bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the TDP-
epi
-vancosaminyltransferase GtfA from the chloroeremomycin biosynthetic pathway
Authors: Mulichak Anne M. , Losey Heather C. , Lu Wei , Wawrzak Zdzislaw , Walsh Christopher T. , Garavito R. Michael
Published: August 2003

During the biosynthesis of the vancomycin-class antibiotic
chloroeremomycin, TDP-
epi
-vancosaminyltransferase GtfA catalyzes the
attachment of 4-
epi
-vancosamine from a TDP donor to the
β-OHTyr-6 of the aglycone cosubstrate. Glycosyltransferases from this
pathway are potential tools for the combinatorial design of new antibiotics
that are effective against vancomycin-resistant bacterial strains. These
enzymes are members of the GT-B glycosyltransferase superfamily, which share a
homologous bidomain topology. We present the 2.8-Å crystal structures of
GtfA complexes with vancomycin and the natural monoglycosylated peptide
substrate, representing the first direct observation of acceptor substrate
binding among closely related glycosyltransferases. The acceptor substrates
bind to the N-terminal domain such that the aglycone substrate's reactive
hydroxyl group hydrogen bonds to the side chains of Ser-10 and Asp-13, thus
identifying these as residues of potential catalytic importance. As well as an
open form of the enzyme, the crystal structures have revealed a closed form in
which a TDP ligand is bound at a donor substrate site in the interdomain
cleft, thereby illustrating not only binding interactions, but the
conformational changes in the enzyme that accompany substrate binding.
Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses
Authors: Belyakov Igor M. , Earl Patricia , Dzutsev Amiran , Kuznetsov Vladimir A. , Lemon Michael , Wyatt Linda S. , Snyder James T. , Ahlers Jeffrey D. , Franchini Genoveffa , Moss Bernard , Berzofsky Jay A.
Published: August 2003

The concern about bioterrorism with smallpox has raised the possibility of widespread vaccination, but the greater prevalence of immunocompromised individuals today requires a safer vaccine, and the mechanisms of protection are not well understood. Here we show that, at sufficient doses, the protection provided by both modified vaccinia Ankara and NYVAC replication-deficient vaccinia viruses, safe in immunocompromised animals, was equivalent to that of the licensed Wyeth vaccine strain against a pathogenic vaccinia virus intranasal challenge of mice. A similar variety and pattern of immune responses were involved in protection induced by modified vaccinia Ankara and Wyeth viruses. For both, antibody was essential to protect against disease, whereas neither effector CD4+nor CD8+T cells were necessary or sufficient. However, in the absence of antibody, T cells were necessary and sufficient for survival and recovery. Also, T cells played a greater role in control of sublethal infection in unimmunized animals. These properties, shared with the existing smallpox vaccine, provide a basis for further evaluation of these replication-deficient vaccinia viruses as safer vaccines against smallpox or against complications from vaccinia virus.
Clusterin as a biomarker in murine and human intestinal neoplasia
Authors: Chen Xiaodi , Halberg Richard B. , Ehrhardt William M. , Torrealba Jose , Dove William F.
Published: August 2003

Early detection of colorectal cancer is critical for the management of this disease. Biomarkers for early detection of several cancers have been developed and applied clinically in recent years. We have sought to discover candidate biomarkers without the restricted choice of markers placed on microarrays, and without the biological complications of genetic and environmental heterogeneity. We have compared by cDNA subtraction two genetically matched sets of mice, one developing multiple intestinal neoplasia (C57BL/6J-ApcMin) and the other tumor-free (C57BL/6J). One prominent candidate biomarker, clusterin, was then subjected to a series of validation steps.In situhybridization and immunohistochemistry were used to analyze clusterin expression at a cellular level on a series of murine intestinal and human colonic neoplasms. Elevated clusterin expression was characterized within certain regions of murine and human tumors regardless of tumor stage, location, or mode of initiation. The cells showing high clusterin levels generally lacked differentiation markers and adenomatous polyposis coli antigen. Tumor cells undergoing apoptosis expressed low levels of clusterin. Its specific expression patterns and correlation with cellular events during tumorigenesis make it a useful diagnostic tool in the mouse and a potential contributor to the set of biomarkers for early detection of human colon cancer.
Conjugation to gold nanoparticles enhances polyethylenimine's transfer of plasmid DNA into mammalian cells
Authors: Thomas Mini , Klibanov Alexander M.
Published: August 2003

Branched polyethylenimine (PEI) chains with an average molecular mass of 2
kDa (PEI2) have been covalently attached to gold nanoparticles (GNPs), and the
potency of the resulting PEI2–GNPs conjugates as vectors for the
delivery of plasmid DNA into monkey kidney (COS-7) cells in the presence of
serum
in vitro
has been systematically investigated. The transfection
efficiencies vary as a function of the PEI/gold molar ratio in the conjugates,
with the best one (PEI2–GNPII) being 12 times more potent than the
unmodified polycation. This potency can be further doubled by adding
amphiphilic
N
-dodecyl–PEI2 during complex formation with DNA.
The resulting ternary complexes are at least 1 order of magnitude more
efficient than the 25-kDa PEI, one of the premier polycationic gene-delivery
vectors. Importantly, although unmodified PEI2 transfects just 4% of the
cells, PEI2–GNPII transfects 25%, and the
PEI2–GNPII/dodecyl–PEI2 ternary complex transfects 50% of the
cells. The intracellular trafficking of the DNA complexes of these vectors,
monitored by transmission electron microscopy, has detected the complexes in
the nucleus <1 h after transfection.
The serum response factor coactivator myocardin is required for vascular smooth muscle development
Authors: Li Shijie , Wang Da-Zhi , Wang Zhigao , Richardson James A. , Olson Eric N.
Published: August 2003

Formation of the vascular system requires differentiation and patterning of
endothelial and smooth muscle cells (SMCs). Although much attention has
focused on development of the vascular endothelial network, the mechanisms
that control vascular SMC development are largely unknown. Myocardin is a
smooth and cardiac muscle-specific transcriptional coactivator of serum
response factor, a ubiquitous transcription factor implicated in smooth muscle
gene expression. When expressed ectopically in nonmuscle cells, myocardin can
induce smooth muscle differentiation by its association with serum response
factor. Here we report that mouse embryos homozygous for a myocardin
loss-of-function mutation die by embryonic day 10.5 and show no evidence of
vascular SMC differentiation. Myocardin is the only transcription factor known
to be necessary and sufficient for vascular SMC differentiation.
Mechanisms of HFE-induced regulation of iron homeostasis: Insights from the W81A HFE mutation
Authors: Zhang An-Sheng , Davies Paige S. , Carlson Hanqian L. , Enns Caroline A.
Published: August 2003

The mechanisms by which the hereditary hemochromatosis protein, HFE,
decreases transferrin-mediated iron uptake were examined.
Coimmunoprecipitation studies using solubilized cell extracts demonstrated
that transferrin (Tf) competed with HFE for binding to the transferrin
receptor (TfR) similar to previous
in vitro
studies using soluble
truncated forms of HFE and the TfR. At concentrations of Tf approaching those
found in the blood, no differences in Tf binding to cells were detected, which
is consistent with the lower binding constant of HFE for TfR versus Tf.
However, cells expressing HFE still showed a decrease in Tf-mediated iron
uptake at concentrations of Tf sufficient to dissociate HFE from the TfR.
These results indicate that the association of HFE with TfR is not essential
for its ability to lower intracellular iron stores. To test the effect of HFE
on lowering intracellular iron levels independently of its association with
TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the TfR
was transfected into tetracycline-controlled transactivator HeLa cells. HeLa
cells expressing fW81AHFE behaved in a similar manner to cells expressing
wild-type HFE with respect to decreased intracellular iron levels measured by
iron regulatory protein gel-shift assays and ferritin levels. The results
indicate that HFE can lower intracellular iron levels independently of its
interaction with the TfR.
Osmotic pressure inhibition of DNA ejection from phage
Authors: Evilevitch Alex , Lavelle Laurence , Knobler Charles M. , Raspaud Eric , Gelbart William M.
Published: August 2003

Bacterial viral capsids in aqueous solution can be opened
in vitro
by addition of their specific receptor proteins, with consequent full ejection
of their genomes. We demonstrate that it is possible to control the extent of
this ejection by varying the external osmotic pressure. In the particular case
of bacteriophage λ, the ejection is 50% inhibited by osmotic pressures
(of polyethylene glycol) comparable to those operative in the cytoplasm of
host bacteria; it is completely suppressed by a pressure of 20 atmospheres.
Furthermore, our experiments monitor directly a dramatic decrease of the
stress inside the unopened phage capsid upon addition of polyvalent cations to
the host solution, in agreement with many recent theories of DNA
interactions.
Molecular genetics and evolution of pheromone biosynthesis in Lepidoptera
Authors: Roelofs Wendell L. , Rooney Alejandro P.
Published: August 2003

A great diversity of pheromone structures are used by moth species (Insecta: Lepidoptera) for long-distance mating signals. The signal/response channel seems to be narrow for each species, and a major conundrum is how signal divergence has occurred in the face of strong selection pressures against small changes in the signal. Observations of various closely related and morphologically similar species that use pheromone components biosynthesized by different enzymes and biosynthetic routes underscore the question as to how major jumps in the biosynthetic routes could have evolved with a mate recognition system that is based on responses to a specific blend of chemicals. Research on the desaturases used in the pheromone biosynthetic pathway for various moth species has revealed that one way to make a major shift in the pheromone blend is by activation of a different desaturase from mRNA that already exists in the pheromone gland. Data will be presented to support the hypothesis that this process was used in the evolution of the Asian corn borer,
Ostrinia furnacalis
species. In that context, moth sex-pheromone desaturase genes seem to be evolving under a birth-and-death process. According to this model of multigene family evolution, some genes are maintained in the genome for long periods of time, whereas others become deleted or lose their functionality, and new genes are created through gene duplication. This mode of evolution seems to play a role in moth speciation, as exemplified by the case of the Asian corn borer and European corn borer,
Ostrinia nubilalis
species.
Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3
Authors: Chen Chi-hong B. , Chernis George A. , Hoang Van Q. , Landgraf Ralf
Published: August 2003

Human epidermal growth factor receptor-3 (HER3) is a member of the type I
receptor tyrosine kinase family. Several members of this family are
overexpressed in various carcinomas. Specifically, HER2 is found to be
overexpressed in 20–30% of breast cancers. In contrast to epidermal
growth factor receptor or HER2, the kinasedeficient HER3 self-associates
readily at low nanomolar concentrations and in the absence of its ligands,
various isoforms of heregulin (hrg). Binding of hrg disrupts HER3
oligomerization and leads to the formation of signaling-competent
heterodimers, preferentially with HER2. Elevated levels of HER3 contribute to
increased drug resistance observed in HER2-overexpressing cells. We have used
the SELEX (systematic evolution of ligands by exponential enrichment)
methodology to select RNA aptamers against the oligomeric state of the
extracellular domains of HER3 (HER3ECD, monomeric molecular mass 82,000 Da).
One of the aptamers, A30, binds with high affinity to a limited number of
binding sites in the oligomeric state of HER3ECD. Binding of A30 and hrg are
not competitive. Instead, the disruption of HER3 oligomers by hrg results in
an ≈10-fold increase in total binding sites, but the newly created binding
sites are of lower affinity. High-affinity binding of A30 inhibits
hrg-dependent tyrosine phosphorylation of HER2 and the hrg-induced growth
response of MCF7 cells. As an example of an aptamer against a large
macromolecular protein complex, A30 can serve as a tool for the analysis of
receptor interactions and may serve as a lead compound for the development of
inhibitors against overexpressed receptor tyrosine kinases in carcinomas.
A biochemical rationale for the discrete behavior of nitroxyl and nitric oxide in the cardiovascular system
Authors: Miranda Katrina M. , Paolocci Nazareno , Katori Tatsuo , Thomas Douglas D. , Ford Eleonora , Bartberger Michael D. , Espey Michael G. , Kass David A. , Feelisch Martin , Fukuto Jon M. , Wink David A.
Published: August 2003

The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine/NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine/NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the O2/\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \pagestyle{empty} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document}couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with O2is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.
A new class of mutations reveals a novel function for the original phosphatidylinositol 3-kinase binding site
Authors: Hong Y. Kate , Mikami Aki , Schaffhausen Brian , Jun Toni , Roberts Thomas M.
Published: August 2003

Previous studies have demonstrated that the specificity of Src homology 2
(SH2) and phosphotyrosine-binding domain interactions are mediated by
phosphorylated tyrosines and their neighboring amino acids. Two of the first
phosphotyrosine-based binding sites were found on middle T antigen of polyoma
virus. Tyr-250 acts as a binding site for ShcA, whereas Tyr-315 forms a
binding site for the SH2 domain of the p85 subunit of phosphatidylinositol
3-kinase. However, genetic analysis of a given phosphotyrosine's role in
signaling can be complicated when it serves as a binding site for multiple
proteins. The situation is particularly difficult when the phosphotyrosine
serves as a secondary binding site for a protein with primary binding
determinates elsewhere. Mutation of a tyrosine residue to phenylalanine blocks
association of all bound proteins. Here we show that the mutation of the amino
acids following the phosphorylated tyrosine to alanine can reveal
phosphotyrosine function as a secondary binding site, while abrogating the
phosphotyrosine motif's role as a primary binding site for SH2 domains. We
tested this methodology by using middle T antigen. Our results suggest that
Tyr-250 is a secondary binding site for phosphatidylinositol 3-kinase, whereas
Tyr-315 is a secondary binding site for a yet-to-be-identified protein, which
is critical for transformation.
Glutamate carboxypeptidase II inhibition protects motor neurons from death in familial amyotrophic lateral sclerosis models
Authors: Ghadge Ghanashyam D. , Slusher Barbara S. , Bodner Amos , Canto Mauro Dal , Wozniak Krystyna , Thomas Ajit G. , Rojas Camilo , Tsukamoto Takashi , Majer Pavel , Miller Richard J. , Monti Anna Liza , Roos Raymond P.
Published: August 2003

Approximately 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and ≈20% of these cases of familial ALS (FALS) are caused by mutations of copper/zinc superoxide dismutase type 1. Glutamate excitotoxicity has been implicated as a mechanism of MN death in both ALS and FALS. In this study, we tested whether a neuroprotective strategy involving potent and selective inhibitors of glutamate carboxypeptidase II (GCPII), which converts the abundant neuropeptideN-acetylaspartylglutamate to glutamate, could protect MNs in anin vitroand animal model of FALS. Data suggest that the GCPII inhibitors prevented MN cell death in both of these systems because of the resultant decrease in glutamate levels. GCPII inhibition may represent a new therapeutic target for the treatment of ALS.
Statistical significance for genomewide studies
Authors: Storey John D. , Tibshirani Robert
Published: August 2003

With the increase in genomewide experiments and the sequencing of multiple
genomes, the analysis of large data sets has become commonplace in biology. It
is often the case that thousands of features in a genomewide data set are
tested against some null hypothesis, where a number of features are expected
to be significant. Here we propose an approach to measuring statistical
significance in these genomewide studies based on the concept of the false
discovery rate. This approach offers a sensible balance between the number of
true and false positives that is automatically calibrated and easily
interpreted. In doing so, a measure of statistical significance called the
q
value is associated with each tested feature. The
q
value
is similar to the well known
p
value, except it is a measure of
significance in terms of the false discovery rate rather than the false
positive rate. Our approach avoids a flood of false positive results, while
offering a more liberal criterion than what has been used in genome scans for
linkage.
The role(s) of lipophosphoglycan (LPG) in the establishment ofLeishmania majorinfections in mammalian hosts
Authors: Späth Gerald F. , Garraway L. A. , Turco Salvatore J. , Beverley Stephen M.
Published: August 2003

The abundant cell surface glycolipid lipophosphoglycan (LPG) was implicated in many steps of theLeishmaniainfectious cycle by biochemical tests. The presence of other abundant surface or secreted glycoconjugates sharing LPG domains, however, has led to uncertainty about the relative contribution of LPGin vivo. Here we used anLeishmania major lpg1-mutant, which lacks LPG alone and shows attenuated virulence, to dissect the role of LPG in the establishment of macrophage infectionsin vivo. lpg1-was highly susceptible to human complement, had lost the ability to inhibit phagolysosomal fusion transiently, and was oxidant sensitive. Studies of mouse mutants defective in relevant defense mechanisms confirmed the role of LPG in oxidant resistance but called into question the importance of transient inhibition of phagolysosomal fusion forLeishmaniamacrophage survival. Moreover, the limited lytic activity of mouse complement appears to be an ineffective pathogen defense mechanismin vitroandin vivo, unlike human hosts. In contrast,lpg1-parasites bound C3b and resisted low pH and proteases normally, entered macrophages efficiently and silently, and continued to inhibit host-signaling pathways. These studies illustrate the value of mechanistic approaches focusing on both parasite and host defense pathways in dissecting the specific biological roles of complex virulence factors such as LPG.
Activity of the
Caenorhabditis elegans
UNC-86 POU transcription factor modulates olfactory sensitivity
Authors: Sze Ji Ying , Ruvkun Gary
Published: August 2003

The activity of transcription factors modulates several neural pathways
that mediate complex behaviors. We describe here the role of the POU
transcription factor UNC-86 in the olfactory behavior of
Caenorhabditis
elegans. unc-86
-null mutants are defective in response to odor
attractants but avoid odor repellents normally. Continuous UNC-86 activity is
necessary for maintenance of odortaxis behavior; hyperactivation of UNC-86 by
fusion to a VP16 activation domain dramatically enhances sensitivity to odor
attractants and promotes odor-attractant adaptation. UNC-86 is not expressed
in olfactory sensory neurons but is expressed throughout the life of the
animal in the AIZ interneurons of the odorsensory pathway. We suggest that
UNC-86 transcriptional activity regulates the expression of genes that mediate
synaptic properties of AIZ and that hyperactive UNC-86::VP16 may enhance the
expression of synaptic components to affect the capacity to analyze and
process sensory information.
Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter
Authors: Yasothornsrikul Sukkid , Greenbaum Doron , Medzihradszky Katalin F. , Toneff Thomas , Bundey Richard , Miller Ruthellen , Schilling Birgit , Petermann Ivonne , Dehnert Jessica , Logvinova Anna , Goldsmith Paul , Neveu John M. , Lane William S. , Gibson Bradford , Reinheckel Thomas , Peters Christoph , Bogyo Matthew , Hook Vivian
Published: August 2003

Multistep proteolytic mechanisms are essential for converting proprotein
precursors into active peptide neurotransmitters and hormones. Cysteine
proteases have been implicated in the processing of proenkephalin and other
neuropeptide precursors. Although the papain family of cysteine proteases has
been considered the primary proteases of the lysosomal degradation pathway,
more recent studies indicate that functions of these enzymes are linked to
specific biological processes. However, few protein substrates have been
described for members of this family. We show here that secretory vesicle
cathepsin L is the responsible cysteine protease of chromaffin granules for
converting proenkephalin to the active enkephalin peptide neurotransmitter.
The cysteine protease activity was identified as cathepsin L by affinity
labeling with an activity-based probe for cysteine proteases followed by mass
spectrometry for peptide sequencing. Production of [Met]enkephalin by
cathepsin L occurred by proteolytic processing at dibasic and monobasic
prohormone-processing sites. Cellular studies showed the colocalization of
cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine
chromaffin cells by immunofluorescent confocal and immunoelectron microscopy.
Functional localization of cathepsin L to the regulated secretory pathway was
demonstrated by its cosecretion with [Met]enkephalin. Finally, in cathepsin L
gene knockout mice, [Met]enkephalin levels in brain were reduced
significantly; this occurred with an increase in the relative amounts of
enkephalin precursor. These findings indicate a previously uncharacterized
biological role for secretory vesicle cathepsin L in the production of
[Met]enkephalin, an endogenous peptide neurotransmitter.
An activated receptor tyrosine kinase, TEL/PDGFβR, cooperates with AML1/ETO to induce acute myeloid leukemia in mice
Authors: Grisolano Jay L. , O'Neal Julie , Cain Jennifer , Tomasson Michael H.
Published: August 2003

The t(8;21)(q22;q22) translocation, occurring in 40% of patients with acute
myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation), results in
expression of the
RUNX1-CBF2T1
[
AML1
-
ETO
(
AE
)] fusion oncogene. AML/ETO may contribute to leukemogenesis by
interacting with nuclear corepressor complexes that include histone
deacetylases, which mediate the repression of target genes. However,
expression of
AE
is not sufficient to transform primary hematopoietic
cells or cause disease in animals, suggesting that additional mutations are
required. Activating mutations in receptor tyrosine kinases (RTK) are present
in at least 30% of patients with AML. To test the hypothesis that activating
RTK mutations cooperate with
AE
to cause leukemia, we transplanted
retrovirally transduced murine bone marrow coexpressing
TEL-PDGFRB
and
AE
into lethally irradiated syngeneic mice. These mice (19/19,
100%) developed AML resembling M2-AML that was transplantable in secondary
recipients. In contrast, control mice coexpressing with
TEL-PDGFRB
and a DNA-binding-mutant of
AE
developed a nontransplantable
myeloproliferative disease identical to that induced by
TEL-PDGFRB
alone. We used this unique model of AML to test the efficacy of
pharmacological inhibition of histone deacetylase activity by using
trichostatin A and suberoylanilide hydroxamic acid alone or in combination
with the tyrosine kinase inhibitor, imatinib mesylate. We found that although
imatinib prolonged the survival of treated mice, histone deacetylase
inhibitors provided no additional survival benefit. These data demonstrate
that an activated RTK can cooperate with
AE
to cause AML in mice, and
that this system can be used to evaluate novel therapeutic strategies.
Estrogen receptor-α mediates the brain antiinflammatory activity of estradiol
Authors: Vegeto Elisabetta , Belcredito Silvia , Etteri Sabrina , Ghisletti Serena , Brusadelli Alessia , Meda Clara , Krust Andrée , Dupont Sonia , Ciana Paolo , Chambon Pierre , Maggi Adriana
Published: August 2003

Beyond the key role in reproductive and cognitive functions, estrogens have
been shown to protect against neurodegeneration associated with acute and
chronic injuries of the adult brain. Current hypotheses reconcile this
activity with a direct effect of 17β-estradiol (E
2
) on
neurons. Here we demonstrate that brain macrophages are also involved in
E
2
action on the brain. Systemic administration of hormone
prevents, in a time- and dose-dependent manner, the activation of microglia
and the recruitment of peripheral monocytes induced by intraventricular
injection of lipopolysaccharide. This effect occurs by limiting the expression
of neuroinflammatory mediators, such as the matrix metalloproteinase 9 and
lysosomal enzymes and complement C3 receptor, as well as by preventing
morphological changes occurring in microglia during the inflammatory response.
By injecting lipopolysaccharide in estrogen receptor (ER)-null mouse brains,
we demonstrate that hormone action is mediated by activation of ERα but
not of ERβ. The specific role of ERα is further confirmed by
comparing the effects of ERs on the matrix metalloproteinase 9 promoter
activity in transient transfection assays. Finally, we report that genetic
ablation of ERα is associated with a spontaneous reactive phenotype of
microglia in specific brain regions of adult ERα-null mice. Altogether,
these results reveal a previously undescribed function for E
2
in
brain and provide a mechanism for its beneficial activity on neuroinflammatory
pathologies. They also underline the key role of ERα in brain macrophage
reactivity and hint toward the usefulness of ERα-specific drugs in
hormone replacement therapy of inflammatory diseases.
Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin
Authors: Yan Elsa C. Y. , Kazmi Manija A. , Ganim Ziad , Hou Jian-Min , Pan Douhai , Chang Belinda S. W. , Sakmar Thomas P. , Mathies Richard A.
Published: August 2003

The biological function of Glu-181 in the photoactivation process of
rhodopsin is explored through spectroscopic studies of site-specific mutants.
Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are
nearly identical to spectra of the native pigment, supporting the view that
Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the
absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is
investigated, revealing a dramatic shift of its Schiff base pK
a
compared with the native pigment. This result is most consistent with the
assignment of Glu-181 as the primary counterion of the retinylidene protonated
Schiff base in the Meta I state, implying that there is a counterion switch
from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where
the counterion switch occurs by transferring a proton from Glu-181 to Glu-113
through an H-bond network formed primarily with residues on extracellular loop
II (EII). The resulting reorganization of EII is then coupled to movements of
helix III through a conserved disulfide bond (Cys110–Cys187); this
process may be a general element of G protein-coupled receptor activation.
Pheromone gland-specific fatty-acyl reductase of the silkmoth,
Bombyx mori
Authors: Moto Ken'ichi , Yoshiga Toyoshi , Yamamoto Masanobu , Takahashi Shunya , Okano Kazuhiro , Ando Tetsu , Nakata Tadashi , Matsumoto Shogo
Published: August 2003

The C
10
-C
18
unsaturated, acyclic, aliphatic compounds
that contain an oxygenated functional group (alcohol, aldehyde, or acetate
ester) are a major class of sex pheromones produced by female moths. In the
biosynthesis of these pheromone components, the key enzyme required to produce
the oxygenated functional groups is fatty-acyl reductase (FAR). This enzyme
converts fatty-acyl pheromone precursors to their corresponding alcohols,
which, depending on the moth species, can then be acetylated or oxidized to
the corresponding aldehydes. Despite the significant role this enzyme has in
generating the species-specific oxygenated constituents of lepidopteran sex
pheromones, the enzyme has yet to be fully characterized and identified. In
experiments designed to characterize a pheromone-gland-specific FAR in the
silkmoth,
Bombyx mori
, we have isolated a cDNA clone encoding a
protein homologous to a FAR from the desert shrub,
Simmondsia
chinensis
, commonly known as jojoba. The deduced amino acid sequence of
this clone predicts a 460-aa protein with a consensus NAD(P)H binding motif
within the amino terminus. Northern blot analysis indicated that 2-kb
transcripts of this gene were specifically expressed in the pheromone gland at
1 day before adult eclosion. Functional expression of this gene in the yeast
Saccharomyces cerevisiae
not only confirmed the long-chain FAR
activity, but also indicated a distinct substrate specificity. Finally, the
transformed yeast cells evoked typical mating behavior in male moths when
cultured with the pheromone precursor fatty acid,
(
E,Z
)-10,12-hexadecadienoic acid.
Controlling the size of nanoscale toroidal DNA condensates with static curvature and ionic strength
Authors: Conwell Christine C. , Vilfan Igor D. , Hud Nicholas V.
Published: August 2003

The process of DNA condensation into nanometer-scale particles has direct
relevance to several fields, including cell biology, virology, and gene
delivery for therapeutic purposes. DNA condensation has also attracted the
attention of polymer physicists, as the collapse of DNA molecules from
solution into well defined particles represents an exquisite example of a
polymer phase transition. Here we present a quantitative study of DNA toroids
formed by condensation of 3 kb DNA with hexammine cobalt (III). The presence
or absence of static loops within this DNA molecule demonstrates the effect of
nucleation loop size on toroid dimensions and that nucleation is principally
decoupled from toroid growth. A comparison of DNA condensates formed at low
ionic strength with those formed in the presence of additional salts (NaCl or
MgCl
2
) shows that toroid thickness is a salt-dependant phenomenon.
Together, these results have allowed the development of models for DNA toroid
formation in which the size of the nucleation loop directly influences the
diameter of the fully formed toroid, whereas solution conditions govern toroid
thickness. The data presented illustrate the potential that exists for
controlling DNA toroid dimensions. Furthermore, this study provides a set of
data that should prove useful as a test for theoretical models of DNA
condensation.
Ligand-dependent and -independent effects of splice variant 1 of growth hormone-releasing hormone receptor
Authors: Kiaris Hippokratis , Chatzistamou Ioulia , Schally Andrew V. , Halmos Gabor , Varga Jozsef L. , Koutselini Helen , Kalofoutis Anastasios
Published: August 2003

Existing evidence indicates that, in addition to its neuroendocrine action, growth hormone-releasing hormone (GHRH) acts directly on several nonpituitary tissues, especially neoplasms, and stimulates cell proliferation. We have recently reported that a splice variant of the receptor (SV1) is expressed in various normal tissues and particularly in tumor tissues, producing mitogenic effects on GHRH binding. By using HEC-1A human endometrial carcinoma cells, which express endogenous SV1, we show that, in addition to its ability to mediate the mitogenic effects of GHRH, SV1 also possesses relatively high intrinsic, ligand-independent activity. By using an antisense RNA-based approach we found that SV1 ablation reduces the efficacy of colony formation and the rate of cell proliferation of HEC-1A cells in the absence of exogenous GHRH, and decreases their sensitivity to GHRH when the neurohormone is added to the culture media. This ligand-independent stimulation of cell proliferation appears to be a characteristic property of the truncated form of the receptor, because the expression of SV1 and not of the full-length GHRH receptor stimulated the proliferation of 3T3 fibroblasts in the absence of exogenous GHRH, whereas both forms mediated the proliferative effects of GHRH. Evaluation of 21 specimens of human primary endometrial carcinoma for expression of SV1 by immunohistochemistry indicated that in contrast to the GHRH receptor, which is absent, SV1 is expressed in ≈43% of the specimens. These findings indicate that SV1 can operate in a ligand-independent as well as a ligand-dependent manner. The overexpression of this form of GHRH receptor may be associated with carcinogenesis.
Transcriptional regulatory cascades in development: Initial rates, not steady state, determine network kinetics
Authors: Bolouri Hamid , Davidson Eric H.
Published: August 2003

A model was built to examine the kinetics of regulatory cascades such as
occur in developmental gene networks. The model relates occupancy of
cis-regulatory target sites to transcriptional initiation rate, and thence to
RNA and protein output. The model was used to simulate regulatory cascades in
which genes encoding transcription factors are successively activated. Using
realistic parameter ranges based on extensive earlier measurements in sea
urchin embryos, we find that transitions of regulatory states occur sharply in
these simulations, with respect to time or changing transcription factor
concentrations. As is often observed in developing systems, the simulated
regulatory cascades display a succession of gene activations separated by
delays of some hours. The most important causes of this behavior are
cooperativity in the assembly of cis-regulatory complexes and the high
specificity of transcription factors for their target sites. Successive
transitions in state occur long in advance of the approach to steady-state
levels of the molecules that drive the process. The kinetics of such
developmental systems thus depend mainly on the initial output rates of genes
activated in response to the advent of new transcription factors.
Toward a protein profile of
Escherichia coli
: Comparison to its transcription profile
Authors: Corbin Rebecca W. , Paliy Oleg , Yang Feng , Shabanowitz Jeffrey , Platt Mark , Lyons Charles E. , Root Karen , McAuliffe Jon , Jordan Michael I. , Kustu Sydney , Soupene Eric , Hunt Donald F.
Published: August 2003

High-pressure liquid chromatography–tandem mass spectrometry was used
to obtain a protein profile of
Escherichia coli
strain MG1655 grown
in minimal medium with glycerol as the carbon source. By using cell lysate
from only 3 × 10
8
cells, at least four different tryptic
peptides were detected for each of 404 proteins in a short 4-h experiment. At
least one peptide with a high reliability score was detected for 986 proteins.
Because membrane proteins were underrepresented, a second experiment was
performed with a preparation enriched in membranes. An additional 161 proteins
were detected, of which from half to two-thirds were membrane proteins.
Overall, 1,147 different
E. coli
proteins were identified, almost 4
times as many as had been identified previously by using other tools. The
protein list was compared with the transcription profile obtained on
Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein
products were found was detected at the mRNA level. The arithmetic mean mRNA
signal intensity for these genes was 3-fold higher than that for all 4,300
protein-coding genes of
E. coli
. Thus, GeneChip data confirmed the
high reliability of the protein list, which contains about one-fourth of the
proteins of
E. coli
. Detection of even those membrane proteins and
proteins of undefined function that are encoded by the same operons
(transcriptional units) encoding proteins on the list remained low.
The p53-dependent effects of macrophage migration inhibitory factor revealed by gene targeting
Authors: Fingerle-Rowson G. , Petrenko O. , Metz C. N. , Forsthuber T. G. , Mitchell R. , Huss R. , Moll U. , Müller W. , Bucala R.
Published: August 2003

Macrophage migration inhibitory factor (MIF) is a mediator of host immunity and functions as a high, upstream activator of cells within the innate and the adaptive immunological systems. Recent studies have suggested a potentially broader role for MIF in growth regulation because of its ability to antagonize p53-mediated gene activation and apoptosis. To better understand MIF's activity in growth control, we generated and characterized a strain of MIF-knockout (MIF-KO) mice in the inbred, C57BL/6 background. Embryonic fibroblasts from MIF-KO mice exhibit p53-dependent growth alterations, increased p53 transcriptional activity, and resistance toras-mediated transformation. Concurrent deletion of the p53 genein vivoreversed the observed phenotype of cells deficient in MIF.In vivostudies showed that fibrosarcomas induced by the carcinogen benzo[α]pyrene are smaller in size and have a lower mitotic index in MIF-KO mice relative to their WT counterparts. The data provide direct genetic evidence for a functional link between MIF and the p53 tumor suppressor and indicate an important and previously unappreciated role for MIF in carcinogenesis.
Crystal structure of an RNA·DNA hybrid reveals intermolecular intercalation: Dimer formation by base-pair swapping
Authors: Han Gye Won , Kopka Mary L. , Langs David , Sawaya Michael R. , Dickerson Richard E.
Published: August 2003

An intermolecular intercalation of base pairs was found at the CA step in
the I222 crystal structure of the RNA·DNA hybrid,
r(CAAAGAAAAG)·d(CTTTTCTTTG), which contains two-thirds of the
polypurine tract sequence of HIV-1 with a substitution of cytosine for the
initial adenine. This sequence crystallized in both
P2
1
2
1
2
1
and I222 space groups, with an rms
difference of only 0.63 Å between residues 3 to 18 of the two forms.
P2
1
2
1
2
1
and I222 helices are both A-like, but
intercalation occurs only in the I222 crystal form. The present structure
shows bases stacked in parallel rather than perpendicular as in intercalated
DNA (I-DNA). The base intercalation is also different from zipper-like meshing
of bases seen in the center of the crystal structure of d(GCGAAAGCT), which
does not have Watson–Crick base pairing. The base-step intercalation
seen here is reminiscent of domain swapping in proteins; therefore, we call
this phenomenon “base-pair swapping.” It involves a highly mobile
CA step and seems to be sequence-specific and electrostatically stable without
disrupting Watson–Crick interactions. It also exhibits a large rise
concurrent with unwinding of the helix (low twist). We present a base-pair
swapping dimer in nucleic acids.
Protein–DNA interaction mapping using genomic tiling path microarrays in
Drosophila
Authors: Sun Ling V. , Chen Liang , Greil Frauke , Negre Nicolas , Li Tong-Ruei , Cavalli Giacomo , Zhao Hongyu , van Steensel Bas , White Kevin P.
Published: August 2003

We demonstrate the use of a chromosomal walk (or “tiling path”)
printed as DNA microarrays for mapping protein–DNA interactions across
large regions of contiguous genomic DNA in
Drosophila melanogaster
.
Microarrays were constructed with genomic DNA fragments 430–920 bp in
length, covering 2.9 million base pairs of the
Adh
–
cactus
region of chromosome 2 and 85,000 base
pairs of the
82F
region of chromosome 3. We performed DNA
localization mapping for the heterochromatin protein HP1 and for the
sequence-specific GAGA transcription factor, producing a comprehensive,
high-resolution map of
in vivo
protein–DNA interactions
throughout these regions of the
Drosophila
genome.
Biosynthesis of isoprenoids: Crystal structure of 4-diphosphocytidyl-2C-methyl-
d
-erythritol kinase
Authors: Miallau Linda , Alphey Magnus S. , Kemp Lauris E. , Leonard Gordon A. , McSweeney Sean M. , Hecht Stefan , Bacher Adelbert , Eisenreich Wolfgang , Rohdich Felix , Hunter William N.
Published: August 2003

4-Diphosphocytidyl-2
C
-methyl-
d
-erythritol kinase, an
essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and
dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent
phosphorylation stage affording
4-diphosphocytidyl-2
C
-methyl-
d
-erythritol-2-phosphate. The
2-Å resolution crystal structure of the
Escherichia coli
enzyme
in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals
the molecular determinants of specificity and catalysis. The enzyme subunit
displays the α/β fold characteristic of the galactose
kinase/homoserine kinase/mevalonate kinase/phosphomevalonate kinase
superfamily, arranged into cofactor and substrate-binding domains with the
catalytic center positioned in a deep cleft between domains. Comparisons with
related members of this superfamily indicate that the core regions of each
domain are conserved, whereas there are significant differences in the
substrate-binding pockets. The nonmevalonate pathway is essential in many
microbial pathogens and distinct from the mevalonate pathway used by mammals.
The high degree of sequence conservation of the enzyme across bacterial
species suggests similarities in structure, specificity, and mechanism. Our
model therefore provides an accurate template to facilitate the
structure-based design of broad-spectrum antimicrobial agents.
Bad
-deficient mice develop diffuse large B cell lymphoma
Authors: Ranger Ann M. , Zha Jiping , Harada Hisashi , Datta Sandeep Robert , Danial Nika N. , Gilmore Andrew P. , Kutok Jeffery L. , Le Beau Michelle M. , Greenberg Michael E. , Korsmeyer Stanley J.
Published: August 2003

The proapoptotic activity of the “BH3-only” molecule BAD can be
differentially regulated by survival factor signaling.
Bad
-deficient
mice lacking both BAD long and BAD short proteins proved viable, and most cell
types appeared to develop normally. BAD did not exclusively account for cell
death after withdrawal of survival factors, but it was an intermediate for
epidermal growth factor- or insulin-like growth factor I-countered apoptosis,
consistent with a “sensitizing” BH3-only molecule. Lymphocytes
developed normally with no premalignant hyperplasia, but they displayed subtle
abnormalities in proliferation and IgG production. Despite the minimal
phenotype,
Bad
-deficient mice progressed, with aging, to diffuse
large B cell lymphoma of germinal center origin. Exposure of
Bad
-null
mice to sublethal γ-irradiation resulted in an increased incidence of
pre-T cell and pro-/pre-B cell lymphoblastic leukemia/lymphoma. Thus,
proapoptotic BAD suppresses tumorigenesis in the lymphocyte lineage.
Executive and social behaviors under nicotinic receptor regulation
Authors: Granon Sylvie , Faure Philippe , Changeux Jean-Pierre
Published: August 2003

Nicotine enhances several cognitive and psychomotor behaviors, and nicotinic antagonists cause impairments in tasks requiring cognitive effort. To explore the contribution of nicotinic receptors to complex cognitive functions, we developed an automated method to investigate sequential locomotor behavior in the mouse and an analysis of social behavior. We show that, in the β2-/-mutant, the high-order spatiotemporal organization of locomotor behavior, together with conflict resolution and social interaction, is selectively dissociated from low-level, more automatic motor behaviors. Such deficits in executive functions resemble the rigid and asocial behavior found in some psychopathological disorders such as autism and attention deficit hyperactivity disorder.
The genome sequence of
Blochmannia floridanus
: Comparative analysis of reduced genomes
Authors: Gil Rosario , Silva Francisco J. , Zientz Evelyn , Delmotte François , González-Candelas Fernando , Latorre Amparo , Rausell Carolina , Kamerbeek Judith , Gadau Jürgen , Hölldobler Bert , van Ham Roeland C. H. J. , Gross Roy , Moya Andrés
Published: August 2003

Bacterial symbioses are widespread among insects, probably being one of the
key factors of their evolutionary success. We present the complete genome
sequence of
Blochmannia floridanus
, the primary endosymbiont of
carpenter ants. Although these ants feed on a complex diet, this symbiosis
very likely has a nutritional basis:
Blochmannia
is able to supply
nitrogen and sulfur compounds to the host while it takes advantage of the host
metabolic machinery. Remarkably, these bacteria lack all known genes involved
in replication initiation (
dna
A,
pri
A, and
rec
A).
The phylogenetic analysis of a set of conserved protein-coding genes shows
that
Bl. floridanus
is phylogenetically related to
Buchnera
aphidicola
and
Wigglesworthia glossinidia
, the other
endosymbiotic bacteria whose complete genomes have been sequenced so far.
Comparative analysis of the five known genomes from insect endosymbiotic
bacteria reveals they share only 313 genes, a number that may be close to the
minimum gene set necessary to sustain endosymbiotic life.