bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface sensing and adhesion of
Escherichia coli
controlled by the Cpx-signaling pathway
Authors: Otto Karen , Silhavy Thomas J.
Published: July 2002

Bacterial adhesion is an important initial step in biofilm formation, which may cause problems in medical, environmental, and industrial settings. In spite of obvious phenotypic differences between attached and planktonic cells, knowledge about the genetic basis for these differences and how adhesion-induced changes are mediated is limited. The Cpx two-component signal transduction pathway responds specifically to stress caused by disturbances in the cell envelope and activates genes encoding periplasmic protein folding and degrading factors. Here, we address the role of the Cpx-signaling pathway in sensing and responding to the physical change occurring during adhesion of
Escherichia coli
to surfaces. We present evidence that the expression of Cpx-regulated genes is induced during initial adhesion of
E. coli
to abiotic surfaces. This induction is specifically observed upon attachment of stationary-phase cells to hydrophobic surfaces. Moreover, surface-induced activity of the Cpx response requires NlpE, an outer membrane lipoprotein, which has previously been shown to induce the Cpx system when overproduced. The importance of a functional Cpx response during adhesion is further supported by the fact that a dramatically lower number of cells attach to the surface and dynamic cell–surface interactions as measured by a quartz crystal microbalance technique are altered when the CpxRA pathway is disrupted. The defects in adhesion exhibited by the
cpxR
and
nlpE
mutants were strikingly similar to those of wild-type cells in which protein synthesis was inhibited, suggesting that the Cpx pathway plays a key role in the regulation of adhesion-induced gene expression.
Thyroid hormone-dependent metamorphosis in a direct developing frog
Authors: Callery Elizabeth M. , Elinson Richard P.
Published: July 2002

The direct developing anuran,
Eleutherodactylus
coqui
, lacks a tadpole, hatching as a tiny frog. We
investigated the role of the metamorphic trigger, thyroid hormone (TH),
in this unusual ontogeny. Expression patterns of the thyroid hormone
receptors, TRα and TRβ, were similar to those of indirect
developers. TRβ mRNA levels increased dramatically around the time of
thyroid maturation, when remodeling events reminiscent of metamorphosis
occur. Treatment with the goitrogen methimazole inhibited this
remodeling, which was reinitiated on cotreatment with TH. Despite their
radically altered ontogeny, direct developers still undergo a
TH-dependent metamorphosis, which occurs before hatching. We propose a
new model for the evolution of anuran direct development.
Akt phosphorylates and regulates the orphan nuclear receptor Nur77
Authors: Pekarsky Yuri , Hallas Cora , Palamarchuk Alexey , Koval Anatoliy , Bullrich Florencia , Hirata Yoko , Bichi Roberta , Letofsky Jean , Croce Carlo M.
Published: July 2002

The immediate early gene
NUR77
(also called
NGFI-B
) is required for T cell antigen receptor-mediated
cell death and is induced to very high levels in immature thymocytes
and T cell hybridomas undergoing apoptosis. The Akt (PKB)
kinase is a key player in transduction of anti-apoptotic and
proliferative signals in T cells. Because Nur77 has a putative Akt
phosphorylation site at Ser-350, and phosphorylation of this residue is
critical for the transactivation activity of Nur77, we investigated
whether Akt regulates Nur77. Coimmunoprecipitation experiments showed
the detection of Nur77 in Akt immune complexes, suggesting that Nur77
and Akt physically interact. We further show that Akt specifically
phosphorylates Ser-350 of the Nur77 protein within its DNA-binding
domain
in vitro
and
in vivo
in 293 and
NIH 3T3 cells. Because phosphorylation of Ser-350 of Nur77 is critical
for its function as a transcription factor, we examined the effect of
Akt on this function. By using luciferase assay experiments, we showed
that phosphorylation of Nur77 by Akt decreased the transcriptional
activity of Nur77 by 50–85%. Thus, we show that Akt interacts with
Nur77 and inactivates Nur77 by phosphorylation at Ser-350 in a
phosphatidylinositol 3-kinase-dependent manner, connecting the
phosphatidylinositol 3-kinase-dependent Akt pathway and a nuclear
receptor pathway.
The p24 proteins are not essential for vesicular transport in
Saccharomyces cerevisiae
Authors: Springer Sebastian , Chen Eric , Duden Rainer , Marzioch Martina , Rowley Adele , Hamamoto Susan , Merchant Sabeeha , Schekman Randy
Published: July 2002

To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of
S. cerevisiae
(p24Δ8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p). The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells. We find that p24Δ8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single
Δemp24
deletion strain. Thus, p24 proteins do not have an essential function in the secretory pathway. Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles.
The Notch ligand
Jagged1
is required for inner ear sensory development
Authors: Kiernan Amy E. , Ahituv Nadav , Fuchs Helmut , Balling Rudi , Avraham Karen B. , Steel Karen P. , Hrabé de Angelis Martin
Published: July 2002

Within the mammalian inner ear there are six separate sensory
regions that subserve the functions of hearing and balance, although
how these sensory regions become specified remains unknown. Each
sensory region is populated by two cell types, the mechanosensory hair
cell and the supporting cell, which are arranged in a mosaic in which
each hair cell is surrounded by supporting cells. The proposed
mechanism for creating the sensory mosaic is lateral inhibition
mediated by the Notch signaling pathway. However, one of the Notch
ligands,
Jagged1
(
Jag1
), does not show an
expression pattern wholly consistent with a role in lateral inhibition,
as it marks the sensory patches from very early in their
development—presumably long before cells make their final fate
decisions. It has been proposed that
Jag1
has a role in
specifying sensory versus nonsensory epithelium within the ear [Adam,
J., Myat, A., Roux, I. L., Eddison, M., Henrique, D.,
Ish-Horowicz, D. & Lewis, J. (1998)
Development (Cambridge,
U.K.)
125, 4645–4654]. Here we provide experimental evidence
that Notch signaling may be involved in specifying sensory regions by
showing that a dominant mouse mutant headturner (
Htu
)
contains a missense mutation in the
Jag1
gene and
displays missing posterior and sometimes anterior ampullae, structures
that house the sensory cristae.
Htu
/+ mutants also
demonstrate a significant reduction in the numbers of outer hair cells
in the organ of Corti. Because lateral inhibition mediated by Notch
predicts that disruptions in this pathway would lead to an increase in
hair cells, we believe these data indicate an earlier role for Notch
within the inner ear.
Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes
Authors: Vosseller Keith , Wells Lance , Lane M. Daniel , Hart Gerald W.
Published: July 2002

Increased flux of glucose through the hexosamine biosynthetic pathway (HSP) is believed to mediate hyperglycemia-induced insulin resistance in diabetes. The end product of the HSP, UDPβ-
N
-acetylglucosamine (GlcNAc), is a donor sugar nucleotide for complex glycosylation in the secretory pathway and for O-linked GlcNAc (O-GlcNAc) addition to nucleocytoplasmic proteins. Cycling of the O-GlcNAc posttranslational modification was blocked by pharmacological inhibition of O-GlcNAcase, the enzyme that catalyzes O-GlcNAc removal from proteins, with
O
-(2-acetamido-2-deoxy-
d
-glucopyranosylidene)amino-
N
-phenylcarbamate (PUGNAc). PUGNAc treatment increased levels of O-GlcNAc and caused insulin resistance in 3T3-L1 adipocytes. Insulin resistance induced through the HSP by glucosamine and chronic insulin treatment correlated with increased O-GlcNAc levels on nucleocytoplasmic proteins. Whereas insulin receptor autophosphorylation and insulin receptor substrate 2 tyrosine phosphorylation were not affected by PUGNAc inhibition of O-GlcNAcase, downstream phosphorylation of Akt at Thr-308 and glycogen synthase kinase 3β at Ser-9 was inhibited. PUGNAc-induced insulin resistance was associated with increased O-GlcNAc modification of several proteins including insulin receptor substrate 1 and β-catenin, two important effectors of insulin signaling. These results suggest that elevation of O-GlcNAc levels attenuate insulin signaling and contribute to the mechanism by which increased flux through the HSP leads to insulin resistance in adipocytes.
The homeobox gene
BREVIPEDICELLUS
is a key regulator of inflorescence architecture in
Arabidopsis
Authors: Venglat S. P. , Dumonceaux T. , Rozwadowski K. , Parnell L. , Babic V. , Keller W. , Martienssen R. , Selvaraj G. , Datla R.
Published: July 2002

Flowering plants display a remarkable range of inflorescence architecture, and pedicel characteristics are one of the key contributors to this diversity. However, very little is known about the genes or the pathways that regulate pedicel development. The
brevipedicellus
(
bp
) mutant of
Arabidopsis thaliana
displays a unique phenotype with defects in pedicel development causing downward-pointing flowers and a compact inflorescence architecture. Cloning and molecular analysis of two independent mutant alleles revealed that
BP
encodes the homeodomain protein KNAT1, a member of the KNOX family.
bp-1
is a null allele with deletion of the entire locus, whereas
bp-2
has a point mutation that is predicted to result in a truncated protein. In both
bp
alleles, the pedicels and internodes were compact because of fewer cell divisions; in addition, defects in epidermal and cortical cell differentiation and elongation were found in the affected regions. The downward-pointing pedicels were produced by an asymmetric effect of the
bp
mutation on the abaxial vs. adaxial sides. Cell differentiation, elongation, and growth were affected more severely on the abaxial than adaxial side, causing the change in the pedicel growth angle. In addition,
bp
plants displayed defects in cell differentiation and radial growth of the style. Our results show that
BP
plays a key regulatory role in defining important aspects of the growth and cell differentiation of the inflorescence stem, pedicel, and style in
Arabidopsis
.
Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain
Authors: Constantinescu Stefan N. , Keren Tzvia , Socolovsky Merav , Nam Hyung-song , Henis Yoav I. , Lodish Harvey F.
Published: July 2002

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is
crucial for production of mature red cells. Although it is well
established that the Epo-bound EpoR is a dimer, it is not clear
whether, in the absence of ligand, the intact EpoR is a monomer or
oligomer. Using antibody-mediated immunofluorescence copatching
(oligomerizing) of epitope-tagged receptors at the surface of live
cells, we show herein that a major fraction of the full-length murine
EpoR exists as preformed dimers/oligomers in BOSC cells, which
are human embryo kidney 293T-derived cells. This observed
oligomerization is specific because, under the same conditions,
epitope-tagged EpoR did not oligomerize with several other tagged
receptors (thrombopoietin receptor, transforming growth factor β
receptor type II, or prolactin receptor). Strikingly, the EpoR
transmembrane (TM) domain but not the extracellular or intracellular
domains enabled the prolactin receptor to copatch with EpoR. Preformed
EpoR oligomers are not constitutively active and Epo binding was
required to induce signaling. In contrast to tyrosine kinase receptors
(e.g., insulin receptor), which cannot signal when their TM domain is
replaced by the strongly dimerizing TM domain of glycophorin A, the
EpoR could tolerate the replacement of its TM domain with that of
glycophorin A and retained signaling. We propose a model in which TM
domain-induced dimerization maintains unliganded EpoR in an inactive
state that can readily be switched to an active state by physiologic
levels of Epo.
Otoancorin, an inner ear protein restricted to the interface between the apical surface of sensory epithelia and their overlying acellular gels, is defective in autosomal recessive deafness DFNB22
Authors: Zwaenepoel Ingrid , Mustapha Mirna , Leibovici Michel , Verpy Elisabeth , Goodyear Richard , Liu Xue Zhong , Nouaille Sylvie , Nance Walter E. , Kanaan Moien , Avraham Karen B. , Tekaia Fredj , Loiselet Jacques , Lathrop Marc , Richardson Guy , Petit Christine
Published: July 2002

A 3,673-bp murine cDNA predicted to encode a glycosylphosphatidylinositol-anchored protein of 1,088 amino acids was isolated during a study aimed at identifying transcripts specifically expressed in the inner ear. This inner ear-specific protein, otoancorin, shares weak homology with megakaryocyte potentiating factor/mesothelin precursor. Otoancorin is located at the interface between the apical surface of the inner ear sensory epithelia and their overlying acellular gels. In the cochlea, otoancorin is detected at two attachment zones of the tectorial membrane, a permanent one along the top of the spiral limbus and a transient one on the surface of the developing greater epithelial ridge. In the vestibule, otoancorin is present on the apical surface of nonsensory cells, where they contact the otoconial membranes and cupulae. The identification of the mutation (IVS12+2T>C) in the corresponding gene
OTOA
in one consanguineous Palestinian family affected by nonsyndromic recessive deafness DFNB22 assigns an essential function to otoancorin. We propose that otoancorin ensures the attachment of the inner ear acellular gels to the apical surface of the underlying nonsensory cells.
Growth of new brainstem connections in adult monkeys with massive sensory loss
Authors: Jain Neeraj , Florence Sherre L. , Qi Hui-Xin , Kaas Jon H.
Published: July 2002

Somatotopic maps in the cortex and the thalamus of adult monkeys and humans reorganize in response to altered inputs. After loss of the sensory afferents from the forelimb in monkeys because of transection of the dorsal columns of the spinal cord, therapeutic amputation of an arm or transection of the dorsal roots of the peripheral nerves, the deprived portions of the hand and arm representations in primary somatosensory cortex (area 3b), become responsive to inputs from the face and any remaining afferents from the arm. Cortical and subcortical mechanisms that underlie this reorganization are uncertain and appear to be manifold. Here we show that the face afferents from the trigeminal nucleus of the brainstem sprout and grow into the cuneate nucleus in adult monkeys after lesions of the dorsal columns of the spinal cord or therapeutic amputation of an arm. This growth may underlie the large-scale expansion of the face representation into the hand region of somatosensory cortex that follows such deafferentations.
Selection–mutation balance in polysomic tetraploids: Impact of double reduction and gametophytic selection on the frequency and subchromosomal localization of deleterious mutations
Authors: Butruille D. V. , Boiteux L. S.
Published: July 2002

We modeled the behavior of recessive mutations with deleterious effects to either the sporophyte or the gametophyte, or both, in polysomic tetraploid populations by allowing for varying levels of double reduction, mutation, and self-fertilization. Double reduction causes a decrease of the equilibrium frequencies of deleterious alleles, and it has much more influence on genes subjected to gametophytic selection than on genes solely under sporophytic selection. With gametophytic selection, low frequencies of double reduction are enough to reduce equilibrium frequencies severalfold. Double reduction occurs when sister alleles migrate to the same gamete during meiosis. It depends on the frequency at which a locus recombines with its centromere, and on the frequency of multivalent formation. Therefore, a greater accumulation of deleterious mutations should occur on polysomic chromosomes with a prevalence of bivalent pairing and in chromosomal regions between centromeres and proximal chiasmata. Proximal loci should have a greater impact in reducing the fitness of a polyploid population being inbred. This prediction can explain observations that homozygosities at different subchromosomal regions have distinct effects on inbreeding depression in polyploids. Furthermore, even mildly deleterious alleles can lead to large amounts of inbreeding depression because of their high equilibrium frequencies. Molecular studies correlating level of heterozygosity and degree of heterosis should take into account this nonuniform distribution of deleterious alleles in polyploid genomes. Preservation or enhancement of heterozygosity would be more critical at proximal regions than at other chromosome regions in polysomic polyploid species.
A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb
Authors: Didier Anne , Carleton Alan , Bjaalie Jan G. , Vincent Jean-Didier , Ottersen Ole Petter , Storm-Mathisen Jon , Lledo Pierre-Marie
Published: July 2002

Neuronal synchronization in the olfactory bulb has been
proposed to arise from a diffuse action of glutamate released from
mitral cells (MC, olfactory bulb relay neurons). According to this
hypothesis, glutamate spills over from dendrodendritic synapses formed
between MC and granule cells (GC, olfactory bulb interneurons) to
activate neighboring MC. The excitation of MC is balanced by a strong
inhibition from GC. Here we show that MC excitation is caused by
glutamate released from bulbar interneurons located in the GC layer.
These reciprocal synapses depend on an unusual,
2-amino-5-phosphonovaleric acid-resistant,
N
-methyl-
d
-aspartate receptor. This type of
feedback excitation onto relay neurons may strengthen the original
sensory input signal and further extend the function of the dendritic
microcircuit within the main olfactory bulb.
Molecular organization of the postsynaptic specialization
Authors: Sheng Morgan
Published: July 2002

A specific set of molecules including glutamate receptors is targeted to the postsynaptic specialization of excitatory synapses in the brain, gathering in a structure known as the postsynaptic density (PSD). Synaptic targeting of glutamate receptors depends on interactions between the C-terminal tails of receptor subunits and specific PDZ domain-containing scaffold proteins in the PSD. These scaffold proteins assemble a specialized protein complex around each class of glutamate receptor that functions in signal transduction, cytoskeletal anchoring, and trafficking of the receptors. Among the glutamate receptor subtypes, the
N
-methyl-
d
-aspartate receptor is relatively stably integrated in the PSD, whereas the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor moves in and out of the postsynaptic membrane in highly dynamic fashion. The distinctive cell biological behaviors of
N
-methyl-
d
-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be explained by their differential interactions with cytoplasmic proteins.
Developmental plasticity of CNS microglia
Authors: Santambrogio L. , Belyanskaya S. L. , Fischer F. R. , Cipriani B. , Brosnan C. F. , Ricciardi-Castagnoli P. , Stern L. J. , Strominger J. L. , Riese R.
Published: July 2002

Microglia arise from CD45
+
bone marrow precursors that
colonize the fetal brain and play a key role in central nervous system
inflammatory conditions. We report that parenchymal microglia are
uncommitted myeloid progenitors of immature dendritic cells and
macrophages by several criteria, including surface expression of
“empty” class II MHC protein and their cysteine protease
(cathepsin) profile. Microglia express receptors for stem cell factor
and can be skewed toward more dendritic cell or macrophage-like
profiles in response to the lineage growth factors
granulocyte/macrophage colony-stimulating factor or macrophage
colony-stimulating factor. Thus, in contrast to other organs, where
terminally differentiated populations of resident dendritic cells
and/or macrophages outnumber colonizing precursors, the majority
of microglia within the brain remain in an undifferentiated
state.
A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity
Authors: Scherf Tali , Kasher Roni , Balass Moshe , Fridkin Mati , Fuchs Sara , Katchalski-Katzir Ephraim
Published: July 2002

Snake-venom α-bungarotoxin is a member of the
α-neurotoxin family that binds with very high affinity to the
nicotinic acetylcholine receptor (AChR) at the neuromuscular junction.
The structure of the complex between α-bungarotoxin and a 13-mer
peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus
inhibiting its interactions with AChR with an IC
50
of 2 nM,
has been solved by
1
H-NMR spectroscopy. The bound peptide
folds into a β-hairpin structure created by two antiparallel
β-strands, which combine with the already existing triple-stranded
β-sheet of the toxin to form a five-stranded intermolecular,
antiparallel β-sheet. Peptide residues Y3
P
,
E5
P
, and L8
P
have the highest intermolecular
contact area, indicating their importance in the binding of
α-bungarotoxin; W1
P
, R2
P
, and Y4
P
also contribute significantly to the binding. A large number of
characteristic hydrogen bonds and electrostatic and hydrophobic
interactions are observed in the complex. The high-affinity peptide
exhibits inhibitory potency that is better than any known peptide
derived from AChR, and is equal to that of the whole α-subunit of
AChR. The high degree of sequence similarity between the peptide and
various types of AChRs implies that the binding mode found within the
complex might possibly mimic the receptor binding to the toxin. The
design of the high-affinity peptide was based on our previous findings:
(
i
) the detection of a lead peptide (MRYYESSLKSYPD) that
binds α-bungarotoxin, using a phage-display peptide library,
(
ii
) the information about the three-dimensional
structure of α-bungarotoxin/lead-peptide complex, and
(
iii
) the amino acid sequence analysis of different
AChRs.
Dendritic K
+
channels contribute to spike-timing dependent long-term potentiation in hippocampal pyramidal neurons
Authors: Watanabe Shigeo , Hoffman Dax A. , Migliore Michele , Johnston Daniel
Published: July 2002

We investigated the role of A-type K
+
channels for the induction of long-term potentiation (LTP) of Schaffer collateral inputs to hippocampal CA1 pyramidal neurons. When low-amplitude excitatory postsynaptic potentials (EPSPs) were paired with two postsynaptic action potentials in a theta-burst pattern,
N
-methyl-
d
-aspartate (NMDA)-receptor-dependent LTP was induced. The amplitudes of the back-propagating action potentials were boosted in the dendrites only when they were coincident with the EPSPs. Mitogen-activated protein kinase (MAPK) inhibitors PD 098059 or U0126 shifted the activation of dendritic K
+
channels to more hyperpolarized potentials, reduced the boosting of dendritic action potentials by EPSPs, and suppressed the induction of LTP. These results support the hypothesis that dendritic K
+
channels and the boosting of back-propagating action potentials contribute to the induction of LTP in CA1 neurons.
Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction
Authors: Panda Dibyendu K. , Miao Dengshun , Tremblay Michel L. , Sirois Jacinthe , Farookhi Riaz , Hendy Geoffrey N. , Goltzman David
Published: July 2002

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)
2
D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)
2
D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the
in vivo
functions of 1,25(OH)
2
D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.
Crystal structure of an anticoagulant protein in complex with the Gla domain of factor X
Authors: Mizuno Hiroshi , Fujimoto Zui , Atoda Hideko , Morita Takashi
Published: July 2002

The γ-carboxyglutamic acid (Gla) domain of blood coagulation factors is responsible for Ca
2+
-dependent phospholipid membrane binding. Factor X-binding protein (X-bp), an anticoagulant protein from snake venom, specifically binds to the Gla domain of factor X. The crystal structure of X-bp in complex with the Gla domain peptide of factor X at 2.3-Å resolution showed that the anticoagulation is based on the fact that two patches of the Gla domain essential for membrane binding are buried in the complex formation. The Gla domain thus is expected to be a new target of anticoagulant drugs, and X-bp provides a basis for designing them. This structure also provides a membrane-bound model of factor X.
Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models
Authors: Cao Brian , Su Yanli , Oskarsson Marianne , Zhao Ping , Kort Eric J. , Fisher Robert J. , Wang Ling-Mei , Vande Woude George F.
Published: July 2002

The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested
in vitro
for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the
in vitro
activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation.
In vivo
, the neutralizing mAb combination inhibited s.c. growth in athymic
nu/nu
mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.
Endogenous
Msx1
antisense transcript:
In vivo
and
in vitro
evidences, structure, and potential involvement in skeleton development in mammals
Authors: Blin-Wakkach C. , Lezot F. , Ghoul-Mazgar S. , Hotton D. , Monteiro S. , Teillaud C. , Pibouin L. , Orestes-Cardoso S. , Papagerakis P. , Macdougall M. , Robert B. , Berdal A.
Published: July 2002

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous
Msx1
antisense RNA (
Msx1
-AS RNA) in mice, rats, and humans.
In situ
analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These
in vivo
data and overexpression of
Msx1
sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two
Msx1
RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the
Msx1
-AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation,
Cbfa1
. All these data strongly suggest that the ratio between
Msx1
sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for
Msx1
-AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of
Msx1
gene expression.
Different modes of hippocampal plasticity in response to estrogen in young and aged female rats
Authors: Adams Michelle M. , Shah Ravi A. , Janssen William G. M. , Morrison John H.
Published: July 2002

Estrogen regulates hippocampal dendritic spine density and synapse number in an
N-
methyl-
d
-aspartate (NMDA) receptor-dependent manner, and these effects may be of particular importance in the context of age-related changes in endocrine status. We investigated estrogen's effects on axospinous synapse density and the synaptic distribution of the NMDA receptor subunit, NR1, within the context of aging. Although estrogen induced an increase in axospinous synapse density in young animals, it did not alter the synaptic representation of NR1, in that the amount of NR1 per synapse was equivalent across groups. Estrogen replacement in aged female rats failed to increase axospinous synapse density; however, estrogen up-regulated synaptic NR1 compared with aged animals with no estrogen. Therefore, the young and aged hippocampi react differently to estrogen replacement, with the aged animals unable to mount a plasticity response generating additional synapses, yet responsive to estrogen with respect to additional NMDA receptor content per synapse. These findings have important implications for estrogen replacement therapy in the context of aging.
Localization of atherosclerosis susceptibility loci to chromosomes 4 and 6 using the
Ldlr
knockout mouse model
Authors: Welch Carrie L. , Bretschger Sara , Latib Nashat , Bezouevski Mikhail , Guo Yan , Pleskac Nick , Liang Chien-Ping , Barlow Courtenay , Dansky Hayes , Breslow Jan L. , Tall Alan R.
Published: July 2002

Atherosclerosis is a complex disease resulting from the interaction of multiple genes. We have used the
Ldlr
knockout mouse model in an interspecific genetic cross to map atherosclerosis susceptibility loci. A total of 174 (MOLF/Ei × B6.129S7-
Ldlr

tm1Her

) × C57BL/6J-
Ldlr

tm1Her

backcross mice, homozygous for the
Ldlr
null allele, were fed a Western-type diet for 3 months and then killed for quantification of aortic lesions. A genome scan was carried out by using DNA pools and microsatellite markers spaced at ≈18-centimorgan intervals. Quantitative trait locus analysis of individual backcross mice confirmed linkages to chromosomes 4 (
Athsq1
, logarithm of odds = 6.2) and 6 (
Athsq2
, logarithm of odds = 6.7).
Athsq1
affected lesions in females only whereas
Athsq2
affected both sexes. Among females, the loci accounted for ≈50% of the total variance of lesion area. The susceptible allele at
Athsq1
was derived from the MOLF/Ei genome whereas the susceptible allele at
Athsq2
was derived from C57BL/6J. Inheritance of susceptible alleles at both loci conferred a 2-fold difference in lesion area, suggesting an additive effect of
Athsq1
and
Athsq2
. No associations were observed between the quantitative trait loci and levels of plasma total cholesterol, high density lipoprotein cholesterol, non-high density lipoprotein cholesterol, insulin, or body weight. We provide strong evidence for complex inheritance of atherosclerosis in mice with elevated plasma low density lipoprotein cholesterol and show a major influence of nonlipoprotein-related factors on disease susceptibility.
Athsq1
and
Athsq2
represent candidate susceptibility loci for human atherosclerosis, most likely residing on chromosomes 1p36–32 and 12p13–12, respectively.
Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity
Authors: Bocchetta Maurizio , Di Resta Ilaria , Powers Amy , Fresco Raoul , Tosolini Alessandra , Testa Joseph R. , Pass Harvey I. , Rizzo Paola , Carbone Michele
Published: July 2002

Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens.
IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production
Authors: Ochi Hiroshi , De Jesus Nidia H. , Hsieh Fred H. , Austen K. Frank , Boyce Joshua A.
Published: July 2002

Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMCin vitro. Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcɛRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-α, IL-5, IL-13, macrophage inflammatory protein 1α, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcɛRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcɛRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.
Lineage-specific loss and divergence of functionally linked genes in eukaryotes
Authors: Aravind L. , Watanabe Hidemi , Lipman David J. , Koonin Eugene V.
Published: July 2002

By comparing 4,344 protein sequences from fission yeast
Schizosaccharomyces pombe
with all available eukaryotic
sequences, we identified those genes that are conserved in
S.
pombe
and nonfungal eukaryotes but are missing or highly
diverged in the baker's yeast
Saccharomyces cerevisiae
.
Since the radiation from the common ancestor with
S.
pombe
,
S. cerevisiae
appears to have lost about
300 genes, and about 300 more genes have diverged by far beyond
expectation. The most notable feature of the set of genes lost in
S. cerevisiae
is the coelimination of functionally
connected groups of proteins, such as the signalosome and the
spliceosome components. We predict similar coelimination of the
components of the posttranscriptional gene-silencing system that
includes the recently identified RNA-dependent RNA polymerase. Because
one of the functions of posttranscriptional silencing appears to be
“taming” of retrotransposons, the loss of this system in yeast
could have triggered massive retrotransposition, resulting in
elimination of introns and subsequent loss of spliceosome components
that become dispensable. As the genome database grows, systematic
analysis of coordinated gene loss may become a general approach for
predicting new components of functional systems or even defining
previously unknown functional complexes.
Altered selectivity in anArabidopsismetal transporter
Authors: Rogers Elizabeth E. , Eide David J. , Guerinot Mary Lou
Published: July 2002

Plants require metals for essential functions ranging from respiration to photosynthesis. These metals also contribute to the nutritional value of plants for both humans and livestock. Additionally, plants have the ability to accumulate nonessential metals such as cadmium and lead, and this ability could be harnessed to remove pollutant metals from the environment. Designing a transporter that specifically accumulates certain cations while excluding others has exciting applications in all of these areas. TheArabidopsisroot membrane protein IRT1 is likely to be responsible for uptake of iron from the soil. Like other Fe(II) transporters identified to date, IRT1 transports a variety of other cations, including the essential metals zinc and manganese as well as the toxic metal cadmium. By heterologous expression in yeast, we show here that the replacement of a glutamic acid residue at position 103 in wild-type IRT1 with alanine increases the substrate specificity of the transporter by selectively eliminating its ability to transport zinc. Two other mutations, replacing the aspartic acid residues at either positions 100 or 136 with alanine, also increase IRT1 metal selectivity by eliminating transport of both iron and manganese. A number of other conserved residues in or near transmembrane domains appear to be essential for all transport function. Therefore, this study identifies at least some of the residues important for substrate selection and transport in a protein belonging to theZIPgene family, a large transporter family found in a wide variety of organisms.
TNF-α-dependent maturation of local dendritic cells is critical for activating the adaptive immune response to virus infection
Authors: Trevejo Jose M. , Marino Michael W. , Philpott Nicola , Josien Regis , Richards Elizabeth C. , Elkon Keith B. , Falck-Pedersen Erik
Published: July 2002

Tumor necrosis factor-α (TNF-α) is well recognized for its role in mediating innate immune responses. However, the mechanisms of TNF-α that influence the adaptive immune response to virus infections are not well understood. In this study, we have investigated the role of TNF-α in activating the cellular and humoral responses to systemic viral challenge with recombinant replication-defective adenovirus (rAd). Evaluation of T cell function in TNF-α-deficient (TNFKO) mice revealed impaired virus-specific proliferation of T cells derived from the draining lymph nodes of the liver. Analysis of dendritic cells (DC) isolated from local draining lymph nodes after systemic challenge showed that DC from TNFKO mice were relatively immature compared with those from strain-matched wild-type mice.In vitro, TNF-α was required to mature DC efficiently during virus-mediated stimulation. Adoptive transfer of primed, mature DC into TNFKO mice restored T cell responses and reconstituted anti-adenovirus antibody responses. Thus, TNF-α plays a significant role in the maturation of DC after adenovirus challenge bothin vitroandin vivo, highlighting the importance of this innate cytokine in activating adaptive immunity to viral challenge.
P{Switch}, a system for spatial and temporal control of gene expression in
Drosophila melanogaster
Authors: Roman Gregg , Endo Keita , Zong Lin , Davis Ronald L.
Published: July 2002

We have developed a method for turning on and off the expression of transgenes within
Drosophila
in both time and space. Two different enhancer detector elements carrying an RU486-inducible form of the yeast transcription factor GAL4 were constructed and used to generate enhancer detector lines. These lines were screened for RU486-inducible reporter gene expression in the adult head. We identified lines that exhibit inducible expression in many cell and tissue types, verifying that the elements respond to nearby enhancers. No expression was detected in the absence of the ligand. The P{Switch1} element responded to genomic enhancers less efficiently than P{Switch2} but produced more specific patterns of expression. Two P{Switch} lines were used to ablate fat body tissue in adult females through the induced expression of diphtheria toxin. These females were sterile, which correlates with fat body loss, and they died prematurely.
Metabolic rate and environmental productivity: Well-provisioned animals evolved to run and idle fast
Authors: Mueller Pamela , Diamond Jared
Published: July 2002

Even among vertebrate species of the same body mass and higher-level taxonomic group, metabolic rates exhibit substantial differences, for which diverse explanatory factors—such as dietary energy content, latitude, altitude, temperature, and rainfall—have been postulated. A unifying underlying factor could be food availability, in turn controlled by net primary productivity (NPP) of the animal's natural environment. We tested this possibility by studying five North American species of
Peromyscus
mice, all of them similar in diet (generalist omnivores) and in gut morphology but differing by factors of up to 13 in NPP of their habitat of origin. We maintained breeding colonies of all five species in the laboratory under identical conditions and consuming identical diets. Basal metabolic rate (BMR) and daily ad libitum food intake both increased with NPP, which explained 88% and 90% of their variances, respectively. High-metabolism mouse species from high-NPP environments were behaviorally more active than were low-metabolism species from low-NPP environments. Intestinal glucose uptake capacity also increased with NPP (and with BMR and food intake), because species of high-NPP environments had larger small intestines and higher uptake rates. For metabolic rates of our five species, the driving environmental variable is environmental productivity itself (and hence food availability), rather than temporal variability of productivity. Thus, species that have evolved in the presence of abundant food run their metabolism “fast,” both while active and while idling, as compared with species of less productive environments, even when all species are given access to unlimited food.
Impairment of MAD2B–PRCC interaction in mitotic checkpoint defective t(X;1)-positive renal cell carcinomas
Authors: Weterman Marian A. J. , van Groningen Jan J. M. , Tertoolen Leon , van Kessel Ad Geurts
Published: July 2002

The papillary renal cell carcinoma (RCC)-associated (X;1)(p11;q21) translocation fuses the genes
PRCC
and
TFE3
and leads to cancer by an unknown molecular mechanism. We here demonstrate that the mitotic checkpoint protein MAD2B interacts with PRCC. The PRCCTFE3 fusion protein retains the MAD2B interaction domain, but this interaction is impaired. In addition, we show that two t(X;1)-positive RCC tumor cell lines are defective in their mitotic checkpoint. Transfection of
PRCCTFE3
, but not the reciprocal product
TFE3PRCC
, disrupts the mitotic checkpoint in human embryonic kidney cells. Our results suggest a dominant-negative effect of the
PRCCTFE3
fusion gene leading to a mitotic checkpoint defect as an early event in papillary RCCs.
Plasma membrane rafts play a critical role in HIV-1 assembly and release
Authors: Ono Akira , Freed Eric O.
Published: July 2002

HIV-1 particle production occurs in a series of steps promoted by the viral Gag protein. Although it is well established that assembly and release take place at the plasma membrane, the nature of membrane assembly sites remains poorly understood. We show here that Gag specifically associates with cholesterol-enriched microdomains (“rafts”) at the plasma membrane. Kinetic studies demonstrate that raft association follows membrane binding, and the analysis of Gag mutants reveals that, whereas the N terminus of Gag mediates raft binding, this association is greatly enhanced by Gag–Gag interaction domains. We observe that depletion of cellular cholesterol markedly and specifically reduces HIV-1 particle production. Furthermore, treatment of virus-producing cells or virus particles with raft-disrupting agents significantly impairs virus infectivity. These results identify the association of Gag with plasma membrane rafts as an important step in HIV-1 replication. These findings may lead to novel strategies for suppressing HIV-1 replication
in vivo
.
Magnetite morphology and life on Mars
Authors: Buseck Peter R. , Dunin-Borkowski Rafal E. , Devouard Bertrand , Frankel Richard B. , McCartney Martha R. , Midgley Paul A. , Pósfai Mihály , Weyland Matthew
Published: July 2002

Nanocrystals of magnetite (Fe
3
O
4
) in a meteorite from Mars provide the strongest, albeit controversial, evidence for the former presence of extraterrestrial life. The morphological and size resemblance of the crystals from meteorite ALH84001 to crystals formed by certain terrestrial bacteria has been used in support of the biological origin of the extraterrestrial minerals. By using tomographic and holographic methods in a transmission electron microscope, we show that the three-dimensional shapes of such nanocrystals can be defined, that the detailed morphologies of individual crystals from three bacterial strains differ, and that none uniquely match those reported from the Martian meteorite. In contrast to previous accounts, we argue that the existing crystallographic and morphological evidence is inadequate to support the inference of former life on Mars.
A RHO GTPase-mediated pathway is required during P cell migration in
Caenorhabditis elegans
Authors: Spencer Andrew G. , Orita Satoshi , Malone Christian J. , Han Min
Published: July 2002

The Rho family of guanine triphosphate hydrolases controls various cellular processes, including cell migration. We describe here the demonstration of a role for a RhoA GTPase homologue during cell migration in
Caenorhabditis elegans.
We show that eliminating or reducing
rho-1
gene function by using a dominant-negative transgene or dsRNA interference results in a severe defect in migration of hypodermal P cells to a ventral position. Biochemical and genetic data also suggest that
unc-73
, which encodes a Trio-like guanine nucleotide exchange factor, may act as an activator of
rho-1
in the migration process. Mutations in
let-502
ROCK, a homologue of a RhoA effector in mammals, also cause defects in P cell migration, suggesting that it may be one of several effectors acting downstream of
rho-1
during P cell migration. Finally, we provide evidence to support the idea that other small Rac subfamily small GTPases act redundantly and in parallel to RHO-1 in this specific cell migration event.