bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid amyloid fiber formation from the fast-folding WW domain FBP28
Authors: Ferguson Neil , Berriman John , Petrovich Miriana , Sharpe Timothy D. , Finch John T. , Fersht Alan R.
Published: August 2003

The WW domains are small proteins that contain a three-stranded,
antiparallel β-sheet. The 40-residue murine FBP28 WW domain rapidly
formed twirling ribbon-like fibrils at physiological temperature and pH, with
morphology typical of amyloid fibrils. These ribbons were unusually wide and
well ordered, making them highly suitable for structural studies. Their x-ray
and electron-diffraction patterns displayed the characteristic amyloid fiber
0.47-nm reflection of the cross-β diffraction signature. Both
conventional and electron cryomicroscopy showed clearly that the ribbons were
composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis
of the fiber. There was a region of lower density along the center of each
filament. Lateral association of these filaments generated twisted, often
interlinked, sheets up to 40 nm wide and many microns in length. The pitch of
the helix varied from 60 to 320 nm, depending on the width of the ribbon. The
wild-type FBP28 fibers were formed under conditions in which multiexponential
folding kinetics is observed in other studies and which was attributed to a
change in the mechanism of folding. It is more likely that those phases result
from initial events in the off-pathway aggregation observed here.
Targeted gene evolution in
Escherichia coli
using a highly error-prone DNA polymerase I
Authors: Camps Manel , Naukkarinen Jussi , Johnson Ben P. , Loeb Lawrence A.
Published: August 2003

We present a system for random mutagenesis in
Escherichia coli
for
the evolution of targeted genes. To increase error rates of DNA polymerase I
(Pol I) replication, we introduced point mutations in three structural domains
that govern Pol I fidelity. Expression of error-prone Pol I
in vivo
results in strong mutagenesis of a target sequence encoded in a Pol
I-dependent plasmid (8.1 × 10
–
4
mutations
per bp, an 80,000-fold increase), with a preference for plasmid relative to
chromosome sequence. Mutagenesis is maximal in cultures maintained at
stationary phase. Mutations are evenly distributed and show a variety of base
pair substitutions, predominantly transitions. Mutagenesis extends at least 3
kb beyond the 400–500 nt reportedly synthesized by Pol I. We demonstrate
that our error-prone Pol I can be used to generate enzymes with distinct
properties by generating TEM-1 β-lactamase mutants able to hydrolyze a
third-generation lactam antibiotic, aztreonam. Three different mutations
contribute to aztreonam resistance. Two are found in the extended-spectrum
β-lactamases most frequently identified in clinical isolates, and the
third (G276R) has not been previously described. Our system of targeted
mutagenesis in
E. coli
should have an impact on enzyme-based
applications in areas such as synthetic chemistry, gene therapy, and molecular
biology. Given the structural conservation between polymerases, this work
should also provide a reference for altering the fidelity of other
polymerases.
Predator diversity hotspots in the blue ocean
Authors: Worm Boris , Lotze Heike K. , Myers Ransom A.
Published: August 2003

Concentrations of biodiversity, or hotspots, represent conservation priorities in terrestrial ecosystems but remain largely unexplored in marine habitats. In the open ocean, many large predators such as tunas, sharks, billfishes, and sea turtles are of current conservation concern because of their vulnerability to overfishing and ecosystem role. Here we use scientific-observer records from pelagic longline fisheries in the Atlantic and Pacific Oceans to show that oceanic predators concentrate in distinct diversity hotspots. Predator diversity consistently peaks at intermediate latitudes (20–30° N and S), where tropical and temperate species ranges overlap. Individual hotspots are found close to prominent habitat features such as reefs, shelf breaks, or seamounts and often coincide with zooplankton and coral reef hotspots. Closed-area models in the northwest Atlantic predict that protection of hotspots outperforms other area closures in safeguarding threatened pelagic predators from ecological extinction. We conclude that the seemingly monotonous landscape of the open ocean shows rich structure in species diversity and that these features should be used to focus future conservation efforts.
Characterization of myotubularin-related protein 7 and its binding partner, myotubularin-related protein 9
Authors: Mochizuki Yasuhiro , Majerus Philip W.
Published: August 2003

Myotubularin-related protein 7 (MTMR7) is a member of the myotubularin
(MTM) family. The cDNA encoding the mouse MTMR7 contains 1,983 bp, and the
predicted protein has a deduced molecular mass of 75.6 kDa. Northern and
Western blot analyses showed that MTMR7 is expressed mainly in brain and mouse
neuroblastoma N1E-115 cells. Recombinant MTMR7 dephosphorylated the D-3
position of phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate
[Ins(1,3)P
2
]. The substrate specificity of MTMR7 is different than
other MTM proteins in that this enzyme prefers the water-soluble substrate.
Immunofluorescence showed that MTMR7 is localized in Golgi-like granules and
cytosol, and subcellular fractionation showed both cytoplasmic and membrane
localization of MTMR7 in N1E-115 cells. An MTMR7-binding protein was found in
an anti-MTMR7 immunoprecipitate from N1E-115 cells and identified as
MTM-related protein 9 (MTMR9) by tandem mass spectrometry. The coiled-coil
domain of MTMR9 was sufficient for binding to MTMR7. The binding of MTMR9
increased the Ins(1,3)P
2
phosphatase activity of MTMR7. Our results
show that MTMR7 forms a complex with MTMR9 and dephosphorylates
phosphatidylinositol 3-phosphate and Ins(1,3)P
2
in neuronal
cells.
Proteasome inhibitors disrupt the unfolded protein response in myeloma cells
Authors: Lee Ann-Hwee , Iwakoshi Neal N. , Anderson Kenneth C. , Glimcher Laurie H.
Published: August 2003

Novel agents that target the proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, have demonstrated remarkable therapeutic efficacy in multiple myeloma, a plasma cell malignancy. However, the mechanism by which these compounds act remains unknown. A signaling pathway called the unfolded protein response (UPR) allows cells to handle the proper folding of proteins. The transcription factor XBP-1, a regulator of the UPR, is also required for plasma cell differentiation, suggesting a link between the UPR and plasma cell differentiation. Here we show that proteasome inhibitors target XBP-1 and the UPR in myeloma cells. Proteasome inhibitors suppress the activity of the translumenal endoplasmic reticulum endoribonuclease/kinase, IRE1α, to impair the generation of the active, spliced XBP-1 species and simultaneously stabilize the unspliced species that acts as a dominant negative. Myeloma cells rendered functionally deficient in XBP-1 undergo increased apoptosis in response to endoplasmic reticulum stress. Identification of compounds that target the activity of IRE1α/XBP-1 may yield novel therapies for the treatment of multiple myeloma and other malignancies that rely on an intact UPR.
Averaging and renormalization for the Korteveg–deVries–Burgers equation
Authors: Chorin Alexandre J.
Published: August 2003

We consider traveling wave solutions of the
Korteveg–deVries–Burgers equation and set up an analogy between
the spatial averaging of these traveling waves and real-space renormalization
for Hamiltonian systems. The result is an effective equation that reproduces
means of the unaveraged, highly oscillatory, solution. The averaging enhances
the apparent diffusion, creating an “eddy” (or renormalized)
diffusion coefficient; the relation between the eddy diffusion coefficient and
the original diffusion coefficient is found numerically to be one of
incomplete similarity, setting up an instance of Barenblatt's renormalization
group. The results suggest a relation between self-similar solutions of
differential equations on one hand and renormalization groups and optimal
prediction algorithms on the other. An analogy with hydrodynamics is pointed
out.
A modular, bifunctional RNA that integrates itself into a target RNA
Authors: Kumar Roshan M. , Joyce Gerald F.
Published: August 2003

Nature often combines independent functional domains to achieve complex
function, but this approach has not been extensively explored with artificial
enzymes. Here, a group I ribozyme, which can act as an endoribonuclease, was
partnered with the R3C ribozyme, which catalyzes the ligation of RNA
molecules. The conjoined ribozymes have the potential to perform successive
RNA cleavage and joining reactions, resulting in their mutual integration into
a target RNA substrate. When simply joined together, however, the ribozymes
were unable to achieve this outcome because of inefficient transfer of the
substrate between the two catalytic subunits.
In vitro
evolution was
used to optimize the behavior of the conjoined ribozymes, resulting in
bifunctional molecules with substantially improved integration activity. The
ligase subunit of these molecules was unchanged, whereas the group I subunit
acquired several mutations, mostly in peripheral regions. The generation and
study of this bifunctional assembly helps shed light on the evolution of
modular enzymes and the obstacles that must be overcome in bringing together
independent functional domains. These molecules also may be useful as tools
for the insertional mutagenesis of target mRNAs.
Generation of systemin signaling in tobacco by transformation with the tomato systemin receptor kinase gene
Authors: Scheer Justin M. , Pearce Gregory , Ryan Clarence A.
Published: August 2003

The tomato systemin receptor, SR160, a plasma membrane-bound, leucine-rich
repeat receptor kinase that signals systemic plant defense, and the
brassinolide (BL) receptor, BRI1, that regulates developmental processes, have
been shown recently to have identical amino acid sequences. We report herein
that tobacco, a solanaceous species that does not express a systemin precursor
gene nor responds to systemin, when transformed with the SR160 receptor gene,
expresses the gene in suspension-cultured cells, evidenced by mRNA and protein
analyses and photoaffinity-labeling experiments. Additionally, systemin
induced an alkalinization response in the transgenic tobacco cells similar to
that found in tomato cells, but not in WT cells. The gain in function in
tobacco cells indicates that early steps of the systemin signaling pathway
found in tomato are present in tobacco cells. A tomato line,
cu-3
, in
which a mutation in the BRI1 gene has rendered the plant nonfunctional in BL
signaling, exhibits a severely reduced response to systemin. In leaves of WT
tomato plants, BL strongly and reversibly antagonized systemic signaling by
systemin. The results suggest that the systemin-mediated systemic defense
response may have evolved in some solanaceous species by co-opting the BRI1
receptor and associated components for defense signaling.
Attenuation of FLOWERING LOCUS C activity as a mechanism for the evolution of summer-annual flowering behavior in
Arabidopsis
Authors: Michaels Scott D. , He Yuehui , Scortecci Katia C. , Amasino Richard M.
Published: August 2003

Plant species have evolved a wide variety of flowering habits, each adapted
to maximize reproductive success in their local environment. Even within a
species, accessions from different environments can exhibit markedly different
flowering behavior. In
Arabidopsis
, some accessions are rapid-cycling
summer annuals, whereas others accessions are late flowering and vernalization
responsive and thus behave as winter annuals. Two genes,
FLOWERING LOCUS
C
(
FLC
) and
FRIGIDA
(
FRI
), interact
synergistically to confer the winter-annual habit. Previous work has shown
that many summer-annual accessions contain null mutations in the
FRI
gene; thus it appears that these summer-annual accessions have arisen from
winter-annual ancestors by losing
FRI
function. In this work we
demonstrate that naturally occurring allelic variation in
FLC
has
provided another route to the evolution of summer-annual flowering behavior in
Arabidopsis
. We have identified two summer-annual accessions, Da
(1)-12 and Shakhdara, that contain functional alleles of
FRI
, but are
early flowering because of weak alleles of
FLC
. We have also
determined that the weak allele of
FLC
found in Landsberg
erecta
is naturally occurring. Unlike accessions that have arisen
because of loss-of-function mutations in
FRI
, the
FLC
alleles from Da (1)-12, Shakhdara, and Landsberg
erecta
are not
nulls; however, they exhibit lower steady-state mRNA levels than strong
alleles of
FLC
. Sequence analysis indicates that these weak alleles
of
FLC
have arisen independently at least twice during the course of
evolution.
Optimal design of a bistable switch
Authors: Brenner Michael P. , Lang Jeffrey H. , Li Jian , Qiu Jin , Slocum Alexander H.
Published: August 2003

Determining optimally designed structures is important for diverse fields
of science and engineering. Here we describe a procedure for calculating the
optimal design of a switch and apply the method to a bistable
microelectromechanical system relay switch. The approach focuses on
characterizing the unstable transition state connecting the two stable
equilibria to control the force displacements. Small modifications in
component shape lead to a substantial improvement in device operation.
Fabrication of the optimized devices confirms the predictions.
Enhanced diffusion-edited NMR spectroscopy of mixtures using chromatographic stationary phases
Authors: Viel Stéphane , Ziarelli Fabio , Caldarelli Stefano
Published: August 2003

We introduce an analytical method that combines in one pot the advantages
of column chromatography separation and NMR structural analysis. The
separation of the NMR spectra of the components of a mixture can be achieved
according to their apparent diffusion rates [James, T. L. and McDonald, G. G.
(1973)
J. Magn. Reson
. 58, 58–61]. We show that the separation
of the spectral components, corresponding to single molecular species, can be
enhanced by order of magnitudes upon addition of a typical stationary phase
used in HPLC. The solid phase imbibed by the mixture for analysis is an
heterogeneous ensemble, so that solid-state NMR methods (high-resolution magic
angle spinning) are necessary to recover high-resolution spectra. We
demonstrate applications of this combination of high-resolution magic angle
spinning and NMR diffusometry on test mixtures for direct (silica gel) and
inverse (C18) columns. However, many common chromatographic supports available
for HPLC should be readily adaptable for use with this technique.
Neurodegeneration and defective neurotransmission in a
Caenorhabditis elegans
model of tauopathy
Authors: Kraemer Brian C. , Zhang Bin , Leverenz James B. , Thomas James H. , Trojanowski John Q. , Schellenberg Gerard D.
Published: August 2003

Frontotemporal dementia with parkinsonism chromosome 17 type (FTDP-17) is
caused by mutations in
MAPT
, the gene encoding tau. FTDP-17 begins
with executive function deficits and other abnormal behaviors, which progress
to dementia. Neurodegenerative changes include accumulation of aggregated tau
as neuronal and glial fibrillary tangles. Aggregated tau is seen in numerous
other neurodegenerative diseases, including Alzheimer's disease (AD). We
expressed normal and FTDP-17 mutant human tau (mutations
P
301
L
and
V
337
M
) in
Caenorhabditis elegans
to model tauopathy disorders. Tau pan-neuronal
expression caused progressive uncoordinated locomotion (Unc), characteristic
of nervous system defects in worms. Subsequently, insoluble tau accumulates
and both soluble and insoluble tau is phosphorylated at many of the sites
hyperphosphorylated in FTDP-17, AD, and other tauopathies. Substantial
neurodegeneration, seen as bulges and gaps in nerve cords followed by loss of
neurons, occurs after insoluble tau begins to accumulate. Axons show vacuoles,
membranous infoldings, and whorls with associated amorphous tau accumulations
and abnormal tau-positive aggregates. FTDP-17 mutation lines had a more severe
Unc phenotype, accumulated more insoluble tau at a younger age, were more
resistant to cholinergic inhibitors, and had more severe axonal degeneration
when compared with lines expressing normal tau. The Unc phenotype is caused by
a presynaptic defect. Postsynaptic transmission is intact. This transgenic
model will enable mechanistic dissection of tau-induced neurodegeneration and
identification of genes and compounds that inhibit pathological tau
formation.
Caspase cleavage of tau: Linking amyloid and neurofibrillary tangles in Alzheimer's disease
Authors: Gamblin T. Chris , Chen Feng , Zambrano Angara , Abraha Aida , Lagalwar Sarita , Guillozet Angela L. , Lu Meiling , Fu Yifan , Garcia-Sierra Francisco , LaPointe Nichole , Miller Richard , Berry Robert W. , Binder Lester I. , Cryns Vincent L.
Published: August 2003

The principal pathological features of Alzheimer's disease (AD) are extracellular amyloid plaques and intracellular neurofibrillary tangles, the latter composed of the microtubule-binding protein tau assembled into paired helical and straight filaments. Recent studies suggest that these pathological entities may be functionally linked, although the mechanisms by which amyloid deposition promotes pathological tau filament assembly are poorly understood. Here, we report that tau is proteolyzed by multiple caspases at a highly conserved aspartate residue (Asp421) in its C terminusin vitroand in neurons treated with amyloid-β (Aβ) (1–42) peptide. Tau is rapidly cleaved at Asp421in Aβ-treated neurons (within 2 h), and its proteolysis appears to precede the nuclear events of apoptosis. We also demonstrate that caspase cleavage of tau generates a truncated protein that lacks its C-terminal 20 amino acids and assembles more rapidly and more extensively into tau filamentsin vitrothan wild-type tau. Using a monoclonal antibody that specifically recognizes tau truncated at Asp421, we show that tau is proteolytically cleaved at this site in the fibrillar pathologies of AD brain. Taken together, our results suggest a novel mechanism linking amyloid deposition and neurofibrillary tangles in AD: Aβ peptides promote pathological tau filament assembly in neurons by triggering caspase cleavage of tau and generating a proteolytic product with enhanced polymerization kinetics.
Rapid evolution of male-biased gene expression inDrosophila
Authors: Meiklejohn Colin D. , Parsch John , Ranz José M. , Hartl Daniel L.
Published: August 2003

A number of genes associated with sexual traits and reproduction evolve at the sequence level faster than the majority of genes coding for non-sex-related traits. Whole genome analyses allow this observation to be extended beyond the limited set of genes that have been studied thus far. We use cDNA microarrays to demonstrate that this pattern holds inDrosophilafor the phenotype of gene expression as well, but in one sex only. Genes that are male-biased in their expression show more variation in relative expression levels between conspecific populations and two closely related species than do female-biased genes or genes with sexually monomorphic expression patterns. Additionally, elevated ratios of interspecific expression divergence to intraspecific expression variation among male-biased genes suggest that differences in rates of evolution may be due in part to natural selection. This finding has implications for our understanding of the importance of sexual dimorphism for speciation and rates of phenotypic evolution.
MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms
Authors: Zeng Yan , Yi Rui , Cullen Bryan R.
Published: August 2003

MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived
by processing of short RNA hairpins, that can inhibit the translation of mRNAs
bearing partially complementary target sequences. In contrast, small
interfering RNAs (siRNAs), which are derived by processing of long
double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing
fully complementary sequences. Here, we demonstrate that an endogenously
encoded human miRNA is able to cleave an mRNA bearing fully complementary
target sites, whereas an exogenously supplied siRNA can inhibit the expression
of an mRNA bearing partially complementary sequences without inducing
detectable RNA cleavage. These data suggest that miRNAs and siRNAs can use
similar mechanisms to repress mRNA expression and that the choice of mechanism
may be largely or entirely determined by the degree of complementary of the
RNA target.
The 15-lipoxygenase-1 product 13-
S
-hydroxyoctadecadienoic acid down-regulates PPAR-δ to induce apoptosis in colorectal cancer cells
Authors: Shureiqi Imad , Jiang Wei , Zuo Xiangsheng , Wu Yuanqing , Stimmel Julie B. , Leesnitzer Lisa M. , Morris Jeffrey S. , Fan Hui-Zhen , Fischer Susan M. , Lippman Scott M.
Published: August 2003

Diminished apoptosis, a critical event in tumorigenesis, is linked to
down-regulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancer
cells. 13-
S
-hydroxyoctadecadienoic acid (13-
S
-HODE), which
is the primary product of 15-LOX-1 metabolism of linoleic acid, restores
apoptosis. Nonsteroidal antiinflammatory drugs (NSAIDs) transcriptionally
up-regulate 15-LOX-1 expression to induce apoptosis. Peroxisome
proliferator-activated receptors (PPARs) are nuclear receptors for linoleic
and arachidonic acid metabolites. PPAR-δ promotes colonic tumorigenesis.
NSAIDs suppress PPAR-δ activity in colon cancer cells. The mechanistic
relationship between 15-LOX-1 and PPAR-δ was previously unknown. Our
current study shows that (
i
) 13-
S
-HODE binds to
PPAR-δ, decreases PPAR-δ activation, and down-regulates
PPAR-δ expression in colorectal cancer cells; (
ii
) the
induction of 15-LOX-1 expression is a critical step in NSAID down-regulation
of PPAR-δ and the resultant induction of apoptosis; and (
iii
)
PPAR-δ is an important signaling receptor for 13-
S
-HODE-induced
apoptosis. The
in vivo
relevance of these mechanistic findings was
demonstrated in our tumorigenesis studies in nude mouse xenograft models. Our
findings indicate that the down-regulation of PPAR-δ by 15-LOX-1 through
13-
S
-HODE is an apoptotic signaling pathway that is activated by
NSAIDs.
The role of Rel
Mtb
-mediated adaptation to stationary phase in long-term persistence of
Mycobacterium tuberculosis
in mice
Authors: Dahl John L. , Kraus Carl N. , Boshoff Helena I. M. , Doan Bernard , Foley Korrie , Avarbock David , Kaplan Gilla , Mizrahi Valerie , Rubin Harvey , Barry Clifton E.
Published: August 2003

Long-term survival of nonreplicating
Mycobacterium tuberculosis
(Mtb) is ensured by the coordinated shutdown of active metabolism through a
broad transcriptional program called the stringent response. In Mtb, this
response is initiated by the enzymatic action of Rel
Mtb
and
deletion of
rel

Mtb

produces a strain
(H37RvΔ
rel

Mtb

) severely compromised in the
maintenance of long-term viability. Although aerosol inoculation of mice with
H37RvΔ
rel

Mtb

results in normal initial
bacterial growth and containment, the ability of this strain to sustain
chronic infection is severely impaired. Significant histopathologic
differences were noted in lungs and spleens of mice infected with
H37RvΔ
rel

Mtb

compared with controls
throughout the course of the infection. Microarray analysis revealed that
H37RvΔ
rel

Mtb

suffers from a generalized
alteration of the transcriptional apparatus, as well as specific changes in
the expression of virulence factors, cell-wall biosynthetic enzymes, heat
shock proteins, and secreted antigens that may alter immune recognition of the
recombinant organism. Thus, Rel
Mtb
is critical for the successful
establishment of persistent infection in mice by altering the expression of
antigenic and enzymatic factors that may contribute to successful latent
infection.
Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases
Authors: Charvin G. , Bensimon D. , Croquette V.
Published: August 2003

Type-II topoisomerases are responsible for untangling DNA during
replication by removing supercoiled and interlinked DNA structures. Using a
single-molecule micromanipulation setup, we follow the real-time decatenation
of two mechanically braided DNA molecules by
Drosophila melanogaster
topoisomerase (Topo) II and
Escherichia coli
Topo IV. Although Topo
II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of
≈2.9 s
–
1
, Topo IV has a marked preference for
L-braids, which it relaxes completely and processively at a rate of ≈2.4
s
–
1
. However, Topo IV can unlink R-braids at about
half that rate when they supercoil to form L-plectonemes. These results imply
that the preferred substrate for unlinking by Topo IV has the symmetry of an
L-crossing and shed new light on the decatenation of daughter strands during
DNA replication, which are usually assumed to be linked in an R-braid.
The sex peptide of
Drosophila melanogaster:
Female post-mating responses analyzed by using RNA interference
Authors: Chapman Tracey , Bangham Jenny , Vinti Giovanna , Seifried Beth , Lung Oliver , Wolfner Mariana F. , Smith Hazel K. , Partridge Linda
Published: August 2003

Mating induces profound changes in female insect behavior and physiology.
In
Drosophila melanogaster
, mating causes a reduction in sexual
receptivity and an elevation in egg production for at least 5 days. Injection
of the seminal fluid sex peptide (SP) induces both responses in virgin
females, but only for 1–2 days. The role of SP in eliciting the
responses to mating remains to be elucidated. Functional redundancy between
seminal fluid components may occur. In addition, mating with spermless males
results in brief (1- to 2-day) post-mating responses, indicating either that
there is a “sperm effect” or that sperm act as carriers for SP or
other seminal fluid components. Here we used RNA interference to suppress SP
expression, to determine whether SP is required to elicit full post-mating
responses, the magnitude of responses due to other seminal fluid components,
and whether SP accounts for the “sperm effect.” Receptivity was
higher and egg production lower in females mated to SP knock-down males than
in controls. Comparison with virgins showed that the responses were brief. SP
is therefore required for normal magnitude and persistence of postmating
responses. Sperm transfer and use were normal in mates of SP knock-down males,
yet their post-mating responses were briefer than after normal matings, and
similar to those reported in mates of spermless
son-of-tudor
males.
The prolonged “sperm effect” on female receptivity and egg
production is therefore entirely attributable to SP, but sperm are necessary
for its occurrence.
Sex-peptide is the molecular basis of the sperm effect in
Drosophila melanogaster
Authors: Liu Huanfa , Kubli Eric
Published: August 2003

Mating elicits two major changes in the reproductive behavior of many
insect females. The egg-laying rate increases and the readiness to accept
males (receptivity) is reduced. These postmating responses last ≈1 week in
Drosophila melanogaster
. Males that do not transfer sperm but
transfer seminal fluid during mating induce a short-term response of 1 day.
The long-term response of 1 week requires the presence of sperm (sperm
effect). Hence, sperm is essential for the long-term persistence of the
postmating responses. Three seminal fluid peptides elicit postmating
responses: ovulin, sex-peptide (SP), and DUP99B. Using the technique of
targeted mutagenesis by homologous recombination, we have produced males with
mutant SP genes. Here, we report that males lacking functional SP elicit only
a weak short-term response. However, these males do transfer sperm. Thus,
(
i
) SP is the major agent eliciting the short-term and the long-term
postmating responses and (
ii
) sperm is merely the carrier for SP. The
second conclusion is supported by the finding that SP binds to sperm. The
36-aa-encoding SP gene is the first small
Drosophila
gene knocked out
with the method of homologous recombination.
Modeling the optimum duration of antibiotic prophylaxis in an anthrax outbreak
Authors: Brookmeyer Ron , Johnson Elizabeth , Bollinger Robert
Published: August 2003

A critical consideration in effective and measured public health responses
to an outbreak of inhalational anthrax is the optimum duration of antibiotic
prophylaxis. We develop a competing-risks model to address the duration of
antibiotic prophylaxis and the incubation period that accounts for the risks
of spore germination and spore clearance. The model predicts the incubation
period distribution, which is confirmed by empirical data. The optimum
duration of antibiotic prophylaxis depends critically on the dose of inhaled
spores. At high doses, we show that exposed persons would need to remain on
antibiotic prophylaxis for at least 4 months, and considerable morbidity would
likely occur before antibiotic prophylaxis could even be initiated. At very
low doses, 60 days of antibiotic prophylaxis is adequate. Exposure doses can
be estimated from the cumulative attack rate up to the point antibiotic
prophylaxis begins. The model explains that whereas ≤60 days of antibiotics
were enough to protect persons in the 2001 U.S. outbreak, because doses were
very low, at moderate or high doses considerably longer durations would be
necessary to adequately protect exposed populations.
When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism
Authors: Roy Hervé , Becker Hubert Dominique , Reinbolt Joseph , Kern Daniel
Published: August 2003

Faithful protein synthesis relies on a family of essential enzymes called
aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the
completed archaeal genomes reveals that all archaea that possess
asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an
AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein
that
Pyrococcus abyssi
AsnRS2, in contrast to AsnRS, does not sustain
asparaginyl-tRNA
Asn
synthesis but is instead capable of converting
aspartic acid into asparagine. Functional analysis and complementation of an
Escherichia coli
asparagine auxotrophic strain show that AsnRS2
constitutes the archaeal homologue of the bacterial ammonia-dependent
asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase
A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal
AspRS ancestor originated AS-AR, which was subsequently transferred into
bacteria by lateral gene transfer in which it underwent structural changes
producing AS-A. This study provides evidence that a contemporary
aminoacyl-tRNA synthetase can be recruited to sustain amino acid
metabolism.
Engineered biosynthesis of an ansamycin polyketide precursor in
Escherichia coli
Authors: Watanabe Kenji , Rude Mathew A. , Walsh Christopher T. , Khosla Chaitan
Published: August 2003

Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an
important class of polyketide natural products. Their biosynthetic pathways
are especially complex because they involve the formation of
3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid
nonribosomal peptide synthetase/polyketide synthase. We have reconstituted the
ability to synthesize
2,6-dimethyl-3,5,7-trihydroxy-7-(3′-amino-5′-hydroxyphenyl)-2,4-heptadienoic
acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively
manipulated strain of
Escherichia coli
. The parent strain, BAP1,
contains the
sfp
phosphopantetheinyl transferase gene from
Bacillus subtilis
, which posttranslationally modifies polyketide
synthase and nonribosomal peptide synthetase modules. AHBA biosynthesis in
this host required introduction of seven genes from
Amycolatopsis
mediterranei
, which produces rifamycin, and
Actinosynnema
pretiosum
, which produces ansamitocin. Because the four-module RifA
protein (530 kDa) from the rifamycin synthetase could not be efficiently
produced in an intact form in
E. coli
, it was genetically split into
two bimodular proteins separated by matched linker pairs to facilitate
efficient inter-polypeptide transfer of a biosynthetic intermediate. A
derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon,
the bicistronic RifA construct and the
pccB
and
accA1
genes
from
Streptomyces coelicolor
, which enable methylmalonyl-CoA
biosynthesis. Fermentation of this strain of
E. coli
yielded P8/1-OG,
an
N
-acetyl P8/1-OG analog, and AHBA. In addition to providing a
fundamentally new route to shikimate and ansamycin-type compounds, this result
enables further genetic manipulation of AHBA-derived polyketide natural
products with unprecedented power.
Molecular cloning and functional expression of the first two specific insect myosuppressin receptors
Authors: Egerod Kristoffer , Reynisson Eyjólfur , Hauser Frank , Cazzamali Giuseppe , Williamson Michael , Grimmelikhuijzen Cornelis J. P.
Published: August 2003

The
Drosophila
Genome Project database contains the sequences of
two genes, CG8985 and CG13803, which are predicted to code for G
protein-coupled receptors. We cloned the cDNAs corresponding to these genes
and found that their gene structures had not been correctly annotated. We
subsequently expressed the coding regions of the two corrected receptor genes
in Chinese hamster ovary cells and found that each of them coded for a
receptor that could be activated by low concentrations of
Drosophila
myosuppressin (EC
50
,4 × 10
–
8
M).
The insect myosuppressins are decapeptides that generally inhibit insect
visceral muscles. Other tested
Drosophila
neuropeptides did not
activate the two receptors. In addition to the two
Drosophila
myosuppressin receptors, we identified a sequence in the genomic database from
the malaria mosquito
Anopheles gambiae
that also very likely codes
for a myosuppressin receptor. To our knowledge, this paper is the first report
on the molecular identification of specific insect myosuppressin
receptors.
Four functionally distinct C-type natriuretic peptides found in fish reveal evolutionary history of the natriuretic peptide system
Authors: Inoue Koji , Naruse Kiyoshi , Yamagami Sayaka , Mitani Hiroshi , Suzuki Norio , Takei Yoshio
Published: August 2003

Natriuretic peptides (NPs) are major cardiovascular and osmoregulatory
hormones in vertebrates. Although tetrapods generally have three subtypes,
atrial NP (ANP), B-type NP (BNP), and C-type NP (CNP), some teleosts lack BNP,
and sharks and hagfish have only one NP. Thus, NPs have diverged during fish
evolution, possibly reflecting changes in osmoregulatory systems. In this
study, we found, by cDNA cloning, four distinct CNPs (1 through 4) in the
medaka (
Oryzias latipes
) and puffer fish (
Takifugu
rubripes
), although to our knowledge no more than two CNPs have been
isolated from a vertebrate species. Predicted mature CNP-1 was most similar,
and CNP-4 was most dissimilar, to mammalian CNPs. However, synthetic CNP-4
most potently activated OlGC1, a medaka CNP-specific receptor (NPR-B)
expressed in cultured cells, whereas CNP-1 and CNP-3 most activated OlGC7 and
OlGC2, two medaka homologues of the ANP/BNP receptor (NPR-A), respectively.
Linkage mapping in medaka followed by comparative genomic analyses among
fishes and humans located four CNP genes in separate medaka chromosomes
corresponding to human chromosomes 1, 2, 12, and 17. From conserved synteny,
the following evolutionary history of NPs was evoked: (
i
) four CNP
genes were generated by chromosomal duplications before the divergence of
elasmobranchs; (
ii
) the CNP-3 gene generated ANP and BNP genes
through tandem duplication before the divergence of tetrapods and teleosts;
(
iii
) CNP-1 and -2 genes were retained in the teleost lineage but not
in the tetrapod lineage; (
iv
) the CNP-3 gene disappeared from the
tetrapod lineage after divergence of amphibians; and (
v
) the CNP-4
gene is retained in humans as CNP.
Identification of a G protein-coupled receptor for pheromone biosynthesis activating neuropeptide from pheromone glands of the moth
Helicoverpa zea
Authors: Choi Man-Yeon , Fuerst Emily-Jean , Rafaeli Ada , Jurenka Russell
Published: August 2003

Pheromone biosynthesis-activating neuropeptide (PBAN), a peptide produced
by the subesophageal ganglion, is used by a variety of moths to regulate
pheromone production. PBAN acts directly on pheromone gland cells by using
calcium and cAMP as second messengers. We have identified a gene encoding a G
protein-coupled receptor (GPCR) from pheromone glands of the female moth
Helicoverpa zea
. The gene was identified based on sequence identity
to a group of GPCRs from
Drosophila
that are homologous to neuromedin
U receptors in vertebrates. The full-length PBAN receptor was subsequently
cloned, expressed in Sf9 insect cells, and shown to mobilize calcium in
response to PBAN. This response was dose-dependent (EC
50
= 25 nM)
with a maximum response at 300 nM and a minimal observable response at 10 nM.
Four additional peptides produced by the PBAN-encoding gene were also tested
for activity, and it was determined that three had similar activity to PBAN
and the other was slightly less active. Peptides belonging to the same family
as PBAN, namely pyrokinins, as well as the vertebrate neuromedin U peptide
also induced a calcium response. We have identified a GPCR for the
PBAN/pyrokinin family of peptides with a known function of stimulating
pheromone biosynthesis in female moths. It is related to several receptors
from insects (
Drosophila
and
Anopheles
) and to neuromedin U
and ghrelin receptors from vertebrates.
Horizontal gene transfer: A critical view
Authors: Kurland C. G. , Canback B. , Berg Otto G.
Published: August 2003

It has been suggested that horizontal gene transfer (HGT) is the
“essence of phylogeny.” In contrast, much data suggest that this
is an exaggeration resulting in part from a reliance on inadequate methods to
identify HGT events. In addition, the assumption that HGT is a ubiquitous
influence throughout evolution is questionable. Instead, rampant global HGT is
likely to have been relevant only to primitive genomes. In modern organisms we
suggest that both the range and frequencies of HGT are constrained most often
by selective barriers. As a consequence those HGT events that do occur most
often have little influence on genome phylogeny. Although HGT does occur with
important evolutionary consequences, classical Darwinian lineages seem to be
the dominant mode of evolution for modern organisms.
Identification ofPlasmodium falciparumantigens by antigenic analysis of genomic and proteomic data
Authors: Doolan Denise L. , Southwood Scott , Freilich Daniel A. , Sidney John , Graber Norma L. , Shatney Lori , Bebris Lolita , Florens Laurence , Dobano Carlota , Witney Adam A. , Appella Ettore , Hoffman Stephen L. , Yates John R. , Carucci Daniel J. , Sette Alessandro
Published: August 2003

The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in the context ofPlasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuatedP. falciparumsporozoites, but not by mock immunized controls, were identified. Several of these were more antigenic than previously identified and well characterizedP. falciparum-derived protein antigens. The data suggest that immune responses toPlasmodiumare dispersed on a relatively large number of parasite antigens. These studies have implications for our understanding of immunodominance and breadth of responses to complex pathogens.
A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome
Authors: Hansen Jens , Floss Thomas , Van Sloun Petra , Füchtbauer Ernst-Martin , Vauti Franz , Arnold Hans-Hennig , Schnütgen Frank , Wurst Wolfgang , von Melchner Harald , Ruiz Patricia
Published: August 2003

A major challenge of the postgenomic era is the functional characterization
of every single gene within the mammalian genome. In an effort to address this
challenge, we assembled a collection of mutations in mouse embryonic stem (ES)
cells, which is the largest publicly accessible collection of such mutations
to date. Using four different gene-trap vectors, we generated 5,142 sequences
adjacent to the gene-trap integration sites (gene-trap sequence tags;
http://genetrap.de
)
from >11,000 ES cell clones. Although most of the gene-trap vector
insertions occurred randomly throughout the genome, we found both
vector-independent and vector-specific integration “hot spots.”
Because >50% of the hot spots were vector-specific, we conclude that the
most effective way to saturate the mouse genome with gene-trap insertions is
by using a combination of gene-trap vectors. When a random sample of gene-trap
integrations was passaged to the germ line, 59% (17 of 29) produced an
observable phenotype in transgenic mice, a frequency similar to that achieved
by conventional gene targeting. Thus, gene trapping allows a large-scale and
cost-effective production of ES cell clones with mutations distributed
throughout the genome, a resource likely to accelerate genome annotation and
the
in vivo
modeling of human disease.
A high-molecular-weight complex of membrane proteins BAP29/BAP31 is involved in the retention of membrane-bound IgD in the endoplasmic reticulum
Authors: Schamel Wolfgang W. A. , Kuppig Stephan , Becker Bernd , Gimborn Kerstin , Hauri Hans-Peter , Reth Michael
Published: August 2003

B cell antigen receptors (BCRs) are multimeric transmembrane protein complexes comprising membrane-bound immunoglobulins (mIgs) and Ig-α/Ig-β heterodimers. In most cases, transport of mIgs from the endoplasmic reticulum (ER) to the cell surface requires assembly with the Ig-α/Ig-β subunits. In addition to Ig-α/Ig-β, mIg molecules also bind two ER-resident membrane proteins, BAP29 and BAP31, and the chaperone heavy chain binding protein (BiP). In this article, we show that neither Ig-α/Ig-β nor BAP29/BAP31 nor BiP bind simultaneously to the same mIgD molecule. Blue native PAGE revealed that only a minor fraction of intracellular mIgD is associated with high-molecular-weight BAP29/BAP31 complexes. BAP-binding to mIgs was found to correlate with ER retention of chimeric mIgD molecules. On high-level expression inDrosophila melanogasterS2 cells, mIgD molecules were detected on the cell surface in the absence of Ig-α/Ig-β. This aberrant transport was prevented by coexpression of BAP29 and BAP31. Thus, BAP complexes contribute to ER retention of mIg complexes that are not bound to Ig-α/Ig-β. Furthermore, the mechanism of ER retention of both BAP31 and mIgD is not through retrieval from a post-ER compartment, but true ER retention. In conclusion, BAP29 and BAP31 might be the long sought after retention proteins and/or chaperones that act on transmembrane regions of various proteins.
Minimal haplotype tagging
Authors: Sebastiani Paola , Lazarus Ross , Weiss Scott T. , Kunkel Louis M. , Kohane Isaac S. , Ramoni Marco F.
Published: August 2003

The high frequency of single-nucleotide polymorphisms (SNPs) in the human
genome presents an unparalleled opportunity to track down the genetic basis of
common diseases. At the same time, the sheer number of SNPs also makes
unfeasible genomewide disease association studies. The haplotypic nature of
the human genome, however, lends itself to the selection of a parsimonious set
of SNPs, called haplotype tagging SNPs (htSNPs), able to distinguish the
haplotypic variations in a population. Current approaches rely on statistical
analysis of transmission rates to identify htSNPs. In contrast to these
approximate methods, this contribution describes an exact, analytical, and
lossless method, called BEST (Best Enumeration of SNP Tags), able to identify
the
minimum
set of SNPs tagging an arbitrary set of haplotypes from
either pedigree or independent samples. Our results confirm that a small
proportion of SNPs is sufficient to capture the haplotypic variations in a
population and that this proportion decreases exponentially as the haplotype
length increases. We used BEST to tag the haplotypes of 105 genes in an
African-American and a European-American sample. An interesting finding of
this analysis is that the vast majority (95%) of the htSNPs in the
European-American sample is a subset of the htSNPs of the African-American
sample. This result seems to provide further evidence that a severe bottleneck
occurred during the founding of Europe and the conjectured “Out of
Africa” event.
Early resistance to cell death and to onset of the mitochondrial permeability transition during hepatocarcinogenesis with 2-acetylaminofluorene
Authors: Klöhn Peter-Christian , Soriano Maria Eugenia , Irwin William , Penzo Daniele , Scorrano Luca , Bitsch Annette , Neumann Hans-Günter , Bernardi Paolo
Published: August 2003

A hallmark of tumorigenesis is resistance to apoptosis. To explore whether
resistance to cell death precedes tumor formation, we have studied the
short-term effects of the hepatocarcinogen 2-acetylaminofluorene (AAF) on
liver mitochondria, on hepatocytes, and on the response to bacterial endotoxin
lipopolysaccharide (LPS) in albino Wistar rats. We show that after as early as
two weeks of AAF feeding liver mitochondria developed an increased resistance
to opening of the permeability transition pore (PTP), an inner membrane
channel that is involved in various forms of cell death. Consistent with a
mitochondrial adaptive response
in vivo
, (
i
) AAF feeding
increased the expression of BCL-2 in mitochondria, and (
ii
)
hepatocytes isolated from AAF-fed rats became resistant to PTP-dependent
depolarization, cytochrome
c
release, and cell death, which were
instead observed in hepatocytes from rats fed a control diet. AAF-fed rats
were fully protected from the hepatotoxic effects of the injection of
20–30 μg of LPS plus 700 mg of
d
-galactosamine
(
d
-GalN) × kg
–
1
of body weight, a
treatment that in control rats readily caused a large increase of terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in
liver cryosections and release of alanine and aspartate aminotransferase into
the bloodstream. Treatment with LPS and
d
-GalN triggered cleavage
of BID, a BCL-2 family member, in the livers of both control- and AAF-fed
animals, whereas caspase 3 was cleaved only in control-fed animals, indicating
that the mitochondrial proapoptotic pathway had been selectively suppressed
during AAF feeding. Phenotypic reversion was observed after stopping the
carcinogenic diet. These results underscore a key role of mitochondria in
apoptosis and demonstrate that regulation of the mitochondrial PTP is altered
early during AAF carcinogenesis, which matches, and possibly causes, the
increased resistance of hepatocytes to death stimuli
in vivo
. Both
events precede tumor formation, suggesting that suppression of apoptosis may
contribute to the selection of a resistant phenotype, eventually increasing
the probability of cell progression to the transformed state.