bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppressive function of androgen receptor in bone resorption
Authors: Kawano Hirotaka , Sato Takashi , Yamada Takashi , Matsumoto Takahiro , Sekine Keisuke , Watanabe Tomoyuki , Nakamura Takashi , Fukuda Toru , Yoshimura Kimihiro , Yoshizawa Tatsuya , Aihara Ken-ichi , Yamamoto Yoko , Nakamichi Yuko , Metzger Daniel , Chambon Pierre , Nakamura Kozo , Kawaguchi Hiroshi , Kato Shigeaki
Published: August 2003

As locally converted estrogen from testicular testosterone contributes to
apparent androgen activity, the physiological significance of androgen
receptor (AR) function in the beneficial effects of androgens on skeletal
tissues has remained unclear. We show here that inactivation of AR in mice
using a Cre-loxP system-mediated gene-targeting technique caused bone loss in
males but not in females. Histomorphometric analyses of 8-week-old male AR
knockout (ARKO) mice showed high bone turnover with increased bone resorption
that resulted in reduced trabecular and cortical bone mass without affecting
bone shape. Bone loss in orchidectomized male ARKO mice was only partially
prevented by treatment with aromatizable testosterone. Analysis of primary
osteoblasts and osteoclasts from ARKO mice revealed that AR function was
required for the suppressive effects of androgens on osteoclastogenesis
supporting activity of osteoblasts but not on osteoclasts. Furthermore,
expression of the receptor activator of NF-κB ligand (
RANKL
)
gene, which encodes a major osteoclastogenesis inducer, was found to be
up-regulated in osteoblasts from AR-deficient mice. Our results indicate that
AR function is indispensable for male-type bone formation and remodeling.
Gene
RB
cloned from
Solanum bulbocastanum
confers broad spectrum resistance to potato late blight
Authors: Song Junqi , Bradeen James M. , Naess S. Kristine , Raasch John A. , Wielgus Susan M. , Haberlach Geraldine T. , Liu Jia , Kuang Hanhui , Austin-Phillips Sandra , Buell C. Robin , Helgeson John P. , Jiang Jiming
Published: August 2003

Late blight, caused by the oomycete pathogen
Phytophthora
infestans
, is the most devastating potato disease in the world. Control
of late blight in the United States and other developed countries relies
extensively on fungicide application. We previously demonstrated that the wild
diploid potato species
Solanum bulbocastanum
is highly resistant to
all known races of
P. infestans
. Potato germplasm derived from
S.
bulbocastanum
has shown durable and effective resistance in the field.
Here we report the cloning of the major resistance gene
RB
in
S.
bulbocastanum
by using a map-based approach in combination with a
long-range (LR)-PCR strategy. A cluster of four resistance genes of the
CC-NBS-LRR (coiled coil–nucleotide binding site–Leu-rich repeat)
class was found within the genetically mapped
RB
region. Transgenic
plants containing a LR-PCR product of one of these four genes displayed broad
spectrum late blight resistance. The cloned
RB
gene provides a new
resource for developing late blight-resistant potato varieties. Our results
also demonstrate that LR-PCR is a valuable approach to isolate genes that
cannot be maintained in the bacterial artificial chromosome system.
Cloning and characterization of the
Drosophila
U7 small nuclear RNA
Authors: Dominski Zbigniew , Yang Xiao-cui , Purdy Matthew , Marzluff William F.
Published: August 2003

Base pairing between the 5′ end of U7 small nuclear RNA (snRNA) and
the histone downstream element (HDE) in replication-dependent histone
pre-mRNAs is the key event in 3′-end processing that leads to generation
of mature histone mRNAs. We have cloned the
Drosophila
U7 snRNA and
demonstrated that it is required for histone pre-mRNA 3′-end processing
in a
Drosophila
nuclear extract. The 71-nt
Drosophila
U7
snRNA is encoded by a single gene that is embedded in the direct orientation
in an intron of the
Eip63E
gene. The U7 snRNA gene contains conserved
promoter elements typical of other
Drosophila
snRNA genes, and the
coding sequence is followed by a 3′ box indicating that the
Drosophila
U7 snRNA gene is an independent transcription unit.
Drosophila
U7 snRNA contains a trimethyl-guanosine cap at the
5′ end and a putative Sm-binding site similar to the unique Sm-binding
site found in other U7 snRNAs.
Drosophila
U7 snRNA is ≈10 nt
longer than mammalian U7 snRNAs because of an extended 5′ sequence and
has only a limited potential to form a stem-loop structure near the 3′
end. The extended 5′ end of
Drosophila
U7 snRNA can base pair
with the HDE in all five
Drosophila
histone pre-mRNAs. Blocking the
5′ end of the U7 snRNA with a complementary oligonucleotide specifically
blocks processing of a
Drosophila
histone pre-mRNA. Changes in the
HDE that abolish or decrease processing efficiency result in a reduced ability
to recruit U7 snRNA to the pre-mRNA.
α-Galactosylceramide (KRN7000) suppression of chemical- and oncogene-dependent carcinogenesis
Authors: Hayakawa Yoshihiro , Rovero Stefania , Forni Guido , Smyth Mark J.
Published: August 2003

Recent studies have revealed significant efficacy of the marine sponge glycolipid, α-galactosylceramide (α-GalCer), in treatment of experimental metastatic cancers, infections, and autoimmune diseases. However, the capacity of α-GalCer to prevent tumor development had never, to our knowledge, been evaluated in mouse models of chemical- and oncogene-dependent carcinogenesis. In this study, we demonstrate that long-term administration of soluble α-GalCer, spanning the time of tumor initiation, inhibits primary tumor formation in three different models: methylcholanthrene-induced sarcomas, mammary carcinomas in Her-2/neu transgenic mice, and spontaneous sarcomas in p53–/–mice. Weekly treatment of mice with α-GalCer maintained lymphoid tissue natural killer cell and T cell activation and elevated serum IFN-γ and IL-4 concentrations. Consistent with the antimetastatic activity of α-GalCer, prevention of methylcholanthrene-induced sarcoma was IFN-γand tumor necrosis factor-related apoptosis-inducing ligand dependent, but not perforin-dependent. Taken together, our results demonstrate that NK1.1+αβTCR+cell-based immune therapy can inhibit primary tumorigenesis.
Bimodal circadian secretion of melatonin from the pineal gland in a living CBA mouse
Authors: Nakahara Daiichiro , Nakamura Masato , Iigo Masayuki , Okamura Hitoshi
Published: August 2003

Circadian melatonin secretion is the best-known output signal from the
circadian pacemaker in the suprachiasmatic nucleus that indicates internal
conditions of the body. We have established a system that enables long-term
monitoring of melatonin secretion by implanting a transverse microdialysis
probe in or near the pineal gland in a freely moving mouse. This
in
vivo
method enabled continuous measurement of melatonin secretion over a
period of >20 days in individual CBA mice, with simultaneous recording of
the locomotor activity. Pineal melatonin secretion was completely matched to
the circadian change of locomotor activity, and for the light-induced phase
shift, the shift of melatonin secretion was clearer than the shift of
locomotor rhythm. This analysis allowed us to detect rhythm with a high
sensitivity: two peaks of daily secretion were observed, with the first small
peak at the day–night transition time and the second large peak at
midnight. The large nighttime peak was suppressed by tetrodotoxin, a
Na
+
channel blocker, and enhanced by both phenylephrine and
isoproterenol, α- and β-adrenergic agonists, whereas daytime
melatonin levels were not affected by tetrodotoxin infusion. This finding
suggests that, in CBA mice, melatonin release at night is activated by
adrenergic signaling from the superior cervical ganglion, but the enhancement
of melatonin during daytime is not mediated by neuronal signaling.
Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest
Authors: Mori-Akiyama Yuko , Akiyama Haruhiko , Rowitch David H. , de Crombrugghe Benoit
Published: August 2003

Sox9 has essential roles in endochondral bone formation during axial and appendicular skeletogenesis. Sox9 is also expressed in neural crest cells, but its function in neural crest remains largely unknown. Because many craniofacial skeletal elements are derived from cranial neural crest (CNC) cells, we asked whether deletion ofSox9in CNC cells by using the Cre recombinase/loxPrecombination system would affect craniofacial development. Inactivation ofSox9in neural crest resulted in a complete absence of cartilages and endochondral bones derived from the CNC. In contrast, all of the mesodermal skeletal elements and intramembranous bones were essentially conserved. The migration and the localization ofSox9-null mutant CNC cells were normal. Indeed, the size of branchial arches and the frontonasal mass of mutant embryos was comparable to that of WT embryos, and the pattern of expression ofAp2, a marker of migrating CNC cells, was normal. Moreover, in mouse embryo chimerasSox9-null mutant cells migrated to their correct location in endochondral skeletal elements; however,Sox9-null CNC cells were unable to contribute chondrogenic mesenchymal condensations. In mutant embryos, ectopic expression of osteoblast marker genes, such asRunx2, Osterix, andCol1a1, was found in the locations where the nasal cartilages exist in WT embryos. These results indicate that inactivation ofSox9causes CNC cells to lose their chondrogenic potential. We hypothesize that these cells change their cell fate and acquire the ability to differentiate into osteoblasts. We conclude that Sox9 is required for the determination of the chondrogenic lineage in CNC cells.
Coevolutionary patterns in plasminogen activation
Authors: Gladysheva Inna P. , Turner Ryan B. , Sazonova Irina Y. , Liu Lin , Reed Guy L.
Published: August 2003

The generation of plasmin by plasminogen (Pg) activators (PAs) is a physiologic process in animals that dissolves blood clots and promotes wound healing, blood vessel growth, and the migration of normal and cancerous cells. Pathogenic bacteria have evolved PAs [e.g., streptokinase (SK) and staphylokinase] that exploit the Pg system to infect animals. Animal PAs have a conserved ability to cleave a wide spectrum of animal Pgs, but the ability of bacterial PAs to cleave different animal Pgs is surprisingly restricted. We show that the spectrum of activity of an archetypal bacterial PA (SK) with animal Pgs can be profoundly altered by mutations that affect intermolecular complementarity at sites that participate in complex formation or substrate binding. Comparative sequence analysis of animal plasmins vs. close structural homologues (trypsin and chymotrypsin) that are not molecular targets for invading bacteria indicates that the sites in plasmin that interact with SK are preferentially targeted for mutation. Conversely, intermolecular contact sites in SKs that activate human Pg are more highly conserved than other loci in the molecule or than the same sites in other SKs that activate non-human Pgs. We propose that active modulation of intermolecular complementarity at sites of contact between SK and Pg may represent a competitive evolutionary strategy in a survival battle, whereby animals seek to evade bacterial invasion, and bacteria endeavor to invade their animal hosts.
Crystal structure of NF-κB (p50)
2
complexed to a high-affinity RNA aptamer
Authors: Huang De-Bin , Vu Don , Cassiday Laura A. , Zimmerman Jeff M. , Maher L. James , Ghosh Gourisankar
Published: August 2003

We have recently identified an RNA aptamer for the transcription factor
NF-κB p50 homodimer [(p50)
2
], which exhibits little sequence
resemblance to the consensus DNA target for (p50)
2
, but binds
(p50)
2
with an affinity similar to that of the optimal DNA target.
We describe here the 2.45-Å resolution x-ray crystal structure of the
p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind
independent of each other to the p50 N-terminal Ig-like domains. The RNA
secondary structure is comprised of a stem and a stem–loop separated by
an internal loop folded into a kinked helix because of the cross-strand
stacking of three internal loop guanines. These guanines, placed at the edge
of the 3′ helix, together with the major groove of the irregular
3′ helix, form the binding surface for p50. Each p50 monomer uses the
same surface to recognize the distorted RNA major groove as observed in the
κB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The
structure reveals how the aptamer specifically selects p50 and discriminates
against p65. We also discuss the physiological implications of RNA binding by
(p50)
2
.
Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy
Authors: Tama Florence , Valle Mikel , Frank Joachim , Brooks Charles L.
Published: August 2003

Combining structural data for the ribosome from x-ray crystallography and
cryo-electron microscopy with dynamic models based on elastic network normal
mode analysis, an atomically detailed picture of functionally important
structural rearrangements that occur during translocation is elucidated. The
dynamic model provides a near-atomic description of the ratchet-like
rearrangement of the 70S ribosome seen in cryo-electron microscopy, and
permits the identification of bridging interactions that either facilitate the
conformational switching or maintain structural integrity of the 50S/30S
interface. Motions of the tRNAs residing in the A and P sites also suggest the
early stages of tRNA translocation as a result of this ratchet-like movement.
Displacement of the L1 stalk, alternately closing and opening the intersubunit
space near the E site, is observed in the dynamic model, in line with growing
experimental evidence for the role of this structural component in
facilitating the exiting of tRNA. Finally, a hinge-like transition in the 30S
ribosomal subunit, similar to that observed in crystal structures of this
complex, is also manifest as a dynamic mode of the ribosome. The coincidence
of these dynamic transitions with the individual normal modes of the ribosome
and the good correspondence between these motions and those observed in
experiment suggest an underlying principle of nature to exploit the shape of
molecular assemblies such as the ribosome to provide robustness to
functionally important motions.
Prediction of clinical drug efficacy by classification of drug-induced genomic expression profiles
in vitro
Authors: Gunther Erik C. , Stone David J. , Gerwien Robert W. , Bento Patricia , Heyes Melvyn P.
Published: August 2003

Assays of drug action typically evaluate biochemical activity. However,
accurately matching therapeutic efficacy with biochemical activity is a
challenge. High-content cellular assays seek to bridge this gap by capturing
broad information about the cellular physiology of drug action. Here, we
present a method of predicting the general therapeutic classes into which
various psychoactive drugs fall, based on high-content statistical
categorization of gene expression profiles induced by these drugs. When we
used the classification tree and random forest supervised classification
algorithms to analyze microarray data, we derived general “efficacy
profiles” of biomarker gene expression that correlate with
anti-depressant, antipsychotic and opioid drug action on primary human neurons
in vitro
. These profiles were used as predictive models to classify
naïve
in vitro
drug treatments with 83.3% (random forest) and
88.9% (classification tree) accuracy. Thus, the detailed information contained
in genomic expression data is sufficient to match the physiological effect of
a novel drug at the cellular level with its clinical relevance. This capacity
to identify therapeutic efficacy on the basis of gene expression signatures
in vitro
has potential utility in drug discovery and drug target
validation.
Zinc and mechanical prowess in the jaws of
Nereis
, a marine worm
Authors: Lichtenegger Helga C. , Schöberl Thomas , Ruokolainen Janne T. , Cross Julie O. , Heald Steve M. , Birkedal Henrik , Waite J. Herbert , Stucky Galen D.
Published: August 2003

Higher animals typically rely on calcification to harden certain tissues
such as bones and teeth. Some notable exceptions can be found in
invertebrates: The fangs, teeth, and mandibles of diverse arthropod species
have been reported to contain high levels of zinc. Considerable quantities of
zinc also occur in the jaws of the marine polychaete worm
Nereis
sp.
High copper levels in the polychaete worm
Glycera dibranchiata
recently were attributed to a copper-based biomineral reinforcing the jaws. In
the present article, we attempt to unravel the role of zinc in
Nereis
limbata
jaws, using a combination of position-resolved state-of-the-art
techniques. It is shown that the local hardness and stiffness of the jaws
correlate with the local zinc concentration, pointing toward a structural role
for zinc. Zinc always is detected in tight correlation with chlorine,
suggesting the presence of a zinc–chlorine compound. No crystalline
inorganic phase was found, however, and results from x-ray absorption
spectroscopy further exclude the presence of simple inorganic
zinc–chlorine compounds in amorphous form. The correlation of local
histidine levels in the protein matrix and zinc concentration leads us to
hypothesize a direct coordination of zinc and chlorine to the protein. A
comparison of the role of the transition metals zinc and copper in the jaws of
two polychaete worm species
Nereis
and
Glycera
,
respectively, is presented.
Stimulation of T cell autoreactivity by anomalous expression of NKG2D and its MIC ligands in rheumatoid arthritis
Authors: Groh Veronika , Brühl Anja , El-Gabalawy Hani , Nelson J. Lee , Spies Thomas
Published: August 2003

Effector T cell responses can be modulated by competing positive or
negative signals transduced by natural killer (NK) cell receptors. This raises
the possibility that dominant T cell stimulation might promote autoimmune
reactions. In rheumatoid arthritis (RA), the severity of autoimmune and
inflammatory joint disease correlates with large numbers of
CD4
+
CD28
–
T cells, which are scarce in healthy
individuals. For poorly defined reasons, these T cells are autoreactive,
implying that they may contribute to disease manifestations.
CD4
+
CD28
–
T cells in peripheral blood and synovial
tissue of RA patients were found to express NKG2D, a costimulatory receptor
that is absent on normal CD4 T cells. NKG2D was induced by tumor necrosis
factor α and IL-15, which are abundant in inflamed synovia and RA
patient sera. RA synoviocytes aberrantly expressed the stress-inducible MIC
ligands of NKG2D, which stimulated autologous
CD4
+
CD28
–
T cell cytokine and proliferative
responses. Peripheral blood serum samples of RA patients contained substantial
amounts of synoviocyte-derived soluble MICA, which failed to induce
down-modulation of NKG2D because of the opposing activity of tumor necrosis
factor α and IL-15. These results suggest that a profound dysregulation
of NKG2D and its MIC ligands may cause autoreactive T cell stimulation, thus
promoting the self-perpetuating pathology in RA and possibly other autoimmune
diseases.
A chemical approach for identifying
O
-GlcNAc-modified proteins in cells
Authors: Vocadlo David J. , Hang Howard C. , Kim Eun-Ju , Hanover John A. , Bertozzi Carolyn R.
Published: August 2003

The glycosylation of serine and threonine residues with a single GlcNAc
moiety is a dynamic posttranslational modification of many nuclear and
cytoplasmic proteins. We describe a chemical strategy directed toward
identifying
O
-GlcNAc-modified proteins from living cells or proteins
modified
in vitro
. We demonstrate,
in vitro
, that each
enzyme in the hexosamine salvage pathway, and the enzymes that affect this
dynamic modification (UDP-GlcNAc:polypeptidtyltransferase and
O
-GlcNAcase), tolerate analogues of their natural substrates in which
the
N
-acyl side chain has been modified to bear a bio-orthogonal
azide moiety. Accordingly, treatment of cells with
N
-azidoacetylglucosamine results in the metabolic incorporation of
the azido sugar into nuclear and cytoplasmic proteins. These
O
-azidoacetylglucosamine-modified proteins can be covalently
derivatized with various biochemical probes at the site of protein
glycosylation by using the Staudinger ligation. The approach was validated by
metabolic labeling of nuclear pore protein p62, which is known to be
posttranslationally modified with
O
-GlcNAc. This strategy will prove
useful for both the identification of
O
-GlcNAc-modified proteins and
the elucidation of the specific residues that bear this saccharide.
A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions
Authors: Raghuram Viswanathan , Hormuth Hayley , Foskett J. Kevin
Published: August 2003

Dynamic regulation of ion channels is critical for maintaining fluid
balance in epithelial tissues. Cystic fibrosis, a genetic disease
characterized by impaired fluid transport in epithelial tissues, is caused by
dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR)
Cl
-
channel activity. Recent studies have shown that binding of
PSD-95/Dlg/ZO-1 (PDZ) domain proteins to CFTR is important for retaining it at
the apical membrane and for regulating its channel activity. Here, we describe
a phosphorylation mechanism that regulates CFTR channel activity, which is
mediated by PDZ domains. The Na
+
/H
+
exchanger regulatory
factor (NHERF) binds to CFTR and increases its open probability
(
P
o
). Protein kinase C disrupts the stimulatory effect of
NHERF on CFTR channel
P
o
. Phosphorylation by PKC of
Ser-162 in the PDZ2 domain of NHERF is critical for this functional effect.
Furthermore, a mutation in PDZ2 that mimics phosphorylation decreases CFTR
binding and disrupts the ability of NHERF PDZ1–2 to stimulate CFTR
channel
P
o
. Our results identify a role for PKC and
suggest that phosphorylation of NHERF PDZ2 domain may be an important
mechanism for regulating CFTR channel activity.
Polyserase-I, a human polyprotease with the ability to generate independent serine protease domains from a single translation product
Authors: Cal Santiago , Quesada Víctor , Garabaya Cecilia , López-Otín Carlos
Published: August 2003

We have identified and cloned a human liver cDNA encoding an unusual mosaic
polyprotein, called polyserase-I (polyserine protease-I). This protein
exhibits a complex domain organization including a type II transmembrane
motif, a low-density lipoprotein receptor A module, and three tandem serine
protease domains. This unusual modular architecture is also present in the
sequences predicted for mouse and rat polyserase-I. Human polyserase-I gene
maps to 19p13, and its last exon overlaps with that corresponding to the
3′ UTR of the gene encoding translocase of mitochondrial inner membrane
13. Northern blot analysis showed the presence of a major polyserase-I
transcript of 5.4 kb in human fetal and adult tissues and in tumor cell lines.
Analysis of processing mechanisms of polyserase-I revealed that it is
synthesized as a membrane-associated polyprotein that is further processed to
generate three independent serine protease units. Two of these domains are
proteolytically active against synthetic peptides commonly used for assaying
serine proteases. These proteolytic activities of the polyserase-I units are
blocked by serine protease inhibitors. We show an example of generation of
separate serine protease domains from a single translation product in human
tissues and illustrate an additional mechanism for expanding the complexity of
the human degradome, the entire protease complement of human cells and
tissues.
Differential regulation of microtubule dynamics by three- and four-repeat tau: Implications for the onset of neurodegenerative disease
Authors: Panda Dulal , Samuel Jonathan C. , Massie Michelle , Feinstein Stuart C. , Wilson Leslie
Published: August 2003

The microtubule (MT)-associated protein tau is important in neuronal
development and in Alzheimer's and other neurodegenerative diseases. Genetic
analyses have established a cause-and-effect relationship between tau
dysfunction/misregulation and neuronal cell death and dementia in
frontotemporal dementia and parkinsonism associated with chromosome 17;
several mutations causing this dementia lead to increased ratios of
four-repeat (4R) to three-repeat (3R) wild-type tau, and an attractive
hypothesis is that the abnormally high ratio of 4R to 3R tau might lead to
neuronal cell death by altering normal tau functions in adult neurons. Thus,
we tested whether 3R and 4R tau might differentially modulate the dynamic
instability of MTs
in vitro
using video microscopy. Although both
isoforms promoted MT polymerization and decreased the tubulin critical subunit
concentration to approximately similar extents, 4R tau stabilized MTs
significantly more strongly that 3R tau. For example, 4R tau suppressed the
shortening rate, whereas 3R tau had little or no detectable effect. Similarly,
3R tau had no effect on the length shortened during a shortening event,
whereas 4R tau strongly reduced this parameter. Further, when MTs were diluted
into buffer containing 4R tau, the MTs were stabilized and shortened slowly.
In contrast, when diluted into 3R tau, the MTs were unstable and shortened
rapidly. Thus, 4R tau stabilizes MTs differently and significantly more
strongly than 3R tau. We suggest a “dosage effect” or
haploinsufficiency model in which both tau alleles must be active and properly
regulated to produce appropriate amounts of each tau isoform to maintain MT
dynamics within a tolerable window of activity.
Microarray analysis of evolution of RNA viruses: Evidence of circulation of virulent highly divergent vaccine-derived polioviruses
Authors: Cherkasova Elena , Laassri Majid , Chizhikov Vladimir , Korotkova Ekaterina , Dragunsky Eugenia , Agol Vadim I. , Chumakov Konstantin
Published: August 2003

Two approaches based on hybridization of viral probes with oligonucleotide
microarrays were developed for rapid analysis of genetic variations during
microevolution of RNA viruses. Microarray analysis of viral recombination and
microarray for resequencing and heterogeneity analysis were able to generate
instant genetic maps of vaccine-derived polioviruses (VDPVs) and reveal the
degree of their evolutionary divergence. Unlike conventional methods based on
cDNA sequencing and restriction fragment length polymorphism, the microarray
approaches are better suited for analysis of heterogeneous populations and
mixtures of different strains. The microarray hybridization profile is very
sensitive to the cumulative presence of small quantities of different
mutations, including those that cannot be revealed by sequencing, making this
approach useful for characterization of profiles of nucleotide sequence
diversity in viral populations. By using these methods, we identified a type-3
VDPV isolated from a healthy person and missed by conventional methods of
screening. The mutational profile of the polio strain was consistent with
>1 yr of circulation in human population and was highly virulent in
transgenic mice, confirming the ability of VDPV to persist in communities
despite high levels of immunity. The proposed methods for fine genotyping of
heterogeneous viral populations can also have utility for a variety of other
applications in studies of genetic changes in viruses, bacteria, and genes of
higher organisms.
Profiling receptor tyrosine kinase activation by using Ab microarrays
Authors: Nielsen Ulrik B. , Cardone Mike H. , Sinskey Anthony J. , MacBeath Gavin , Sorger Peter K.
Published: August 2003

Signal transduction in mammalian cells is mediated by complex networks of
interacting proteins. Understanding these networks at a circuit level requires
devices to measure the amounts and activities of multiple proteins in a rapid
and accurate manner. Ab microarrays have previously been applied to the
quantification of labeled recombinant proteins and proteins in serum. The
development of methods to analyze intracellular signaling molecules on
microarrays would make Ab arrays widely useful in systems biology. Here we
describe the fabrication of multiplex Ab arrays sensitive to the amounts and
modification states of signal transduction proteins in crude cell lysates and
the integration of these arrays with 96-well microtiter plate technology to
create microarrays in microplates. We apply the Ab arrays to monitoring the
activation, uptake, and signaling of ErbB receptor tyrosine kinases in human
tumor cell lines. Data obtained from multicolor ratiometric microarrays
correlate well with data obtained by using traditional approaches, but the
arrays are faster and simpler to use. The integration of microplate and
microarray methods for crude cell lysates should make it possible to identify
and analyze small molecule inhibitors of signal transduction processes with
unprecedented speed and precision. We demonstrate the future potential of this
approach by characterizing the action of the epidermal growth factor receptor
inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based
screening in 96- and 384-well plates should allow small molecules to be
identified with specific inhibitory profiles against a signaling network.
Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA
Authors: Fan Chunhai , Plaxco Kevin W. , Heeger Alan J.
Published: August 2003

We report a strategy for the reagentless transduction of DNA hybridization
into a readily detectable electrochemical signal by means of a conformational
change analogous to the optical molecular beacon approach. The strategy
involves an electroactive, ferrocene-tagged DNA stem–loop structure that
self-assembles onto a gold electrode by means of facile gold-thiol chemistry.
Hybridization induces a large conformational change in this surface-confined
DNA structure, which in turn significantly alters the electron-transfer
tunneling distance between the electrode and the redoxable label. The
resulting change in electron transfer efficiency is readily measured by cyclic
voltammetry at target DNA concentrations as low as 10 pM. In contrast to
existing optical approaches, an electrochemical DNA (E-DNA) sensor built on
this strategy can detect femtomoles of target DNA without employing cumbersome
and expensive optics, light sources, or photodetectors. In contrast to
previously reported electrochemical approaches, the E-DNA sensor achieves this
impressive sensitivity without the use of exogenous reagents and without
sacrificing selectivity or reusability. The E-DNA sensor thus offers the
promise of convenient, reusable detection of picomolar DNA.
The cell death regulator GRIM-19 is an inhibitor of signal transducer and activator of transcription 3
Authors: Zhang Jun , Yang Jinbo , Roy Sanjit K. , Tininini Silvia , Hu Jiadi , Bromberg Jacqueline F. , Poli Valeria , Stark George R. , Kalvakolanu Dhananjaya V.
Published: August 2003

GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated
as a cell death activator in a genetic screen used to define mechanisms
involved in IFN-β- and retinoic acid-induced cell death, codes for a
≈16-kDa protein that induces apoptosis in a number of cell lines. Antisense
ablation of GRIM-19 caused resistance to cell death induced by IFN plus
retinoic acid and conferred a growth advantage to cells. To understand the
molecular bases for its cell death regulatory activity, we used a yeast
two-hybrid screen and identified that the transcription factor STAT3 (signal
transducer and activator of transcription 3) binds to GRIM-19. GRIM-19
inhibits transcription driven by activation of STAT3, but not STAT1. It
neither inhibits the ligand-induced activation of STAT3 nor blocks its ability
to bind to DNA. Mutational analysis indicates that the transactivation domain
of STAT3, especially residue S727, is required for GRIM-19 binding. Because
GRIM-19 does not bind significantly to other STATs, our studies identify a
specific inhibitor of STAT3. Because constitutively active STAT3 up-regulates
antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also
demonstrates an antioncogenic effect exerted by biological therapeutics.
Role of extracellular subdomains of p185
c-neu
and the epidermal growth factor receptor in ligand-independent association and transactivation
Authors: Kumagai Toru , Katsumata Makoto , Hasegawa Akihiro , Furuuchi Keiji , Funakoshi Toshiki , Kawase Ichiro , Greene Mark I.
Published: August 2003

We investigated the assembly and activation of the epidermal growth factor
receptor (EGFR)–p185
c-neu
heterodimer by using a sequential
immunoprecipitation methodology. Using this approach we detected heterodimers
and also higher-ordered oligomeric complexes. Phosphorylated
EGFR–p185
c-neu
heterodimeric forms were detected in the
absence of EGF, but the species became highly phosphorylated after EGF
stimulation. To evaluate heterodimer formation and additional transactivation
by EGF, we investigated the roles of the four extracellular subdomains of
p185
c-neu
and the EGFR. Subdomains I–IV of the EGFR dimerized
with subdomains I–IV of p185
c-neu
, respectively, in a
parallel manner. In addition, subdomains I–IV of the EGFR also
associated with p185
c-neu
subdomains III, IV, I, and II,
respectively. A lack of one of the p185
c-neu
cysteine-rich domains
(subdomains II or IV) resulted in a loss of EGF-induced transactivation. These
data suggest that two cysteine-rich domains play defining roles in
ligand-dependent transactivation and that both of these cysteine-rich
extracellular subdomains as well as non-cysteine-rich extracellular subdomains
are involved in ligand-independent interactions with the EGFR. Our studies
provide biochemical evidence of the role of the cysteine-rich domains of
p185
c-neu
in assembly and transactivation of erbB complexes and
also indicate that these subdomains might be useful clinical targets.
Long-distance conformational changes in a protein engineered by modulated sequence duplication
Authors: Sagermann Martin , Gay Leslie , Matthews Brian W.
Published: August 2003

There are few, if any, known instances in which a biological signal is
transmitted via a large conformational change through the body of a protein.
We describe here a mutant of T4 lysozyme that was engineered to permit
structural change at a distance. The design uses a tandem sequence repeat that
makes it possible to transmit large-scale structural changes from one end of
an α-helix to the other over a distance of 17–25 Å. The
method should be of general applicability and may make it possible to
introduce a mutation at one site in a protein that will induce large-scale
changes in the structure at a spatially remote site.
The nested assembly of plant–animal mutualistic networks
Authors: Bascompte Jordi , Jordano Pedro , Melián Carlos J. , Olesen Jens M.
Published: August 2003

Most studies of plant–animal mutualisms involve a small number of species. There is almost no information on the structural organization of species-rich mutualistic networks despite its potential importance for the maintenance of diversity. Here we analyze 52 mutualistic networks and show that they are highly nested; that is, the more specialist species interact only with proper subsets of those species interacting with the more generalists. This assembly pattern generates highly asymmetrical interactions and organizes the community cohesively around a central core of interactions. Thus, mutualistic networks are neither randomly assembled nor organized in compartments arising from tight, parallel specialization. Furthermore, nestedness increases with the complexity (number of interactions) of the network: for a given number of species, communities with more interactions are significantly more nested. Our results indicate a nonrandom pattern of community organization that may be relevant for our understanding of the organization and persistence of biodiversity.
Nitric oxide negatively regulates mammalian adult neurogenesis
Authors: Packer Michael A. , Stasiv Yuri , Benraiss Abdellatif , Chmielnicki Eva , Grinberg Alexander , Westphal Heiner , Goldman Steven A. , Enikolopov Grigori
Published: August 2003

Neural progenitor cells are widespread throughout the adult central nervous
system but only give rise to neurons in specific loci. Negative regulators of
neurogenesis have therefore been postulated, but none have yet been identified
as subserving a significant role in the adult brain. Here we report that
nitric oxide (NO) acts as an important negative regulator of cell
proliferation in the adult mammalian brain. We used two independent approaches
to examine the function of NO in adult neurogenesis. In a pharmacological
approach, we suppressed NO production in the rat brain by intraventricular
infusion of an NO synthase inhibitor. In a genetic approach, we generated a
null mutant neuronal NO synthase knockout mouse line by targeting the exon
encoding active center of the enzyme. In both models, the number of new cells
generated in neurogenic areas of the adult brain, the olfactory subependyma
and the dentate gyrus, was strongly augmented, which indicates that division
of neural stem cells in the adult brain is controlled by NO and suggests a
strategy for enhancing neurogenesis in the adult central nervous system.
Differential binding of NAD
+
and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor
Authors: Fjeld Clark C. , Birdsong William T. , Goodman Richard H.
Published: August 2003

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor
originally identified through its ability to interact with adenovirus E1A. The
finding that CtBP–E1A interactions were regulated by the nicotinamide
adeninine dinucleotides NAD
+
and NADH raised the possibility that
CtBP could serve as a nuclear redox sensor. This model requires differential
binding affinities of NAD
+
and NADH, which has been controversial.
The structure of CtBP determined by x-ray diffraction revealed a tryptophan
residue adjacent to the proposed nicotinamide adenine dinucleotide binding
site. We find that this tryptophan residue shows strong fluorescence resonance
energy transfer to bound NADH. In this report, we take advantage of these
findings to measure the dissociation constants for CtBP with NADH and
NAD
+
. The affinity of NADH was determined by using fluorescence
resonance energy transfer. The binding of NADH to protein is associated with
an enhanced intensity of NADH fluorescence and a blue shift in its maximum.
NAD
+
affinity was estimated by measuring the loss of the
fluorescence blue shift as NADH dissociates on addition of NAD
+
.
Our studies show a >100-fold higher affinity of NADH than NAD
+
,
consistent with the proposed function of CtBP as a nuclear redox sensor.
Moreover, the concentrations of NADH and NAD
+
required for
half-maximal binding are approximately the same as their concentrations in the
nuclear compartment. These findings support the possibility that changes in
nuclear nicotinamide adenine dinucleotides could regulate the functions of
CtBP in cell differentiation, development, or transformation.
The lipoxygenase gene
ALOXE3
implicated in skin differentiation encodes a hydroperoxide isomerase
Authors: Yu Zheyong , Schneider Claus , Boeglin William E. , Marnett Lawrence J. , Brash Alan R.
Published: August 2003

Lipoxygenase (LOX) enzymes form fatty acid hydroperoxides used in membrane
remodeling and cell signaling. Mammalian epidermal LOX type 3 (eLOX3) is
distinctive in totally lacking this typical oxygenase activity. Surprisingly,
genetic evidence has linked mutations in eLOX3 or a colocalizing enzyme,
12
R
-LOX, to disruption of the normal permeability barrier of the skin
[Jobard, F., Lefèvre, C., Karaduman, A., Blanchet-Bardon, C., Emre, S.,
Weissenbach, J., Özgüc, M., Lathrop, M., Prud'homme, J. F. &
Fischer, J. (2002)
Hum. Mol. Genet.
11, 107–113]. Herein we
identify a logical link of the biochemistry to the genetics. eLOX3 functions
as a hydroperoxide isomerase (epoxyalcohol synthase) by using the product of
12
R
-LOX as the preferred substrate.
12
R
-Hydroperoxyeicosatetraenoic acid (12
R
-HPETE) is
converted to
8
R
-hydroxy-11
R
,12
R
-epoxyeicosa-5
Z
,9
E
,14
Z
-trienoic
acid, one of the isomers of hepoxilin A
3
, and to
12-ketoeicosatetraenoic acid in a 2:1 ratio. Other hydroperoxides, including
8
R
-HPETE, 12
S
-HPETE, and 15
S
-HPETE, as well as the
13
S
- and 13
R
-hydroperoxides of linoleic acid are converted
less efficiently. Mass spectrometric analysis of the epoxyalcohol formed from
[
18
O]15
S
-HPETE showed that both hydroperoxy oxygens are
retained in the product. We propose that the ferrous form of eLOX3 initiates a
redox cycle, unprecedented among LOX in being autocatalytic, in which the
hydroperoxy substrate is isomerized to the epoxyalcohol or keto product. Our
results provide strong biochemical evidence for a functional linkage of
12
R
-LOX and eLOX3 and clues into skin biochemistry and the etiology
of ichthyosiform diseases in humans.
A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition
Authors: Condon Jennifer C. , Jeyasuria Pancharatnam , Faust Julie M. , Wilson James W. , Mendelson Carole R.
Published: August 2003

The molecular events that lead to the onset of labor in humans and in other
mammalian species remain unclear. We propose that a decline in coactivators
containing histone acetylase activity in myometrium may contribute to the
onset of labor by impairing the function of the
progesterone–progesterone receptor (PR) complex. As assessed by
semiquantitative and real-time RT-PCR, immunohistochemistry, and
immunoblotting, expression of the PR coactivators cAMP-response
element-binding protein (CREB)-binding protein and steroid receptor
coactivators 2 and 3 was decreased in fundal uterine tissue of women in labor.
Using the mouse as an animal model, we also found decreased coactivator levels
in uterine tissues at term. In both human and mouse, the levels of acetylated
histone H3 were also decreased in uterine tissues at term. Administration of
trichostatin A, a specific and potent histone deacetylase inhibitor, to
pregnant mice late in gestation increased histone acetylation and delayed the
initiation of parturition by 24–48 h, suggesting the functional
importance of the decline in histone acetylation in the initiation of labor.
These findings suggest that the decline in PR coactivator expression and in
histone acetylation in the uterus near term may impair PR function by causing
a functional progesterone withdrawal. The resulting decrease in expression of
PR-responsive genes should increase sensitivity of the uterus to contractile
stimuli.
Activated integrin αvβ3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells
Authors: Rolli Melanie , Fransvea Emilia , Pilch Jan , Saven Alan , Felding-Habermann Brunhilde
Published: August 2003

Expression of adhesion receptor integrin αvβ3 in an activated
functional form strongly promotes metastasis in human breast cancer cells.
Here, we report that αvβ3 cooperates with matrix metalloproteinase
type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated
by the activation state of the integrin. Expression of activated
αvβ3 in metastatic variants of MDA-MB 435 human breast cancer cells
and primary metastatic cells from breast cancer patients strongly enhanced
migration toward vitronectin and fibrinogen. This enhancement was mediated by
a soluble factor produced by breast cancer cells expressing activated
αvβ3. When transferred, this factor also up-regulated
αvβ3-dependent migration of breast cancer cells that express the
nonactivated integrin. The factor was identified as metalloproteinase MMP-9.
Whereas all tested breast cancer cell variants produced latent MMP-9, only
those with activated αvβ3 produced the mature form of this
metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either
form of MMP-2, enhanced αvβ3-dependent breast cancer cell
migration. The migratory response was inhibited by tissue inhibitors of
metalloproteinase or when MMP-9 was depleted from the inducing supernatants.
The results indicate a causal relationship between the expression of activated
integrin αvβ3 and production of enzymatically active MMP-9 in
metastatic breast cancer cells. These molecules cooperate to enhance breast
cancer cell migration toward specific matrix proteins, and this may contribute
to the strongly enhanced metastatic capacity of breast cancer cells that
express activated αvβ3.
A Ras activation pathway dependent on Syk phosphorylation of protein kinase C
Authors: Kawakami Yuko , Kitaura Jiro , Yao Libo , McHenry Robert W. , Kawakami Yu , Newton Alexandra C. , Kang Shin , Kato Roberta M. , Leitges Michael , Rawlings David J. , Kawakami Toshiaki
Published: August 2003

Protein kinase C (PKC) and Syk protein tyrosine kinase play critical roles in immune cell activation including that through the high-affinity IgE receptor, FcεRI. Mechanisms by which PKC activation leads to the activation of Ras, a family of GTPases essential for immune cell activation, have been elusive. We present evidence that Tyr-662 and Tyr-658 of PKCβI and PKCα, respectively, are phosphorylated by Syk in the membrane compartment of FcεRI-stimulated mast cells. These phosphorylations require prior PKC autophosphorylation of the adjacent serine residues (Ser-661 and Ser-657, respectively) and generate a binding site for the SH2 domain of the adaptor protein Grb-2. By recruiting the Grb-2/Sos complex to the plasma membrane, these conventional PKC isoforms contribute to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway in FcεRI-stimulated mast cells.
Repression of
c-Myc
and inhibition of G
1
exit in cells conditionally overexpressing
p300
that is not dependent on its histone acetyltransferase activity
Authors: Baluchamy Sudhakar , Rajabi Hasan N. , Thimmapaya Rama , Navaraj Arunasalam , Thimmapaya Bayar
Published: August 2003

p300 and cAMP response element binding protein (CREB)-binding protein (CBP)
are two highly homologous, conserved transcriptional coactivators, and histone
acetyltransferases (HATs) that link chromatin remodeling with transcription.
Cell transformation by viral oncogene products such as adenovirus E1A and SV40
large T antigen depends on their ability to inactivate p300 and CBP. To
investigate the role of p300 in cell-cycle progression, we constructed stable
rat cell lines, which conditionally overexpress
p300
from a
tetracycline-responsive promoter. When
p300
was induced in these
cells, serum-stimulated S-phase entry was significantly inhibited. The
inhibition of S-phase induction was associated with down-regulation of
c-Myc
, but not of
c-Fos
or
c-Jun
. Simultaneous
overexpression of
c-Myc
and
p300
before serum stimulation
reversed the inhibition of S-phase induction to a significant level,
indicating that the inhibition of
c-Myc
to a large extent is
responsible for the p300 inhibition of G
1
exit. Similar studies
with stable rat cell lines that overexpress a mutant p300, which lacks the HAT
activity, showed that the intrinsic HAT activity of p300 is not required for
the negative regulation of
c-Myc
or G
1
. These findings,
and our previously published results (Kolli, S., Buchmann, A. M., Williams,
J., Weitzman, S. & Thimmapaya, B. (2001)
Proc. Natl. Acad. Sci.
USA
98, 4646–4651), establish an important negative regulatory role
for p300 in
c-Myc
expression that may be important in maintaining the
cells in the G
0
/G
1
phase of the cell cycle.