bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.
Authors: Pertsemlidis A , Saxena A M , Soper A K , Head-Gordon T , Glaeser R M
Published: July 2002

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.
The structural
H19
gene is required for transgene imprinting
Authors: Pfeifer Karl , Leighton Philip A. , Tilghman Shirley M.
Published: July 2002

The product of the
H19
gene is an untranslated RNA
that is expressed exclusively from the maternal chromosome during
mammalian development. The
H19
gene and its 5′-flanking
sequence are required for the genomic imprinting of two paternally
expressed genes,
Ins-2
(encodes insulin-2) and
Igf-2
(encodes insulin-like growth factor-2), that lie
90 and 115 kb 5′ to the
H19
gene, respectively. In this
report, the role of the
H19
gene in its own imprinting
is investigated by introducing a
Mus spretus H19
gene
into heterologous locations in the mouse genome. Multiple copies of the
transgene were sufficient for its paternal silencing and DNA
methylation. Replacing the
H19
structural gene with a
luciferase reporter gene resulted in loss of imprinting of the
transgene. That is, high expression and low levels of DNA methylation
were observed upon both paternal and maternal inheritance. The removal
of 701 bp at the 5′ end of the structural gene resulted in a similar
loss of paternal-specific DNA methylation, arguing that those sequences
are required for both the establishment and maintenance of the
sperm-specific gametic mark. The
M. spretus H19
transgene could not rescue the loss of
Igf-2
imprinting
in trans in
H19
deletion mice, implying a cis
requirement for the
H19
gene. In contrast to a previous
report in which overexpression of a marked
H19
gene was
a prenatal lethal, expression of the
M. spretus
transgene had no deleterious effect, leading to the conclusion that the
20-base insertion in the marked gene created a neomorphic mutation.
Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells.
Authors: McBride A E , Schlegel A , Kirkegaard K
Published: July 2002

A HeLa cDNA expression library was screened for human polypeptides that interacted with the poliovirus RNA-dependent RNA polymerase, 3D, using the two-hybrid system in the yeast Saccharomyces cerevisiae. Sam68 (Src-associated in mitosis, 68 kDa) emerged as the human cDNA that, when fused to a transcriptional activation domain, gave the strongest 3D interaction signal with a LexA-3D hybrid protein. 3D polymerase and Sam68 coimmunoprecipitated from infected human cell lysates with antibodies that recognized either protein. Upon poliovirus infection, Sam68 relocalized from the nucleus to the cytoplasm, where poliovirus replication occurs. Sam68 was isolated from infected cell lysates with an antibody that recognizes poliovirus protein 2C, suggesting that it is found on poliovirus-induced membranes upon which viral RNA synthesis occurs. These data, in combination with the known RNA- and protein-binding properties of Sam68, make Sam68 a strong candidate for a host protein with a functional role in poliovirus replication.
A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.
Authors: O'Bryan J P , Songyang Z , Cantley L , Der C J , Pawson T
Published: July 2002

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.
Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors.
Authors: Amalfitano A , Begy C R , Chamberlain J S
Published: July 2002

Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases.
ECA1 complements yeast mutants defective in Ca
2+
pumps and encodes an endoplasmic reticulum-type Ca
2+
-ATPase in
Arabidopsis thaliana
Authors: Liang Feng , Cunningham Kyle W. , Harper Jeffrey F. , Sze Heven
Published: July 2002

To understand the structure, role, and regulation of individual Ca
2+
pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from
Arabidopsis thaliana
(ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca
2+
pumps (EC
3.6.1.38
). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca
2+
pumps. Yeast mutants defective in a Golgi Ca
2+
pump (
pmr1
) or both Golgi and vacuolar Ca
2+
pumps (
pmr1 pmc1 cnb1
) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn
2+
. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the
pmr1 pmc1 cnb1
mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca
2+
-pumping ATPases. In the presence of [γ-
32
P]ATP, ECA1p formed a Ca
2+
-dependent [
32
P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca
2+
pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from
Arabidopsis
plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca
2+
pump in
Arabidopsis
. The ability of ECA1p to restore growth of mutant
pmr1
on medium containing Mn
2+
, and the formation of a Mn
2+
-dependent phosphoprotein suggested that ECA1p may also regulate Mn
2+
homeostasis by pumping Mn
2+
into endomembrane compartments of plants.
Cloning and functional characterization of a subunit of the transporter associated with antigen processing
Authors: Li Suling , Sjögren Hans-Olov , Hellman Ulf , Pettersson Ralf F. , Wang Ping
Published: July 2002

The transporter associated with antigen processing (TAP) is essential for the transport of antigenic peptides across the membrane of the endoplasmic reticulum. In addition, TAP interacts with major histocompatibility complex class I heavy chain (HC)/β2-microglobulin (β2-m) dimers. We have cloned a cDNA encoding a TAP1/2-associated protein (TAP-A) corresponding in size and biochemical properties to tapasin, which was recently suggested to be involved in class I–TAP interaction (Sadasivan, B., Lehner, P. J., Ortmann, B., Spies, T. & Cresswell, P. (1996)Immunity5, 103–114). The cDNA encodes a 448-residue-long ORF, including a signal peptide. The protein is predicted to be a type I membrane glycoprotein with a cytoplasmic tail containing a double-lysine motif (-KKKAE-COOH) known to maintain membrane proteins in the endoplasmic reticulum. Immunoprecipitation with anti-TAP1 or anti-TAP-A antisera demonstrated a consistent and stoichiometric association of TAP-A with TAP1/2. Class I HC and β2-m also were coprecipitated with these antisera, indicating the presence of a pentameric complex. In pulse–chase experiments, class I HC/β2-m rapidly dissociated from TAP1/2-TAP-A. We propose that TAP is a trimeric complex consisting of TAP1, TAP2, and TAP-A that interacts transiently with class I HC/β2-m. In peptide-binding assays using cross-linkable peptides and intact microsomes, TAP-A bound peptides only in the presence of ATP whereas binding of peptides to TAP1/2 was ATP-independent. This suggests a direct role of TAP-A in peptide loading onto class I HC/β2-m dimer.
Mutations of
Arabidopsis thaliana
that transform leaves into cotyledons
Authors: Conway Laura J. , Poethig R. Scott
Published: July 2002

We describe mutations of three genes in
Arabidopsis thaliana
—
extra cotyledon1
(
xtc1
),
extra cotyledon2
(
xtc2
), and
altered meristem programming1
(
amp1
)—that transform leaves into cotyledons. In all three of these mutations, this transformation is associated with a change in the timing of events in embryogenesis.
xtc1
and
xtc2
delay the morphogenesis of the embryo proper at the globular-to-heart transition but permit the shoot apex to develop to an unusually advanced stage late in embryogenesis. Both mutations have little or no effect on seed maturation and do not affect the viability of the shoot or the rate of leaf initiation after germination.
amp1
perturbs the pattern of cell division at an early globular stage, dramatically increases the size of the shoot apex and, like
xtc1
and
xtc2
, produces enlarged leaf primordia during seed development. These unusual phenotypes suggest that these genes play important regulatory roles in embryogenesis and demonstrate that the development of the shoot apical meristem and the development of the embryo proper are regulated by independent processes that must be temporally coordinated to ensure normal organ identity.
Analysis of exocytosis mutants indicates close coupling between regulated secretion and transcription activation in
Tetrahymena
Authors: Haddad Alex , Turkewitz Aaron P.
Published: July 2002

Stimulation of regulated secretory cells promotes protein release via the fusion of cytoplasmic storage vesicles with the plasma membrane. In
Tetrahymena thermophila
, brief exposure to secretagogue results in synchronous fusion of the entire set of docked dense-core granules with the plasma membrane. We show that stimulation is followed by rapid new dense-core granule synthesis involving gene induction. Two genes encoding granule matrix proteins,
GRL1
and
GRL4
, are shown to undergo induction following stimulation, resulting in ≈10-fold message accumulation within 1 h. The mechanism of induction involves transcriptional regulation, and the upstream region of
GRL1
functions
in vivo
as an inducible promoter in a heterologous reporter construct using the gene encoding green fluorescent protein. Taking advantage of the characterized exocytosis (exo
−
) mutants available in this system, we asked whether the signals for regranulation were generated directly by the initial stimulation, or whether downstream events were required for transcription activation. Three mutants, with defects at three distinct stages in the regulated secretory pathway, failed to show induction of
GRL1
and
GRL4
after exposure to secretagogue. These results argue that regranulation depends upon signals generated by the final steps in exocytosis.
Protein S is inducible by interleukin 4 in T cells and inhibits lymphoid cell procoagulant activity
Authors: Smiley Stephen T. , Boyer Sarah N. , Heeb Mary J. , Griffin John H. , Grusby Michael J.
Published: July 2002

Extravascular procoagulant activity often accompanies cell-mediated immune responses and systemic administration of pharmacologic anticoagulants prevents cell-mediated delayed-type hypersensitivity reactions. These observations suggest a direct association between coagulation and cell-mediated immunity. The cytokine interleukin (IL)-4 potently suppresses cell-mediated immune responses, but its mechanism of action remains to be determined. Herein we demonstrate that the physiologic anticoagulant protein S is IL-4-inducible in primary T cells. Although protein S was known to inhibit the classic factor Va-dependent prothrombinase assembled by endothelial cells and platelets, we found that protein S also inhibits the factor Va-independent prothrombinase assembled by lymphoid cells. Thus, protein S-mediated down-regulation of lymphoid cell procoagulant activity may be one mechanism by which IL-4 antagonizes cell-mediated immunity.
Killing K Channels with TEA
+
Authors: Khodakhah Kamran , Melishchuk Alexey , Armstrong Clay M.
Published: July 2002

Tetraethylammonium (TEA
+
) is widely used for reversible blockade of K channels in many preparations. We noticed that intracellular perfusion of voltage-clamped squid giant axons with a solution containing K
+
and TEA
+
irreversibly decreased the potassium current when there was no K
+
outside. Five minutes of perfusion with 20 mM TEA
+
, followed by removal of TEA
+
, reduced potassium current to <5% of its initial value. The irreversible disappearance of K channels with TEA
+
could be prevented by addition of ≥ 10 mM K
+
to the extracellular solution. The rate of disappearance of K channels followed first-order kinetics and was slowed by reducing the concentration of TEA
+
. Killing is much less evident when an axon is held at −110 mV to tightly close all of the channels. The longer-chain TEA
+
derivative decyltriethylammonium (C10
+
) had irreversible effects similar to TEA
+
. External K
+
also protected K channels against the irreversible action of C10
+
. It has been reported that removal of all K
+
internally and externally (dekalification) can result in the disappearance of K channels, suggesting that binding of K
+
within the pore is required to maintain function. Our evidence further suggests that the crucial location for K
+
binding is external to the (internal) TEA
+
site and that TEA
+
prevents refilling of this location by intracellular K
+
. Thus in the absence of extracellular K
+
, application of TEA
+
(or C10
+
) has effects resembling dekalification and kills the K channels.
Torsional directed walks, entropic elasticity, and DNA twist stiffness
Authors: Moroz J. David , Nelson Philip
Published: July 2002

DNA and other biopolymers differ from classical polymers because of their torsional stiffness. This property changes the statistical character of their conformations under tension from a classical random walk to a problem we call the “torsional directed walk.” Motivated by a recent experiment on single lambda-DNA molecules [Strick, T. R., Allemand, J.-F., Bensimon, D., Bensimon, A. & Croquette, V. (1996)
Science
271, 1835–1837], we formulate the torsional directed walk problem and solve it analytically in the appropriate force regime. Our technique affords a direct physical determination of the microscopic twist stiffness
C
and twist-stretch coupling
D
relevant for DNA functionality. The theory quantitatively fits existing experimental data for relative extension as a function of overtwist over a wide range of applied force; fitting to the experimental data yields the numerical values
C
= 120 nm and
D
= 50 nm. Future experiments will refine these values. We also predict that the phenomenon of reduction of effective twist stiffness by bend fluctuations should be testable in future single-molecule experiments, and we give its analytic form.
The bovine papillomavirus E6 oncoprotein interacts with paxillin and disrupts the actin cytoskeleton
Authors: Tong Xiao , Howley Peter M.
Published: July 2002

The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) has been shown to transform cells through a p53-independent pathway, but its transforming mechanism is unknown. Here we demonstrate
in vitro
and
in vivo
interactions between BPV-1 E6 and the focal adhesion protein paxillin. The ability of BPV-1 E6 to complex with paxillin correlated with its ability to transform; E6 mutant proteins impaired in their transformation function also were impaired in their abilities to bind paxillin. E6 binding to paxillin also may contribute to the carcinogenic potential of the human papillomavirus (HPV); we were able to show
in vitro
binding of paxillin to the E6 proteins of the cancer-associated type HPV 16 but not of the nononcogenic types 6 and 11. The association of E6 with paxillin was affected by depolymerization of the actin fiber network, and overexpression of BPV-1 E6 led to disruption of actin fiber formation. Disruption of the actin cytoskeleton is a characteristic of many transformed cells, and, in BPV-1 transformed cells, may be mediated by BPV-1 E6 through its interaction with paxillin.
RETRACTED: Mutations in mitochondrial cytochrome
c
oxidase genes segregate with late-onset Alzheimer disease
Authors: Davis Robert E. , Miller Scott , Herrnstadt Corinna , Ghosh Soumitra S. , Fahy Eoin , Shinobu Leslie A. , Galasko Douglas , Thal Leon J. , Beal M. Flint , Howell Neil , Parker W. Davis
Published: July 2002

Mounting evidence suggests that defects in energy metabolism contribute to the pathogenesis of Alzheimer disease (AD). Cytochrome
c
oxidase (CO) is kinetically abnormal, and its activity is decreased in brain and peripheral tissue in late-onset AD. CO is encoded by both the mitochondrial and the nuclear genomes. Its catalytic centers, however, are encoded exclusively by two mitochondrial genes,
CO1
and
CO2
(encoding CO subunits I and II, respectively). We searched these genes, as well as other mitochondrial genes, for mutations that might alter CO activity and cosegregate with AD. In the present study, specific missense mutations in the mitochondrial
CO1
and
CO2
genes but not the
CO3
gene were found to segregate at a higher frequency with AD compared with other neurodegenerative or metabolic diseases. These mutations appear together in the same mitochondrial DNA molecule and define a unique mutant mitochondrial genome. Asymptomatic offspring of AD mothers had higher levels of these mutations than offspring of AD fathers, suggesting that these mutations can be maternally inherited. Cell lines expressing these mutant mitochondrial DNA molecules exhibited a specific decrease in CO activity and increased production of reactive oxygen species. We suggest that specific point mutations in the
CO1
and
CO2
genes cause the CO defect in AD. A CO defect may represent a primary etiologic event, directly participating in a cascade of events that results in AD.
Synergistic activation of p53 by inhibition of
MDM2
expression and DNA damage
Authors: Chen Lihong , Agrawal Sudhir , Zhou Wenqiang , Zhang Ruiwen , Chen Jiandong
Published: July 2002

The
MDM2
oncogene encodes an inhibitor of the p53 tumor suppressor protein that regulates p53 in a negative feedback loop.
MDM2
gene amplification and overexpression occur in several types of tumors and are often associated with poor prognosis. An
MDM2
antisense phosphorothioate oligodeoxynucleotide has been identified that effectively inhibits
MDM2
expression in tumor cells containing
MDM2
gene amplifications. Antisense inhibition of
MDM2
is associated with a decrease in MDM2–p53 complex formation, increase in p53-inducible gene expression, increase in p53 transcriptional activity, and apoptosis. Significantly, inhibition of
MDM2
expression enhances the activation of p53 by a DNA-damaging cancer chemotherapy agent in a synergistic fashion. Therefore, the
MDM2
negative feedback pathway is an important limiting factor in DNA damage-induced p53 activation.
MDM2
antisense oligonucleotides may be useful as antitumor agents alone or as enhancers of other conventional DNA-damaging drugs.
Modulation of an early step in the secretory machinery in hippocampal nerve terminals
Authors: Trudeau Louis-Eric , Fang Yu , Haydon Philip G.
Published: July 2002

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca
2+
responsiveness of the secretory machinery by partially uncoupling Ca
2+
-sensing from fusion
per se
. This is expressed as a shift toward higher Ca
2+
levels of the Ca
2+
to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca
2+
-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca
2+
to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca
2+
detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.
Dopamine-dependent responses to morphine depend on glucocorticoid receptors
Authors: Marinelli Michela , Aouizerate Bruno , Barrot Michel , Le Moal Michel , Piazza Pier Vincenzo
Published: July 2002

Previous work has shown that glucocorticoid hormones facilitate the behavioral and dopaminergic effects of morphine. In this study we examined the possible role in these effects of the two central corticosteroid receptor types: mineralocorticoid receptor (MR), and glucocorticoid receptor (GR). To accomplish this, specific antagonists of these receptors were infused intracerebroventricularly and 2 hr later we measured: (
i
) locomotor activity induced by a systemic injection of morphine (2 mg/kg); (
ii
) locomotor activity induced by an infusion of morphine (1 μg per side) into the ventral tegmental area, which is a dopamine-dependent behavioral response to morphine; (
iii
) morphine-induced dopamine release in the nucleus accumbens, a dopaminergic projection site mediating the locomotor and reinforcing effects of drugs of abuse. Blockade of MRs by spironolactone had no significant effects on locomotion induced by systemic morphine. In contrast, blockade of GRs by either RU38486 or RU39305, which is devoid of antiprogesterone effects, reduced the locomotor response to morphine, and this effect was dose dependent. GR antagonists also reduced the locomotor response to intraventral tegmental area morphine as well as the basal and morphine-induced increase in accumbens dopamine, as measured by microdialysis in freely moving rats. In contrast, spironolactone did not modify dopamine release. In conclusion, glucocorticoids, via GRs, facilitate the dopamine-dependent behavioral effects of morphine, probably by facilitating dopamine release. The possibility of decreasing the behavioral and dopaminergic effects of opioids by an acute administration of GR antagonists may open new therapeutic strategies for treatment of drug addiction.
The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme
Authors: Schwarz Sylvia E. , Matuschewski Kai , Liakopoulos Dimitris , Scheffner Martin , Jentsch Stefan
Published: July 2002

The ubiquitin-like protein SMT3 fromSaccharomyces cerevisiaeand SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugationin vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3/SUMO-1 conjugation pathway analogous to ubiquitin-conjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.
Extension of the thermodynamics of small systems to open metastable states: An example
Authors: Hill Terrell L. , Chamberlin Ralph V.
Published: July 2002

By using a simplified model of small open liquid-like clusters with surface effects, in the gas phase, it is shown how the statistical thermodynamics of small systems can be extended to include metastable supersaturated gaseous states not too far from the gas–liquid equilibrium transition point. To accomplish this, one has to distinguish between mathematical divergence and physical convergence of the open-system partition function.
Cleavage planes in frog eggs are altered by strong magnetic fields
Authors: Denegre James M. , Valles James M. , Lin Kevin , Jordan W. B. , Mowry Kimberly L.
Published: July 2002

Early cleavages of
Xenopus
embryos were oriented in strong, static magnetic fields. Third-cleavage planes, normally horizontal, were seen to orient to a vertical plane parallel with a vertical magnetic field. Second cleavages, normally vertical, could also be oriented by applying a horizontal magnetic field. We argue that these changes in cleavage-furrow geometries result from changes in the orientation of the mitotic apparatus. We hypothesize that the magnetic field acts directly on the microtubules of the mitotic apparatus. Considerations of the length of the astral microtubules, their diamagnetic anisotropy, and flexural rigidity predict the required field strength for an effect that agrees with the data. This observation provides a clear example of a static magnetic-field effect on a fundamental cellular process, cell division.
Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: Potential role for coiled coil interaction
Authors: Stefancsik Raymund , Jha Prakash K. , Sarkar Satyapriya
Published: July 2002

Troponin T (TnT), a thin filament myofibrillar protein, is essential for the Ca
2+
regulation of striated muscle contraction in vertebrates, both
in vivo
and
in vitro
. To understand the role of TnT in this process, its interaction with two other troponin components, troponin I (TnI) and troponin C (TnC) was examined by using the yeast two hybrid system, which is a genetic approach to detect protein-protein interactions. Computer assisted analysis of phylogenetically distant TnT amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential for α-helical coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TnI sequences. These protein motifs appeared to be the regions where TnI-TnT interaction may take place. Deletions and point mutations in TnT, which disrupted its HR motif, severely reduced or abolished TnI binding, but binding to TnC was not affected, indicating that the TnT-TnI and TnT-TnC binary interactions can be uncoupled. Remarkably, the truncated fragments of TnT and TnI in which the HR motifs were retained showed binary interaction in the yeast two hybrid system. It was also observed that the formation of the TnT-TnI heterodimers is favored over the homodimers TnT-TnT and TnI-TnI. These results indicate that the evolutionarily conserved HR motifs may play a role in TnT-TnI dimerization, presumably through the formation of α-helical coiled coils.
The paired Ig-like receptor PIR-B is an inhibitory receptor that recruits the protein-tyrosine phosphatase SHP-1
Authors: Bléry Mathieu , Kubagawa Hiromi , Chen Ching-Cheng , Vély Frédéric , Cooper Max D. , Vivier Eric
Published: July 2002

An emerging family of cell surface inhibitory receptors is characterized by the presence of intracytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIM). These ITIM-bearing inhibitory receptors, which are typically paired with activating isoforms, associate with Src homology domain 2-containing phosphatases following ITIM tyrosine phosphorylation. Two categories of phosphatases are recruited by the ITIM-bearing receptors: the protein-tyrosine phosphatases, SHP-1 and SHP-2, and the polyphosphate inositol 5-phosphatase, SHIP. The dynamic equilibrium of B cell activation is partially controlled by two well known ITIM-bearing receptors, CD22 and FcγRIIB, a low affinity receptor for IgG. We describe here that a murine ITIM-bearing molecule, PIR-B, can also negatively regulate B cell activation. Tyrosine-phosphorylated ITIMs allow PIR-B to associate with SHP-1 but not with SHIP. Engagement of PIR-B thereby initiates a SHP-1-dependent inhibitory pathway that may play an important role in regulating B lymphocyte activation.
RETRACTED: Prevention of renovascular and cardiac pathophysiological changes in hypertension by angiotensin II type 1 receptor antisense gene therapy
Authors: Martens Jeffrey R. , Reaves Phyllis Y. , Lu Di , Katovich Michael J. , Berecek Kathleen H. , Bishop Sanford P. , Raizada Mohan K. , Gelband Craig H.
Published: July 2002

Hypertension produces pathophysiological changes that are often responsible for the mortality associated with the disease. However, it is unclear whether normalizing blood pressure (BP) with conventional therapy is effective in reversing the pathophysiological damage. The duration and initiation of treatment, site of administration, and agent used all appear to influence the reversal of the pathophysiological alterations associated with hypertension. We have previously established that retrovirally mediated delivery of angiotensin II type 1 receptor antisense (AT
1
R-AS) attenuates the development of high BP in the spontaneously hypertensive (SH) rat model of human essential hypertension. Our objective was to determine whether this attenuation of high BP is associated with prevention of other pathophysiological changes induced by the hypertensive state. Intracardiac delivery of AT
1
R-AS in neonates prevented the development of hypertension in SH rats for at least 120 days. Contractile experiments demonstrated an impaired endothelium-dependent vascular relaxation (acetylcholine) and an enhanced contractile response to vasoactive agents (phenylephrine and KCl) in the SH rat renal vasculature. In addition, the voltage-dependent K
+
current density, which is believed to contribute to smooth muscle resting membrane potential and basal tone, was decreased in renal resistance artery cells of the SH rat. AT
1
R-AS treatment prevented each of these renal vascular alterations. Finally, AT
1
R-AS delivery prevented the pathological alterations observed in the SH rat myocardium, including left ventricular hypertrophy, multifocal fibrosis, and perivascular fibrosis. These observations demonstrate that viral-mediated delivery of AT
1
R-AS attenuates the development of hypertension on a long term basis, and this is associated with prevention of pathophysiological changes in SH rats.
T cell positive selection by a high density, low affinity ligand
Authors: Liu Chih-Pin , Crawford Frances , Marrack Philippa , Kappler John
Published: July 2002

Interaction of the αβ T cell receptor (TCR) with major histocompatibility (MHC) molecules occupied with any of a large collection of peptides derived from self proteins is a critical step in driving T cell “positive” selection in the thymus. Interaction with this same pool of self-peptide/MHC ligands deletes T cells with potential self-reactivity. To examine how T cells survive both of these processes to form a self-tolerant mature repertoire, mice were constructed whose entire class II MHC IEkspecific repertoire was positively selected on a single peptide covalently attached to the IEkmolecule. In these mice T cells were identified that could respond to a variant of the positively selecting peptide bound to IEk. The affinities of the TCRs from these T cells for the positively selecting ligand were extremely low and at least 10-fold less than those for the activating ligand. These results support the theory that positive selection is driven by TCR affinities lower than those involved in T cell deletion or activation and that, if present at high concentration, even very low affinity ligands can positively select.
A new pathway for the secretion of virulence factors by bacteria: The flagellar export apparatus functions as a protein-secretion system
Authors: Young Glenn M. , Schmiel Deborah H. , Miller Virginia L.
Published: July 2002

Biogenesis of the flagellum, a motive organelle of many bacterial species, is best understood for members of the
Enterobacteriaceae
. The flagellum is a heterooligomeric structure that protrudes from the surface of the cell. Its assembly initially involves the synthesis of a dedicated protein export apparatus that subsequently transports other flagellar proteins by a type III mechanism from the cytoplasm to the outer surface of the cell, where oligomerization occurs. In this study, the flagellum export apparatus was shown to function also as a secretion system for the transport of several extracellular proteins in the pathogenic bacterium
Yersinia enterocolitica
. One of the proteins exported by the flagellar secretion system was the virulence-associated phospholipase, YplA. These results suggest type III protein secretion by the flagellar system may be a general mechanism for the transport of proteins that influence bacterial–host interactions.
GCN5
-dependent histone H3 acetylation and
RPD3
-dependent histone H4 deacetylation have distinct, opposing effects on
IME2
transcription, during meiosis and during vegetative growth, in budding yeast
Authors: Burgess Sean M. , Ajimura Masahiro , Kleckner Nancy
Published: July 2002

Diploid yeast undergo meiosis under certain conditions of nutrient limitation, which trigger a transcriptional cascade involving two key regulatory genes.
IME1
is a positive activator of
IME2
, which activates downstream genes. We report that Gcn5, a histone H3 acetylase, plays a central role in initiation of meiosis via effects on
IME2
expression. An allele,
gcn5–21
, was isolated as a mutant defective in spore formation.
gcn5–21
fails to carry out meiotic DNA replication, recombination, or meiotic divisions. This mutant also fails to induce
IME2
transcription;
IME1
transcription, however, is essentially normal. Further investigation shows that during wild-type meiosis the
IME2
promoter undergoes an increase in the level of bound acetylated histone H3. This increase is contemporaneous with meiotic induction of
IME2
transcription and is absent in
gcn5–21
. In contrast, the
RPD3
gene, which encodes a histone H4 deacetylase and is known to be required for repression of basal
IME2
transcription in growing yeast cells, is not involved in induction of
IME2
transcription or
IME2
histone acetlyation during meiosis. These and other results suggest that Gcn5 and Rpd3 play distinct roles, modulating transcription initiation in opposite directions under two different cellular conditions. These roles are implemented via opposing effects of the two gene products on acetylation of two different histones. Finally, we find that
gcn5
and
rpd3
single mutants are not defective in meiosis if acetate is absent and respiration is promoted by a metabolically unrelated carbon source. Perhaps intracellular acetate levels regulate meiosis by controlling histone acetylation patterns.
UmuD′
2
C is an error-prone DNA polymerase,
Escherichia coli
pol V
Authors: Tang Mengjia , Shen Xuan , Frank Ekaterina G. , O’Donnell Mike , Woodgate Roger , Goodman Myron F.
Published: July 2002

The damage-inducible UmuD′ and UmuC proteins are required for most SOS mutagenesis in
Escherichia coli
. Our recent assay to reconstitute this process
in vitro
, using a native UmuD′
2
C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD′
2
C.
E. coli
dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD′
2
C, the fifth DNA polymerase discovered in
E. coli
, be designated as
E. coli
pol V. In the presence of RecA, β sliding clamp, γ clamp loading complex, and
E. coli
single-stranded binding protein (SSB), pol V’s polymerase activity is highly “error prone” at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the “A-rule,” dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD′
2
C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3′-OH template-primer ends. We show, however, that despite competition for primer-3′-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.
Binding of elongin A or a von Hippel–Lindau peptide stabilizes the structure of yeast elongin C
Authors: Botuyan Maria Victoria , Koth Christopher M. , Mer Georges , Chakrabartty Avi , Conaway Joan W. , Conaway Ronald C. , Edwards Aled M. , Arrowsmith Cheryl H. , Chazin Walter J.
Published: July 2002

Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel–Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.
Ligand-induced conformational changes observed in single RNA molecules
Authors: Ha Taekjip , Zhuang Xiaowei , Kim Harold D. , Orr Jeffrey W. , Williamson James R. , Chu Steven
Published: July 2002

We present the first demonstration that fluorescence resonance energy transfer can be used to track the motion of a single molecule undergoing conformational changes. As a model system, the conformational changes of
individual
three-helix junction RNA molecules induced by the binding of ribosomal protein S15 or Mg
2+
ions were studied by changes in single-molecule fluorescence. The transition from an open to a folded configuration was monitored by the change of fluorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Averaged behavior of RNA molecules closely resembles that of unlabeled molecules in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein–nucleic acids interactions. Surprisingly, we observed an anomalously broad distribution of RNA conformations at intermediate ion concentrations that may be attributed to foldability differences among RNA molecules. In addition, an experimental scheme was developed where the real-time response of single molecules can be followed under changing environments. As a demonstration, we repeatedly changed Mg
2+
concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instantaneous Mg
2+
concentration with 20-ms time resolution, making it the world’s smallest Mg
2+
meter.
RETRACTED: A new mechanism of neurodegeneration: A proinflammatory cytokine inhibits receptor signaling by a survival peptide
Authors: Venters Homer D. , Tang Qingsong , Liu Qiang , VanHoy Roger W. , Dantzer Robert , Kelley Keith W.
Published: July 2002

Heightened expression of both a proinflammatory cytokine, tumor necrosis factor α (TNF-α), and a survival peptide, insulin-like growth factor I (IGF-I), occurs in diverse diseases of the central nervous system, including Alzheimer’s disease, multiple sclerosis, the AIDS-dementia complex, and cerebral ischemia. Conventional roles for these two proteins are neuroprotection by IGF-I and neurotoxicity by TNF-α. Although the mechanisms of action for IGF-I and TNF-α in the central nervous system originally were established as disparate and unrelated, we hypothesized that the signaling pathways of these two cytokines may interact during neurodegeneration. Here we show that concentrations of TNF-α as low as 10 pg/ml markedly reduce the capacity of IGF-I to promote survival of primary murine cerebellar granule neurons. TNF-α suppresses IGF-I-induced tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2) and inhibits IRS-2-precipitable phosphatidylinositol 3′-kinase activity. These experiments indicate that TNF-α promotes IGF-I receptor resistance in neurons and inhibits the ability of the IGF-I receptor to tyrosine-phosphorylate the IRS-2 docking molecule and to subsequently activate the critical downstream enzyme phosphatidylinositol 3′-kinase. This intracellular crosstalk between discrete cytokine receptors reveals a novel pathway that leads to neuronal degeneration whereby a proinflammatory cytokine inhibits receptor signaling by a survival peptide.
Chromatin remodeling directly activates V(D)J recombination
Authors: Cherry Sara R. , Baltimore David
Published: July 2002

V(D)J recombination substrate choice is regulated to ensure that the appropriate gene segments are rearranged during lymphocyte development. It has been proposed that regulation of substrate usage is determined by changes in accessibility of the DNA targets. We show that Rag-mediated recombination of an episomal substrate in cells is affected by its packaging into chromatin. Chromatinized substrates were inefficiently rearranged, and methylation further reduced recombination. Disruption of nucleosomes by using butyrate on methylated substrates was sufficient to activate recombination, and dexamethasone could activate recombination in the absence of detectable transcription. Therefore, chromatin structure, and its manipulation by altering nucleosome positioning, can directly affect recombination efficiencies.
Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia
Authors: Jiang Xiaoyan , Lopez Angel , Holyoake Tessa , Eaves Allen , Eaves Connie
Published: July 2002

Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph+/BCR–ABL+chronic myeloid leukemia display an abnormal ability to proliferatein vitroin the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34+leukemic cells. When these cells differentiate into CD34−cellsin vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growthin vitrodespite their continued expression of BCR–ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewalin vitroand slow accumulationin vivo, in spite of an increased cycling activity and selective expansion of later compartments.
RETRACTED: Monarch butterflies (
Danaus plexippus
L.) use a magnetic compass for navigation
Authors: Etheredge Jason A. , Perez Sandra M. , Taylor Orley R. , Jander Rudolf
Published: July 2002

Fall migratory monarch butterflies, tested for their directional responses to magnetic cues under three conditions, amagnetic, normal, and reversed magnetic fields, showed three distinct patterns. In the absence of a magnetic field, monarchs lacked directionality as a group. In the normal magnetic field, monarchs oriented to the southwest with a group pattern typical for migrants. When the horizontal component of the magnetic field was reversed, the butterflies oriented to the northeast. In contrast, nonmigratory monarchs lacked directionality in the normal magnetic field. The results are a direct demonstration of magnetic compass orientation in migratory insects.