suggest a developmental role for the OXT/OXTR system in shaping adult aggressive behavior. Our studies demonstrate that OXTR plays a critical role in regulating several aspects of social behavior and may have important implications for developmental psychiatric disorders characterized by deficits in social behavior.
Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA
Authors: Gowers Darren M. , Wilson Geoffrey G. , Halford Stephen E.
Published: October 2005

Proteins that act at specific DNA sequences bind DNA randomly and then translocate to the target site. The translocation is often ascribed to the protein sliding along the DNA while maintaining continuous contact with it. Proteins also can move on DNA by multiple cycles of dissociation/reassociation within the same chain. To distinguish these pathways, a strategy was developed to analyze protein motion between DNA sites. The strategy reveals whether the protein maintains contact with the DNA as it transfers from one site to another by sliding or whether it loses contact by a dissociation/reassociation step. In reactions at low salt, the test protein stayed on the DNA as it traveled between sites, but only when the sites were <50 bp apart. Transfers of >30 bp at
in vivo
salt, and over distances of >50 bp at any salt, always included at least one dissociation step. Hence, for this enzyme, 1D sliding operates only over short distances at low salt, and 3D dissociation/reassociation is its main mode of translocation.
Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim
Authors: Zhao Yan , Tan Jing , Zhuang Li , Jiang Xia , Liu Edison T. , Yu Qiang
Published: October 2005

Inhibitors of histone deacetylases (HDACIs) are a new generation of anticancer agents that selectively kill tumor cells. However, the molecular basis for their tumor selectivity is not well understood. We investigated the effects of HDACIs on the oncogenic Rb-E2F1 pathway, which is frequently deregulated in human cancers. Here, we report that cancer cells with elevated E2F1 activity, caused either by enforced E2F1 expression, or by E1A oncogene expression, are highly susceptible to HDACI-induced cell death. This E2F1-mediated apoptosis is neither p53- nor p73-dependent but proceeds through selective induction of proapoptotic BH3-only protein Bim. We show that Bim is a direct target of E2F1 and that HDAC inhibition promotes the recruitment of E2F1 to the Bim promoter. Moreover, silencing of Bim by specific small interfering RNA (siRNA) effectively abolishes the E2F1-mediated cell death sensitization to HDACIs. These findings suggest that the oncogenic E2F1 pathway participates in HDACIs-induced apoptosis in cancer cells and underscore the importance of Bim as a key mediator of oncogene-induced apoptosis. Our study provides an important insight into the molecular mechanism of tumor selectivity of HDACIs and predicts that, clinically, HDACIs will be more effective in tumors with high E2F1 activity.
Central role of a bacterial two-component gene regulatory system of previously unknown function in pathogen persistence in human saliva
Authors: Shelburne Samuel A. , Sumby Paul , Sitkiewicz Izabela , Granville Chanel , DeLeo Frank R. , Musser James M.
Published: October 2005

The molecular genetic mechanisms used by bacteria to persist in humans are poorly understood. Group A
Streptococcus
(GAS) causes the majority of bacterial pharyngitis cases in humans and is prone to persistently inhabit the upper respiratory tract. To gain information about how GAS survives in and infects the oropharynx, we analyzed the transcriptome of a serotype M1 strain grown in saliva. The dynamic pattern of changes in transcripts of genes [
spy0874/0875
, herein named
sptR
and
sptS
(sptR/S), for saliva persistence] encoding a two-component gene regulatory system of unknown function suggested that SptR/S contributed to persistence of GAS in saliva. Consistent with this idea, an isogenic nonpolar mutant strain (Δ
sptR
) was dramatically less able to survive in saliva compared with the parental strain. Iterative expression microarray analysis of bacteria grown in saliva revealed that transcripts of several known and putative GAS virulence factor genes were decreased significantly in the Δ
sptR
mutant strain. Compared with the parental strain, the isogenic mutant strain also had altered transcripts of multiple genes encoding proteins involved in complex carbohydrate acquisition and utilization pathways. Western immunoblot analysis and real-time PCR analysis of GAS in throat swabs taken from humans with pharyngitis confirmed the findings. We conclude that SptR/S optimizes persistence of GAS in human saliva, apparently by strategically influencing metabolic pathways and virulence factor production. The discovery of a genetic program that significantly increased persistence of a major human pathogen in saliva enhances understanding of how bacteria survive in the host and suggests new therapeutic strategies.
Light scattered by model phantom bacteria reveals molecular interactions at their surface
Authors: Ghetta A. , Prosperi D. , Mantegazza F. , Panza L. , Riva S. , Bellini T.
Published: October 2005

Testing molecular interactions is an ubiquitous need in modern biology and molecular medicine. Here, we present a qualitative and quantitative method rooted in the basic properties of the scattering of light, enabling detailed measurement of ligand-receptor interactions occurring on the surface of colloids. The key factor is the use of receptor-coated nanospheres matched in refractive index with water and therefore optically undetectable (“phantom”) when not involved in adhesion processes. At the occurrence of ligand binding at the receptor sites, optically unmatched material adsorbs on the nanoparticle surface, giving rise to an increment in their scattering cross section up to a maximum corresponding to saturated binding sites. The analysis of the scattering growth pattern enables extracting the binding affinity. This label-free method has been assessed through the determination of the binding constant of the antibiotic vancomycin with the tripeptide
l
-Lys-
d
-Ala-
d
-Ala and of the vancomycin dimerization constant. We shed light on the role of chelate effect and molecular hindrance in the activity of this glycopeptide.
Cooperative effects of Rho and mechanical stretch on stress fiber organization
Authors: Kaunas Roland , Nguyen Phu , Usami Shunichi , Chien Shu
Published: October 2005

The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rho-kinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Δ1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional ≈3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2Δ1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.
Molecular-level secondary structure, polymorphism, and dynamics of full-length α-synuclein fibrils studied by solid-state NMR
Authors: Heise Henrike , Hoyer Wolfgang , Becker Stefan , Andronesi Ovidiu C. , Riedel Dietmar , Baldus Marc
Published: October 2005

The 140-residue protein α-synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly
13
C/
15
N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V, were analyzed.
13
C and
15
N signals exhibited linewidths of <0.7 ppm. Sequential assignments were obtained for 48 residues in the hydrophobic core region. We identified two different types of fibrils displaying chemical-shift differences of up to 13 ppm in the
15
N dimension and up to 5 ppm for backbone and side-chain
13
C chemical shifts. EM studies suggested that molecular structure is correlated with fibril morphology. Investigation of the secondary structure revealed that most amino acids of the core region belong to β-strands with similar torsion angles in both conformations. Selection of regions with different mobility indicated the existence of monomers in the sample and allowed the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues were mobile and lacked a defined secondary structure, whereas the N terminus was rigid starting from residue 22. Our findings agree well with the overall picture obtained with other methods and provide insight into the amyloid fibril structure and dynamics with residue-specific resolution.
Recruitment of governing elements for electron transfer in the nitric oxide synthase family
Authors: Jáchymová M. , Martásek P. , Panda S. , Roman L. J. , Panda M. , Shea T. M. , Ishimura Y. , Kim J.-J. P. , Masters B. S. S.
Published: October 2005

At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome
c
, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O
2
was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and/or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin–NADP
+
reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.
Crystal structure of theBacillus subtilisanti-alpha, global transcriptional regulator, Spx, in complex with the α C-terminal domain of RNA polymerase
Authors: Newberry Kate J. , Nakano Shunji , Zuber Peter , Brennan Richard G.
Published: October 2005

Spx, a global transcription regulator inBacillus subtilis, interacts with the C-terminal domain of the α subunit (αCTD) of RNA polymerase to control gene expression under conditions of disulfide stress, which is sensed by disulfide bond formation between Spx residues C10 and C13. Here, we describe the crystal structure of theB. subtilisαCTD bound to oxidized Spx. Analysis of the complex reveals interactions between three regions of “anti-alpha” Spx and helix α1 and the “261” determinant of αCTD. The former contact could disrupt the interaction between αCTD and activator proteins or alter the DNA-bound conformation of αCTD, thereby repressing activator-stimulated transcription. Binding to the 261 determinant would prevent interaction between αCTD and region 4 of σA. Intriguingly, the Spx disulfide bond is far from the αCTD–Spx interface, suggesting that Spx regulates transcription allosterically or through the redox-dependent creation or destruction of binding sites for additional components of the transcription machinery.
pH sensing in the two-pore domain K
+
channel, TASK2
Authors: Morton Michael J. , Abohamed Abdulrahman , Sivaprasadarao Asipu , Hunter Malcolm
Published: October 2005

TASK2 is a member of the two-pore domain K
+
channel family that plays a role in acid–base homeostasis; TASK2 knockout animals have plasma electrolyte patterns typical of the human clinical condition of renal tubular acidosis. It is expressed preferentially in epithelia, including the proximal tubules of the kidney. In common with the other TASK channels, TASK2 is sensitive to changes in extracellular pH, although the molecular mechanism of such pH sensing is not understood. We have examined the role of charged residues in the extracellular domains in pH sensing using a mutational approach. Mutant channels were expressed in CHO cells and studied by whole-cell and single-channel patch clamp. Neutralization of no single amino acid in isolation gave complete loss of pH sensitivity. However, the combined removal of five charged amino acids in the large extracellular loop linking the first transmembrane and pore domains, the M1–P1 loop, resulted in an essentially pH-insensitive channel, stabilized in the open state. Wild-type channels contain two such loops, but a concatemeric construct, comprised of one wild-type subunit and one containing the five mutations, was fully pH-sensitive, indicating that only one M1–P1 loop is required to yield a fully pH-sensitive channel, demonstrating a regulatory role of this distinctive structure in two-pore domain K
+
channels. Thus, pH sensing in TASK2 channels is conferred by the combined action of several charged residues in the large extracellular M1–P1 loop.
The highly conserved cardiac glycoside binding site of Na,K-ATPase plays a role in blood pressure regulation
Authors: Dostanic-Larson Iva , Van Huysse James W. , Lorenz John N. , Lingrel Jerry B
Published: October 2005

The Na,K-ATPase contains a binding site for cardiac glycosides, such as ouabain, digoxin, and digitoxin, which is highly conserved among species ranging fromDrosophilato humans. Although advantage has been taken of this site to treat congestive heart failure with drugs such as digoxin, it is unknown whether this site has a natural functionin vivo. Here we show that this site plays an important role in the regulation of blood pressure, and it specifically mediates adrenocorticotropic hormone (ACTH)-induced hypertension in mice. We used genetically engineered mice in which the Na,K-ATPase α2 isoform, which is normally sensitive to cardiac glycosides, was made resistant to these compounds. Chronic administration of ACTH caused hypertension in WT mice but not in mice with an ouabain-resistant α2 isoform of Na,K-ATPase. This finding demonstrates that the cardiac glycoside binding site of the Na,K-ATPase plays an important role in blood pressure regulation, most likely by responding to a naturally occurring ligand. Because the α1 isoform is sensitive to cardiac glycosides in humans, we developed mice in which the naturally occurring ouabain-resistant α1 isoform was made ouabain-sensitive. Mice with the ouabain-sensitive “human-like” α1 isoform and an ouabain-resistant α2 isoform developed ACTH-induced hypertension to greater extent than WT animals. This result indicates that the cardiac glycoside binding site of the α1 isoform can also mediate ACTH-induced hypertension. Taken together these results demonstrate that the cardiac glycoside binding site of the α isoforms of the Na,K-ATPase have a physiological function and supports the hypothesis for a role of the endogenous cardiac glycosides.
Cofilin expression induces cofilin-actin rod formation and disrupts synaptic structure and function in
Aplysia
synapses
Authors: Jang Dong-Hyuk , Han Jin-Hee , Lee Seung-Hee , Lee Yong-Seok , Park Hyungju , Lee Sue-Hyun , Kim Hyoung , Kaang Bong-Kiun
Published: October 2005

Cofilin-actin rods are inclusion-like structures that are induced by certain chemical or physical stresses in cultured cells, and the rods formed in neurons are thought to be associated with neurodegeneration. Here, we cloned an
Aplysia
cofilin homolog and overexpressed it in cultured neurons. Overexpressed cofilin formed rod-like structures that included actin. The overall neuronal morphology was unaffected by cofilin overexpression; however, a decrease in number of synaptic varicosities was observed. Consistent with this structural change by cofilin overexpression, the synaptic strength was reduced, and furthermore, the long-term facilitation elicited by repeated pulses of 5-hydroxytryptamine was impaired in sensory-to-motor synapses. However, cofilin overexpression did not induce programmed cell death. These findings suggest that the formation of cofilin-actin rod-like structures can lead to neurodegeneration, and this might be a mechanism of rundown of neuronal and synaptic function without cell death in neurodegenerative diseases.
Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry
Authors: Ehrich Mathias , Nelson Matthew R. , Stanssens Patrick , Zabeau Marc , Liloglou Triantafillos , Xinarianos George , Cantor Charles R. , Field John K. , van den Boom Dirk
Published: October 2005

Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the
de novo
discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2/H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology.
Spatial regulation of microRNA gene expression in the
Drosophila
embryo
Authors: Biemar Frédéric , Zinzen Robert , Ronshaugen Matthew , Sementchenko Victor , Manak J. Robert , Levine Michael S.
Published: October 2005

MicroRNAs (miRNAs) regulate posttranscriptional gene activity by binding to specific sequences in the 3′ UTRs of target mRNAs. A number of metazoan miRNAs have been shown to exhibit tissue-specific patterns of expression. Here, we investigate the possibility that localized expression is mediated by tissue-specific enhancers, comparable to those seen for protein-coding genes. Two miRNA loci in
Drosophila melanogaster
are investigated, the
mir-309–6
polycistron (8-miR) and the
mir-1
gene. The 8-miR locus contains a cluster of eight distinct miRNAs that are transcribed in a common precursor RNA. The 8-miR primary transcript displays a dynamic pattern of expression in early embryos, including repression at the anterior and posterior poles. An 800-bp 5′ enhancer was identified that recapitulates this complex pattern when attached to a RNA polymerase II core promoter fused to a
lacZ
-reporter gene. The miR-1 locus is specifically expressed in the mesoderm of gastrulating embryos. Bioinformatics methods were used to identify a mesoderm-specific enhancer located ≈5 kb 5′ of the miR-1 transcription unit. Evidence is presented that the 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist. These results provide evidence that restricted activities of the 8-miR and miR-1 miRNAs are mediated by classical tissue-specific enhancers.
Adaptation and incipient sympatric speciation of
Bacillus simplex
under microclimatic contrast at “Evolution Canyons” I and II, Israel
Authors: Sikorski Johannes , Nevo Eviatar
Published: October 2005

The microevolutionary dynamics of prokaryotes in natural habitats, such as soil, is poorly understood in contrast to our increasing knowledge on their immense diversity. We performed microevolutionary analyses on 945 soil isolates of
Bacillus simplex
from “Evolution Canyons” I (Carmel, Israel) and II (Galilee, Israel). These canyons represent similar ecological replicates, separated by 40 km, with highly contrasting interslope abiotic and biotic conditions in each (within a distance of only 100–400 m). Strains representing genetic groups were identical in their 16S sequences, suggesting high genetic similarity and monophyletic origin. Parallel and nested phylogenetic structures correlated with ecological contrasts rather than geographical distance. Additionally, slope-specific populations differed substantially in their diversity. The levels of DNA repair (determined by UV sensitivity) and spontaneous mutation rate (resistance to rifampicin) relate to ecological stress and phylogeny. Altogether, the results suggest adaptive radiation at a microscale. We discuss the observed adaptive population structures in the context of incipient sympatric speciation in soil bacteria. We conclude that, despite different biology, prokaryotes, like sexually reproducing eukaryotes, may consist of true species and parallel ecological speciation in eukaryotes.
Involvement of
winged eye
encoding a chromatin-associated bromo-adjacent homology domain protein in disc specification
Authors: Katsuyama Tomonori , Sugawara Tomo , Tatsumi Masanobu , Oshima Yoshiteru , Gehring Walter J. , Aigaki Toshiro , Kurata Shoichiro
Published: October 2005

How organ identity is determined is a fundamental question in developmental biology. In
Drosophila
, field-specific selector genes, such as
eyeless
(
ey
) for eyes and
vestigial
(
vg
) for wings, participate in the determination of imaginal disc-specific identity. We performed gain-of-function screening and identified a gene named
winged eye
(
wge
), which encodes a bromo-adjacent homology domain protein that localizes at specific sites on chromosomes in a bromo-adjacent homology domain-dependent manner. Overexpression of
wge
-induced ectopic wings with antero-posterior and dorso-ventral axes in the eye field in a region-specific Hox gene-(
Antennapedia
) independent manner. Overexpression of
wge
was sufficient for ectopic expression of
vg
in eye discs. A context-dependent requirement of
wge
was demonstrated for
vg
expression in wing discs and for expression of
eyes absent
(
eya
), a control gene for eye development downstream of
ey
, in eye discs. In contrast to
vg
, however, overexpression of
wge
inhibited EY-mediated expression of
eya
. Consistent with colocalization on polytene chromosomes of WGE and Posterior sex combs (PSC), a Polycomb group gene product, we demonstrated an antagonistic genetic interaction between
wge
and
Psc
. These findings suggest that
wge
functions in the determination of disc-specific identity, downstream of Hox genes.
Diapause in the mosquito
Culex pipiens
evokes a metabolic switch from blood feeding to sugar gluttony
Authors: Robich Rebecca M. , Denlinger David L.
Published: October 2005

A key characteristic of overwintering dormancy (diapause) in the mosquito
Culex pipiens
is the switch in females from blood feeding to sugar gluttony. We present evidence demonstrating that genes encoding enzymes needed to digest a blood meal (
trypsin
and a
chymotrypsin-like protease
) are down-regulated in diapause-destined females, and that concurrently, a gene associated with the accumulation of lipid reserves (
fatty acid synthase
) is highly up-regulated. As the females then enter diapause,
fatty acid synthase
is only sporadically expressed, and expression of
trypsin
and
chymotrypsin-like
remains undetectable. Late in diapause (2–3 months at 18°C), the genes encoding the digestive enzymes begin to be expressed as the female prepares to take a blood meal upon the termination of diapause. Our results thus underscore a molecular switch that either capacitates the mosquito for blood feeding (nondiapause) or channels the adult mosquito exclusively toward sugar feeding and lipid sequestration (diapause).
Metastatic properties and genomic amplification of the tyrosine kinase gene
ACK1
Authors: van der Horst Edward Htun , Degenhardt Yan Y. , Strelow Astrid , Slavin Anthony , Chinn Lawrence , Orf Jessica , Rong Minqing , Li Shyun , See Lei-Hoon , Nguyen Ken Q. C. , Hoey Timothy , Wesche Holger , Powers Scott
Published: October 2005

Metastasis of primary tumors leads to a very poor prognosis for patients suffering from cancer. Although it is well established that not every tumor will eventually metastasize, it is less clear whether primary tumors acquire genetic alterations in a stochastic process at a late stage, which make them invasive, or whether genetic alterations acquired early in the process of tumor development drive primary tumor growth and determine whether this tumor is going to be metastatic. To address this issue, we tested genes identified in a large-scale comparative genomic hybridization analysis of primary tumor for their ability to confer metastatic properties on a cancer cell. We identified amplification of the
ACK1
gene in primary tumors, which correlates with poor prognosis. We further show that overexpression of Ack1 in cancer cell lines can increase the invasive phenotype of these cells both
in vitro
and
in vivo
and leads to increased mortality in a mouse model of metastasis. Biochemical studies show that Ack1 is involved in extracellular matrix-induced integrin signaling, ultimately activating signaling processes like the activation of the small GTPase Rac. Taken together, this study supports a theory from Bernards and Weinberg [Bernards, R. & Weinberg, R. A. (2002)
Nature
418, 823], which postulates that the tendency to metastasize is largely predetermined.
Pivotal role of
Harakiri
in the induction and prevention of gentamicin-induced hearing loss
Authors: Kalinec Gilda M. , Fernandez-Zapico Martin E. , Urrutia Raul , Esteban-Cruciani Nora , Chen Shanping , Kalinec Federico
Published: October 2005

Gentamicin is a widely used ototoxic agent. In this study, we shed light on the mechanisms underlying gentamicin-induced hearing loss. More importantly, we demonstrate
in vivo
and
in vitro
the effectiveness of a strategy for preventing drug-induced hearing loss using
l
-carnitine (LCAR), a safe micronutrient that plays a key role in energy metabolism and detoxification [Rebouche, C. J. & Seim, H. (1998)
Annu. Rev. Nutr.
18, 39-61]. We show that LCAR prevents changes in hearing threshold and cochlear damage in newborn guinea pigs exposed to gentamicin
in utero
. Mechanistically, gentamicin-induced apoptosis of auditory cells is mediated by the extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) pathway through up-regulation of the proapoptotic factor
Harakiri
(
Hrk
). Most important, small interfering RNA (siRNA) experiments demonstrate that
Hrk
up-regulation is crucial for gentamicin-induced apoptosis. LCAR, in contrast, prevents both gentamicin-induced
Hrk
up-regulation and apoptosis acting by means of c-Jun N-terminal kinase (JNK). Together, these results outline pathways for gentamicin-induced hearing loss and its prevention and assign a key role to
Hrk
in these processes. Thus, our data offer a conceptual framework for designing clinical trials using a safe micronutrient, LCAR, as a simple preventive strategy for iatrogenically induced ototoxicity.
Spectral and redox characterization of the heme
c
i
of the cytochrome
b
6
f
complex
Authors: Alric Jean , Pierre Yves , Picot Daniel , Lavergne Jérôme , Rappaport Fabrice
Published: October 2005

Absorption spectra of the purified cytochrome
b
6
f
complex from
Chlamydomonas reinhardtii
were monitored as a function of the redox potential. Four spectral and redox components were identified: in addition to heme
f
and the two
b
hemes, the fourth component must be the new heme
c
i
(also denoted
x
) recently discovered in the crystallographic structures. This heme is covalently attached to the protein, but has no amino acid axial ligand. It is located in the plastoquinone-reducing site Q
i
in the immediate vicinity of a
b
heme. Each heme titrated as a one-electron Nernst curve, with midpoint potentials at pH 7.0 of -130 mV and -35 mV (hemes
b
), +100 mV (heme
c
i
), and +355 mV (heme
f
). The reduced minus oxidized spectrum of heme
c
i
consists of a broad absorption increase centered ≈425 nm. Its potential has a dependence of -60 mV/pH unit, implying that the reduced form binds one proton in the pH 6-9 range. The Q
i
site inhibitor 2-
n
-nonyl-4-hydroxyquinoline
N
-oxide, a semiquinone analogue, induces a shift of this potential by about -225 mV. The spectrum of
c
i
matches the absorption changes previously observed
in vivo
for an unknown redox center denoted “G.” The data are discussed with respect to the effect of the membrane potential on the electron transfer equilibrium between G and heme
b
H
found in earlier experiments.
Haploinsufficiency of t
e
lomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita
Authors: Armanios Mary , Chen Jiunn-Liang , Chang Yen-Pei Christy , Brodsky Robert A. , Hawkins Anita , Griffin Constance A. , Eshleman James R. , Cohen Alan R. , Chakravarti Aravinda , Hamosh Ada , Greider Carol W.
Published: October 2005

Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase,
hTERT
, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.
Centromere-proximal differentiation and speciation in
Anopheles gambiae
Authors: Stump Aram D. , Fitzpatrick Meagan C. , Lobo Neil F. , Traoré Sékou , Sagnon N′Fale , Costantini Carlo , Collins Frank H. , Besansky Nora J.
Published: October 2005

The M and S molecular forms of
Anopheles gambiae
are undergoing speciation as they adapt to heterogeneities in the environment, spreading malaria in the process. We hypothesized that their divergence despite gene flow is facilitated by reduced recombination at the centromeric (proximal) end of the X chromosome. We sequenced introns from 22 X chromosome genes in M and S from two locations of West Africa where the forms are sympatric. Generally, in both forms nucleotide diversity was high distally, lower proximally, and very low nearest the centromere. Conversely, differentiation between the forms was virtually zero distally and very high proximally. Pairwise comparisons to a close relative, the sibling species
Anopheles arabiensis
, demonstrated uniformly high divergence regardless of position along the X chromosome, suggesting that this pattern is not purely mechanical. Instead, the pattern observed for M and S suggests the action of divergent natural selection countering gene flow only at the proximal end of the X chromosome, where recombination is reduced. Comparison of sites with fixed differences between M and S to the corresponding sites in
A. arabiensis
revealed that derived substitutions had been fixed in both forms, further supporting the hypothesis that both have been under selection. These derived substitutions are fixed in the two West African samples and in samples of S from western and coastal Kenya, suggesting that selection occurred before the forms expanded to their current ranges. Our findings are consistent with a role for suppressed genetic recombination in speciation of
A. gambiae
.
Evidence for maternally transmitted small interfering RNA in the repression of transposition in
Drosophila virilis
Authors: Blumenstiel Justin P. , Hartl Daniel L.
Published: October 2005

Hybrid dysgenesis in
Drosophila
is a syndrome of gonadal atrophy, sterility, and male recombination, and it occurs in the progeny of crosses between males that harbor certain transposable elements (TEs) and females that lack them. Known examples of hybrid dysgenesis in
Drosophila melanogaster
result from mobilization of individual families of TEs, such as the
P
element, the
I
element, or
hobo
. An example of hybrid dysgenesis in
Drosophila virilis
is unique in that multiple, unrelated families of TEs become mobilized, but a TE designated
Penelope
appears to play a major role. In all known examples of hybrid dysgenesis, the paternal germ line transmits the TEs in an active state, whereas the female germ line maintains repression of the TEs. The mechanism of maternal maintenance of repression is not known. Recent evidence suggests that the molecular machinery of RNA interference may function as an important host defense against TEs. This protection is mediated by the action of endogenous small interfering RNAs (siRNAs) composed of dsRNA molecules of 21-25 nt that can target complementary transcripts for destruction. In this paper, we demonstrate that endogenous siRNA derived from the
Penelope
element is maternally loaded in embryos through the female germ line in
D. virilis
. We also present evidence that the maternal inheritance of these endogenous siRNAs may contribute to maternal repression of
Penelope
.
3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography
Authors: Nicastro Daniela , McIntosh J. Richard , Baumeister Wolfgang
Published: October 2005

We have used cryo-electron tomography to investigate the 3D structure and macromolecular organization of intact, frozen-hydrated sea urchin sperm flagella in a quiescent state. The tomographic reconstructions provide information at a resolution better than 6 nm about thein situarrangements of macromolecules that are key for flagellar motility. We have visualized the heptameric rings of the motor domains in the outer dynein arm complex and determined that they lie parallel to the plane that contains the axes of neighboring flagellar microtubules. Both the material associated with the central pair of microtubules and the radial spokes display a plane of symmetry that helps to explain the planar beat pattern of these flagella. Cryo-electron tomography has proven to be a powerful technique for helping us understand the relationships between flagellar structure and function and the design of macromolecular machinesin situ.
Early Epidermal Destruction with Subsequent Epidermal Hyperplasia Is a Unique Feature of the Papilloma-Independent Squamous Cell Carcinoma Phenotype in PKCε Overexpressing Transgenic Mice
Authors: Li Yafan , Wheeler Deric L. , Alters Wade , Chaiswing Luksana , Verma Ajit K. , Oberley Terry D.
Published: October 2005

Protein kinase C epsilon (PKCε) overexpressing transgenic (PKCεTg) mice develop papilloma-independent squamous cell carcinomas (SCC) elicited by 7,12-dimethylbenz[a]anthracene (DMBA) tumor initiation and 12- O-tetradecanoylphorbol-13-acetate (TPA) tumor promotion. We examined whether epidermal cell turnover kinetics was altered during the development of SCC in PKCε Tg mice. Dorsal skin samples were fixed for histological examination. A single application of TPA resulted in extensive infiltration of polymorphonuclear neutrophils (PMNs) into the epidermis at 24 h after TPA treatment in PKCε Tg mice while wild-type (WT) mouse skin showed focal infiltration by PMNs. Complete epidermal necrosis was observed at 48 h in PKCε Tg mice only; at 72 h, epidermal cell regeneration beginning from hair follicles was observed in PKCε Tg mice. Since the first TPA treatment to DMBA-initiated PKCε Tg mouse skin led to epidermal destruction analogous to skin abrasion, we propose the papilloma-independent phenotype may be explained by death of initiated interfollicular cells originally destined to become papillomas. Epidermal destruction did not occur after multiple doses of TPA, presumably reflecting adaptation of epidermis to chronic TPA treatment. Prolonged hyperplasia in the hair follicle may result in the early neoplastic lesions originally described by Jansen et al. (2001) by expanding initiated cells in the hair follicles resulting in the subsequent development of SCC.
Transgenic Disruption of Gap Junctional Intercellular Communication Enhances Early but Not Late Stage Hepatocarcinogenesis in the Rat
Authors: Hokaiwado Naomi , Asamoto Makoto , Ogawa Kumiko , Shirai Tomoyuki
Published: October 2005

Much experimental evidence supports the conclusion that loss of gap junctional intercellular communication (GJIC) contributes to carcinogenesis. Transgenic rats featuring a dominant negative mutant of the connexin 32 gene under albumin promoter control (Cx32ΔTg-High and Cx32ΔTg-Low lines, respectively with high and low copy numbers of the transgene) have disrupted GJIC, as demonstrated by scrape dye-transfer assay in vivo as previous report by Asamoto et al. (2004) . In the present study, we investigated the susceptibility of these transgenic rats to a single intraperitoneal administration of diethylnitrosamine (DEN), and found a significant increase in preneoplastic glutathione S-transferase placental form (GST-P) positive lesions in the livers of Cx32ΔTg-High but not Cx32ΔTg-Low rats. However, incidences of adenomas and hepatocellular carcinomas were not elevated at the end of the experiment (52 weeks). In addition, we investigated the promotional effect of phenobarbital (PB) on Cx32ΔTg-High rats pretreated with DEN and found enhanced formation of GST-P positive lesions, in contrast to the lack of promoting effects reported for Cx32 deficient mice. The results indicate that although both high and low expression of the dominant negative connexin 32 mutant gene in our rats is able to inhibit gap junctional capacity, only high expression is effective at enhancing susceptibility to early stage DEN-induced liver carcinogenesis.
Global flows: Terror, oil & strategic philanthropy
Authors: Barnes Sandra T.
Published: October 2005

US involvement in Africa is growing following threats of terrorism and interruptions in oil production and because of desires by foreign corporations to expand their activities on the continent. The response of American policymakers has been to establish a stronger military presence that will engage in counterterrorism initiatives and police oil installations. The goals and extent of this buildup, and the ideology legitimating it, are new. They are departures from Cold War policies. Similarly, the response of American business leaders to weaknesses in the infrastructure and political order of African states leads them to establish their own forms of community development, known as strategic philanthropy, in order to protect and expand local markets. Despite these major developments, the media are not informing the public. This article examines the implications of these military and business initiatives for African nations and the reasons for lack of information about them.

Editor's Note: This article was delivered as the presidential address to the African Studies Association, New Orleans, 12 November 2004. It first appeared in the African Studies Review, Vol. 47, No. 5, April 2005:1–22, the principal scholarly journal of that Association. On the role of the US in Africa, also see Daniel Volman, ‘US Military Involvement in Africa’ and Michel Chossudovsky, ‘New Undeclared Arms Race: America's Agenda for Global Military Domination’ in ROAPE 103, March 2005.