bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.
Authors: Holländer G A , Castigli E , Kulbacki R , Su M , Burakoff S J , Gutierrez-Ramos J C , Geha R S
Published: July 2002

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.
Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia.
Authors: Müllbacher A , Ebnet K , Blanden R V , Hla R T , Stehle T , Museteanu C , Simon M M
Published: July 2002

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.
Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA.
Authors: MA D , Olave I , Merino A , Reinberg D
Published: July 2002

Human transcription factor IIA (TFIIA) is composed of three subunits (alpha, beta, and gamma). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the alpha subunit was isolated from HeLa cells. This "mini-TFIIA" interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function.
In vivo tissue distribution of CD4 lymphocytes in mice determined by radioimmunoscintigraphy with an 111In-labeled anti-CD4 monoclonal antibody.
Authors: Rubin R H , Baltimore D , Chen B K , Wilkinson R A , Fischman A J
Published: July 2002

The tissue distribution of CD4 lymphocytes in normal C57/BL mice and CD4 knockout mice was determined by biodistribution measurements and gamma camera imaging with an 111In-labeled rat IgG2b monoclonal antibody directed against the murine CD-4 antigen. In normal mice high concentrations of antibody accumulated in the spleen and lymph nodes. At 45 hr after injection, the concentration of radiolabel in the spleen and lymph nodes of normal mice were 10- to 20-fold greater than in the corresponding tissue of the CD4 knockout mice and nonlymphoid tissues of both types of mice. At 24 and 45 hr, gamma camera images showed high concentrations of radiolabeled antibody in lymph node and spleen of normal but not knockout mice. These results indicate that radioimmunoscintigraphy with 111In-anti-CD4 is an excellent method for studying tissue distribution of CD lymphocytes in mice. Using an equivalent anti-human CD antibody, this method might be useful for studying the pathophysiology of conditions in which these cells play a critical role and for monitoring therapies for these disorders.
Duplication and functional divergence in the chalcone synthase gene family of Asteraceae: evolution with substrate change and catalytic simplification.
Authors: Helariutta Y , Kotilainen M , Elomaa P , Kalkkinen N , Bremer K , Teeri T H , Albert V A
Published: July 2002

Plant-specific polyketide synthase genes constitute a gene superfamily, including universal chalcone synthase [CHS; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing) (EC 2.3.1.74)] genes, sporadically distributed stilbene synthase (SS) genes, and atypical, as-yet-uncharacterized CHS-like genes. We have recently isolated from Gerbera hybrida (Asteraceae) an unusual CHS-like gene, GCHS2, which codes for an enzyme with structural and enzymatic properties as well as ontogenetic distribution distinct from both CHS and SS. Here, we show that the GCHS2-like function is encoded in the Gerbera genome by a family of at least three transcriptionally active genes. Conservation within the GCHS2 family was exploited with selective PCR to study the occurrence of GCHS2-like genes in other Asteraceae. Parsimony analysis of the amplified sequences together with CHS-like genes isolated from other taxa of angiosperm subclass Asteridae suggests that GCHS2 has evolved from CHS via a gene duplication event that occurred before the diversification of the Asteraceae. Enzyme activity analysis of proteins produced in vitro indicates that the GCHS2 reaction is a non-SS variant of the CHS reaction, with both different substrate specificity (to benzoyl-CoA) and a truncated catalytic profile. Together with the recent results of Durbin et al. [Durbin, M. L., Learn, G. H., Jr., Huttley, G. A. & Clegg, M. T. (1995) Proc. Natl. Acad. Sci. USA 92, 3338-3342], our study confirms a gene duplication-based model that explains how various related functions have arisen from CHS during plant evolution.
Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.
Authors: Di Marco A , Gloaguen I , Graziani R , Paonessa G , Saggio I , Hudson K R , Laufer R
Published: July 2002

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.
Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement.
Authors: Ding X , Shintaku M H , Carter S A , Nelson R S
Published: July 2002

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.
Two eukaryote-specific regions of Hsp82 are dispensable for its viability and signal transduction functions in yeast
Authors: Louvion Jean-François , Warth Rainer , Picard Didier
Published: July 2002

The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that
is very abundant even at normal temperature. It is highly conserved and
essential for viability in yeast. To delineate functional domains of
Hsp90, we have performed a deletion analysis of one of the two Hsp90
isoforms from budding yeast, Hsp82. The Hsp82 derivatives were tested
for complementation of a Hsp90-deficient yeast strain and for their
ability to function in two signal transduction pathways that depend on
Hsp90. Surprisingly, we found that two salient features of Hsp90
sequences from eukaryotes, the N-terminal charged domain and the
extremely conserved C-terminal pentapeptide MEEVD, are dispensable for
viability as well as for proper regulation of vertebrate steroid
receptors and for pheromone signaling. Moreover, we describe, to our
knowledge, the first dominant negative mutant of Hsp90: A Hsp82
derivative that lacks amino acids 538–552 fails to complement but has
a dominant negative effect on viability of wild-type strains at
moderately elevated temperatures. This mutant may become a valuable
tool to study Hsp90 functions both in yeast and in mammalian cells.
Melanoma angiogenesis and metastasis modulated by ribozyme targeting of the secreted growth factor pleiotrophin
Authors: Czubayko Frank , Schulte Anke M. , Berchem Guy J. , Wellstein Anton
Published: July 2002

Clinical and experimental evidence suggests that spreading of
malignant cells from a localized tumor (metastasis) is directly related
to the number of microvessels in the primary tumor. This tumor
angiogenesis is thought to be mediated by tumor-cell-derived growth
factors. However, most tumor cells express a multitude of candidate
angiogenesis factors and it is difficult to decipher which of these are
rate-limiting factors
in vivo
. Herein we use ribozyme
targeting of pleiotrophin (PTN) in metastatic human melanoma cells to
assess the significance of this secreted growth factor for angiogenesis
and metastasis. As a model we used human melanoma cells (1205LU) that
express high levels of PTN and metastasize from subcutaneous tumors to
the lungs of experimental animals. In these melanoma cells, we reduced
PTN mRNA and growth factor activity by transfection with PTN-targeted
ribozymes and generated cell lines expressing different levels of PTN.
We found that the reduction of PTN does not affect growth of the
melanoma cells
in vitro
. In nude mice, however, tumor
growth and angiogenesis were decreased in parallel with the reduced PTN
levels and apoptosis in the tumors was increased.
Concomitantly, the metastatic spread of the tumors from the
subcutaneous site to the lungs was prevented. These studies support a
direct link between tumor angiogenesis and metastasis through a
secreted growth factor and identify PTN as a candidate factor that may
be rate-limiting for human melanoma metastasis.
A novel modifier gene for plasma von Willebrand factor level maps to distal mouse chromosome 11
Authors: Mohlke Karen L. , Nichols William C. , Westrick Randal J. , Novak Edward K. , Cooney Kathleen A. , Swank Richard T. , Ginsburg David
Published: July 2002

Type 1 von Willebrand disease (VWD), characterized by reduced
levels of plasma von Willebrand factor (VWF), is the most common
inherited bleeding disorder in humans. Penetrance of VWD is incomplete,
and expression of the bleeding phenotype is highly variable. In
addition, plasma VWF levels vary widely among normal individuals. To
identify genes that influence VWF level, we analyzed a genetic cross
between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold
difference in plasma VWF level. DNA samples from F
2
progeny
demonstrating either extremely high or extremely low plasma VWF levels
were pooled and genotyped for 41 markers spanning the autosomal genome.
A novel locus accounting for 63% of the total variance in VWF level
was mapped to distal mouse chromosome 11, which is distinct from the
murine
Vwf
locus on chromosome 6. We designated this
locus
Mvwf
for “modifier of VWF.” Additional
genotyping of as many as 2407 meioses established a high resolution
genetic map with gene order
Cola1
-
Itg3a
-
Ngfr
-
Mvwf
/
Gip
-
Hoxb9
-
Hoxb1
-
Cbx
·
rs2
-
Cox5a
-
Gfap
.
The
Mvwf
candidate interval between
Ngfr
and
Hoxb9
is ≈0.5 centimorgan (cM). These results
demonstrate that a single dominant gene accounts for the low VWF
phenotype of RIIIS/J mice in crosses with several other strains. The
pattern of inheritance suggests a gain-of-function mutation in a unique
component of VWF biosynthesis or processing. Characterization of the
human homologue for
Mvwf
may have relevance for a subset
of type 1 VWD cases and may define an important genetic factor
modifying penetrance and expression of mutations at the
VWF
locus.
Protein synthesis editing by a DNA aptamer.
Authors: Hale S P , Schimmel P
Published: July 2002

Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response.
Bidirectional uncoating of the genomic RNA of a helical virus.
Authors: Wu X , Shaw J
Published: July 2002

An essential step in the initiation of a virus infection is the release of the viral genome from the other constituents of the virus particle, a process referred to as uncoating. We have used reverse transcription and polymerase chain reaction amplification procedures to determine the rate and direction of in vivo uncoating of the rod-shaped tobacco mosaic virus. The virus particles contain a single 6.4-kb RNA molecule that lies between successive turns of a helical arrangement of coat protein subunits. When the particles are introduced into plant cells, the subunits are removed via a bidirectional uncoating mechanism. Within 2-3 min, the part of the viral RNA from the 5' end to a position >70% toward the 3' end has been freed of coat protein subunits. This is followed by removal of subunits from the 3' end of the RNA and sequential uncoating of the RNA in a 3'-to-5' direction. An internal region of the viral RNA is the final part to be uncoated. Progeny virus particles are detected in the cells 35-40 min after inoculation.
Earthquake prediction: the interaction of public policy and science.
Authors: Jones L M
Published: July 2002

Earthquake prediction research has searched for both informational phenomena, those that provide information about earthquake hazards useful to the public, and causal phenomena, causally related to the physical processes governing failure on a fault, to improve our understanding of those processes. Neither informational nor causal phenomena are a subset of the other. I propose a classification of potential earthquake predictors of informational, causal, and predictive phenomena, where predictors are causal phenomena that provide more accurate assessments of the earthquake hazard than can be gotten from assuming a random distribution. Achieving higher, more accurate probabilities than a random distribution requires much more information about the precursor than just that it is causally related to the earthquake.
Metabolism of Alzheimer beta-amyloid precursor protein: regulation by protein kinase A in intact cells and in a cell-free system.
Authors: Xu H , Sweeney D , Greengard P , Gandy S
Published: July 2002

Various compounds that affect signal transduction regulate the relative utilization of alternative processing pathways for the beta-amyloid precursor protein (beta APP) in intact cells, increasing the production of nonamyloidogenic soluble beta APP (s beta APP) and decreasing that of amyloidogenic beta-amyloid peptide. In a recent study directed toward elucidating the mechanisms underlying phorbol ester-stimulated s beta APP secretion from cells, it was demonstrated that protein kinase C increases the formation from the trans-Golgi network (TGN) of beta APP-containing secretory vesicles. Here we present evidence that forskolin increases s beta APP production from intact PC12 cells, and protein kinase A stimulates formation from the TGN of beta APP-containing vesicles. Although protein kinase A and protein kinase C converge at the level of formation from the TGN of beta APP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct.
Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of
Schizosaccharomyces pombe
Authors: Lee Joon-Kyu , Huberman Joel A. , Hurwitz Jerard
Published: July 2002

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from
Schizosaccharomyces pombe
. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and
S. pombe
Abp1, and this gene was designated as
cbh
+
(
C
ENP-
B
h
omologue). Cbh protein specifically interacts
in vitro
with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in
S. pombe
.
Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of developmental arrest
Authors: Melamed Doron , Nemazee David
Published: July 2002

In pre-B lymphocytes, productive rearrangement of Ig light chain genes allows assembly of the B cell receptor (BCR), which selectively promotes further developmental maturation through poorly defined transmembrane signaling events. Using a novelin vitrosystem to study immune tolerance during development, we find that BCR reactivity to auto-antigen blocks this positive selection, preventing down-regulation of light chain gene recombination and promoting secondary light chain gene rearrangements that often alter BCR specificity, a process called receptor editing. Under these experimental conditions, self-antigen induces secondary light chain gene rearrangements in at least two-thirds of autoreactive immature B cells, but fails to accelerate cell death at this stage. These data suggest that in these cells the mechanism of immune tolerance is receptor selection rather than clonal selection.
Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from
Rhodobacter sphaeroides
Authors: Artz K. , Williams J. C. , Allen J. P. , Lendzian F. , Rautter J. , Lubitz W.
Published: July 2002

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll
a
molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll–protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to ≈60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron–nuclear–nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from ≈2:1 to ≈5:1 in the mutants compared with ≈2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation–reduction midpoint potential.
Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase
Authors: Kash Shera F. , Johnson Randall S. , Tecott Laurence H. , Noebels Jeffrey L. , Mayfield R. Dayne , Hanahan Douglas , Baekkeskov Steinunn
Published: July 2002

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms,
GAD65
and
GAD67
. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that
GAD65
may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding
GAD65
and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to
GAD67−/−
animals, which are born with developmental abnormalities and die shortly after birth,
GAD65−/−
mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5′ phosphate-inducible apo-enzyme reservoir is significantly decreased.
GAD65−/−
mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in
GAD65−/−
mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the
GAD65−/−
mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by
GAD65
is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present
GAD65−/−
animals as a model of epilepsy involving GABA-ergic pathways.
Cloning of murine RNA polymerase I-specific TAF factors: Conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1
Authors: Heix Jutta , Zomerdijk Joost C. B. M. , Ravanpay Ali , Tjian Robert , Grummt Ingrid
Published: July 2002

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP–TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAF
I
s) and compared the amino acid sequences of the murine TAF
I
s with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAF
I
s, demonstrating that differences in the primary structure of human and mouse TAF
I
s do not dramatically alter the network of protein–protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP–TAF
I
complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.
The quinone reductase gene: A unique estrogen receptor-regulated gene that is activated by antiestrogens
Authors: Montano Monica M. , Katzenellenbogen Benita S.
Published: July 2002

Antiestrogens are thought to exert most of their beneficial effects in breast cancer by antagonizing the actions of estrogen. We report here that antiestrogens also stimulate the expression of quinone reductase (QR) [NAD(P)H:quinone oxidoreductase, EC
1.6.99.2
], which may provide protective effects against the toxicity and mutagenicity caused by quinones. QR is up-regulated by low concentrations of antiestrogens (trans-hydroxytamoxifen, tamoxifen, and ICI182,780) in estrogen receptor (ER)-containing breast cancer cells, and this increase is suppressed by estrogen via an ER-dependent mechanism. Since regulation of the QR gene, as well as other genes involved in detoxification such as the glutathione
S
-transferase Ya subunit (GST Ya) gene, is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE), we examined the effects of antiestrogens on a 41-bp electrophile responsive region derived from the GST Ya gene. Transfection of this EpRE-containing region into ER-negative breast cancer cells in the presence or absence of an expression vector for the human ER, as well as mutagenesis studies, revealed that the EpRE-containing construct was activated by antiestrogen to the same extent as by
tert
-butylhydroquinone (TBHQ), a known activator of EpREs; however, only the stimulation by antiestrogen, and not TBHQ, required ER and was repressed by estradiol, although activation by both inducers mapped to the same 10-bp EpRE consensus sequence. Thus, there appear to be two pathways for QR induction, one that is activated by electrophile inducers such as TBHQ and is ER independent, and a second that is antiestrogen regulated and ER dependent; both pathways act through the EpRE. The anticancer action of antiestrogens may thus derive not only from the already well-known repression of estrogen-stimulated activities but also from the activation of detoxifying enzymes, such as QR, that may contribute to the beneficial antioxidant activity of antiestrogens.
Membrane-promoted unfolding of acetylcholinesterase: A possible mechanism for insertion into the lipid bilayer
Authors: Shin Irina , Kreimer David , Silman Israel , Weiner Lev
Published: July 2002

Acetylcholinesterase fromTorpedo californicapartially unfolds to a state with the physicochemical characteristics of a “molten globule” upon mild thermal denaturation or upon chemical modification of a single nonconserved buried cysteine residue, Cys231. The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys231by mercuric chloride produces initially a quasi-native state ofTorpedoacetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1–2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteinsin situ.
Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme
Authors: Shanklin John , Achim Catalina , Schmidt Hermann , Fox Brian G. , Münck Eckard
Published: July 2002

The gene encoding the alkane ω-hydroxylase (AlkB; EC
1.14.15.3
) from
Pseudomonas oleovorans
was expressed in
Escherichia coli
. The integral-membrane protein was purified as nearly homogeneous protein vesicles by differential ultracentrifugation and HPLC cation exchange chromatography without the detergent solubilization normally required for membrane proteins. Purified AlkB had specific activity of up to 5 units/mg for octane-dependent NADPH consumption. Mössbauer studies of AlkB showed that it contains an exchange-coupled dinuclear iron cluster of the type found in soluble diiron proteins such as hemerythrin, ribonucleotide reductase, methane monooxygenase, stearoyl-acyl carrier protein (ACP) Δ
9
desaturase, rubrerythrin, and purple acid phosphatase. In the as-isolated enzyme, the cluster contains an antiferromagnetically coupled pair of high-spin Fe(III) sites, with an occupancy of up to 0.9 cluster per AlkB. The diferric cluster could be reduced by sodium dithionite, and the diferrous state was found to be stable in air. When both O
2
and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluster occupies the active site. Mössbauer data on reduced AlkB are consistent with a cluster coordination rich in nitrogen-containing ligands. New sequence analyses indicate that at least 11 nonheme integral-membrane enzymes, including AlkB, contain the 8-histidine motif required for catalytic activity in stearoyl-CoA desaturase. Based on our Mössbauer studies of AlkB, we propose that the integral-membrane enzymes in this family contain diiron clusters. Because these enzymes catalyze a diverse range of oxygenation reactions, this proposal suggests a greatly expanded role for diiron clusters in O
2
-activation biochemistry.
In differentiated CD4+T Cells, interleukin 4 production is cytokine-autonomous, whereas interferon γ production is cytokine-dependent
Authors: Hu-Li Jane , Huang Hua , Ryan John , Paul William E.
Published: July 2002

CD4+T cells from T cell receptor transgenic mice that have been vigorously primed to be interleukin (IL)-4 producers (TH2cells) are capable of producing IL-4 even if restimulated in the absence of IL-4 and in the presence of IL-12. T cells vigorously primed in the absence of IL-4 (TH1cells) fail to produce IL-4 even if restimulated under conditions that would cause a naive T cell to produce IL-4. In contrast, interferon γ (IFN-γ) production is highly cytokine-regulated. T cells primed in the presence of IL-4 develop into IFN-γ producers if IFN-γ is included in the priming culture and if the cells are challenged in the presence of IL-12, presumably reflecting the role of IFN-γ in inducing responsiveness to IL-12. Cells primed in the absence of IL-4 become highly responsive to IL-12 if IFN-γ is included in the priming culture, and these cells are excellent IFN-γ producers upon challenge; IL-12 considerably enhances their production of IFN-γ. If cells are primed in the absence of IL-4 and IFN-γ, they show very weak responsiveness to IL-12 as determined by STAT-4 activation. However, these cells acquire IL-12 responsiveness if cultured with IFN-γ for a period as short as 4 hr. Thereafter, they produce large amounts of IFN-γ upon challenge with antigen in the presence of IL-12. These results indicate that in primed CD4+T cells, IL-4 production is largely cytokine-autonomous, whereas IFN-γ production is highly cytokine-regulated.
Measuring the topology of the universe
Authors: Cornish Neil J. , Spergel David N. , Starkman Glenn D.
Published: July 2002

Observations of microwave background fluctuations can yield
information not only about the geometry of the universe but potentially
about the topology of the universe. If the universe is negatively
curved, then the characteristic scale for the topology of the universe
is the curvature radius. Thus, if we are seeing the effects of the
geometry of the universe, we can hope to soon see signatures of the
topology of the universe. The cleanest signature of the topology of the
universe is written on the microwave sky: There should be thousands of
pairs of matched circles. These circles can be used to determine the
precise topology and volume of the universe. Because we see hundreds of
slices through the fundamental domain of the universe, we can use the
microwave observations to reconstruct the initial conditions of the
entire universe on the scale of a few megaparsecs.
New methods of structure refinement for macromolecular structure determination by NMR
Authors: Clore G. Marius , Gronenborn Angela M.
Published: July 2002

Recent advances in multidimensional NMR methodology have permitted
solution structures of proteins in excess of 250 residues to be solved.
In this paper, we discuss several methods of structure refinement that
promise to increase the accuracy of macromolecular structures
determined by NMR. These methods include the use of a conformational
database potential and direct refinement against three-bond coupling
constants, secondary
13
C shifts,
1
H shifts,
T
1
/T
2
ratios, and residual dipolar couplings.
The latter two measurements provide long range restraints that are not
accessible by other solution NMR parameters.
A strategy for organ allografts without using immunosuppressants or irradiation
Authors: Morita Haruo , Sugiura Kikuya , Inaba Muneo , Jin Tienan , Ishikawa Junji , Lian Zhexiong , Adachi Yasushi , Sogo Shinji , Yamanishi Kazuya , Taki Hideo , Adachi Masakazu , Noumi Takato , Kamiyama Yasuo , Good Robert A. , Ikehara Susumu
Published: July 2002

A strategy to achieve regular and long lasting organ and tissue allografts without using immunosuppressants and/or irradiation has been established for mice. One hundred percent of skin allografts can be induced to survive >350 days after transplantation if spleen cells from the same donors are first injected into the portal vein of the recipients. The mechanisms underlying this long-term tolerance induction can be described as follows: (i) donor T cells from the spleen of the donor facilitate the acceptance of the allogeneic engraftment, (ii) donor-specific anergy is induced in the cytotoxic T-lymphocytes of the recipients, (iii) T helper type 2 cells become the dominant T cells in the recipients that are accepting the skin transplants, and (iv) a lasting chimerism (microchimerism) is established in these recipients. This strategy, perhaps with minor modifications, might permit one also to overcome major barriers to organ allografting in humans. If this were the case, it could represent production of long lasting immunologic tolerance without need for irradiation or cytotoxic chemo-preparative regimen and as such could greatly facilitate allotransplantation free of episodes of chronic or acute rejection or toxic and damaging preparatory regimens.
Structural basis of an embryonically lethal single Ala → Thr mutation in the vnd/NK-2 homeodomain
Authors: Xiang Bosong , Weiler Solly , Nirenberg Marshall , Ferretti James A.
Published: July 2002

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to −5°C in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25°C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix–turn–helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.
Mesolimbic dopaminergic decline after cannabinoid withdrawal
Authors: Diana Marco , Melis Miriam , Muntoni Anna Lisa , Gessa Gian Luigi
Published: July 2002

The mesolimbic dopamine system has recently been implicated in the long-term aversive consequences of withdrawal from major drugs of abuse. In the present study we sought to determine whether mesolimbic dopamine neurons are involved in the neurobiologic mechanisms underlying withdrawal from chronic cannabinoid exposure. Rats were treated chronically with the major psychoactive ingredient of hashish and marijuana, Δ
9
-tetrahydrocannabinol (Δ
9
-THC). Administration of the cannabinoid antagonist SR 141716A precipitated an intense behavioral withdrawal syndrome, whereas abrupt Δ
9
-THC suspension failed to produce overt signs of abstinence. In contrast, both groups showed a reduction in dopamine cells activity as indicated by extracellular single unit recordings from antidromically identified meso-accumbens dopamine neurons. The administration of Δ
9
-THC to spontaneously withdrawn rats restored neuronal activity. Conversely, SR 141716A produced a further decrease of spontaneous activity in cannabinoid-treated although it was ineffective in control rats. These data indicate that withdrawal from chronic cannabinoid administration is associated with reduced dopaminergic transmission in the limbic system, similar to that observed with other addictive drugs; these changes in neuronal plasticity may play a role in drug craving and relapse into drug addiction.
Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses
Authors: Masuta Chikara , Ueda Shigenori , Suzuki Masashi , Uyeda Ichiro
Published: July 2002

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the
Cucumovirus
genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created
in vitro
by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto
Nicotiana benthamiana
, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3′-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.
Transient expression and transport of brain-derived neurotrophic factor in the male zebra finch’s song system during vocal development
Authors: Akutagawa Eugene , Konishi Masakazu
Published: July 2002

The distribution of brain-derived neurotrophic factor (BDNF) in the song system of male zebra finches changes with posthatching age. At day 20, the hyperstriatum ventrale, pars caudale is the only song nucleus in which neurons showed BDNF immunoreactivity. At day 45, the staining in hyperstriatum ventrale, pars caudale was denser than at day 20 and the robust nucleus of the archistriatum, another song nucleus, showed BDNF labeling. By day 65, two additional song nuclei, area X and the lateral magnocellular nucleus of the anterior neostriatum, have become immunoreactive. In the adult, however, the amount of BDNF labeling in all of these brain nuclei is sharply reduced. These sequential events, the anatomical connections between these song nuclei, and the labeling of relevant axons and terminals suggest anterograde transport of BDNF. Furthermore, the timing of BDNF expression coincident with the development of singing behavior suggests that this neurotrophin may be directly involved with the differentiation of the song system.
An ion channel of the degenerin/epithelial sodium channel superfamily controls the defecation rhythm in
Caenorhabditis elegans
Authors: Take-uchi Masaya , Kawakami Minoru , Ishihara Takeshi , Amano Tosikazu , Kondo Kazunori , Katsura Isao
Published: July 2002

Ultradian rhythms are widespread phenomena found in various biological organisms. A typical example is the defecation behavior of the nematode
Caenorhabditis elegans,
which repeats at about 45-sec intervals. To elucidate the mechanism, we studied
flr-1
mutants, which show very short defecation cycle periods. The mutations also affect some food-related functions, including growth rate, the expulsion step of defecation behavior, and the regulation of the dauer larva (a nonfeeding, special third-stage larva) formation in the
unc-3
(Olf-1/EBF homolog) background. The
flr-1
gene encodes a novel ion channel belonging to the DEG/ENaC (
C. elegans
degenerin and mammalian epithelial sodium channel) superfamily. A
flr-1
∷GFP (green fluorescent protein) fusion gene that can rescue the
flr-1
mutant phenotypes is expressed only in the intestine from embryos to adults. These results suggest that FLR-1 may be a component of an intestinal regulatory system that controls the defecation rhythm as well as other functions.
13
C/
31
P NMR studies of glucose transport in human skeletal muscle
Authors: Roussel Ronan , Carlier Pierre G. , Robert Jean-Jacques , Velho Gilberto , Bloch Gilles
Published: July 2002

The muscle intracellular (IC) free glucose concentration and the rate of muscle glycogen synthesis were measured by using
in vivo
13
C and
31
P NMR spectroscopy in normal volunteers under hyperinsulinemic (≈300 pM) clamp conditions at the following three plasma glucose levels: euglycemia (≈6 mM), mild (≈10 mM), and high (≈16 mM) hyperglycemia. In keeping with biopsy studies, muscle IC free glucose concentration at euglycemia (−0.03 ± 0.03 mmol/kg of muscle, mean ± SEM,
n
= 10) was not statistically different from zero. A small but statistically significant amount of IC free glucose was observed during mild and high hyperglycemia: 0.15 ± 0.08 (
n
= 5) and 0.43 ± 0.20 mmol/kg of muscle (
n
= 5), respectively. Muscle glycogen synthesis rate, in mmol per kg of muscle per min, was 111 ± 11 at euglycemia (
n
= 10), 263 ± 29 during mild hyperglycemia (
n
= 5), and 338 ± 42 during high hyperglycemia (
n
= 5), these three rates being significantly different from each other. As previous
in vitro
and
in vivo
studies, these rates suggest a
K
m
(concentration at which unidirectional glucose transport reaches half-maximal rate) of the muscle glucose transport system in the 15–25 mM range under hyperinsulinemic conditions. The low concentrations of muscle IC free glucose observed under hyperinsulinemic conditions were interpreted, with this estimate and in the framework of metabolic control theory, as glucose transport being the predominant step controlling muscle glucose flux not only at euglycemia but also during hyperglycemia.
Event-related functional MRI: Past, present, and future
Authors: Rosen Bruce R. , Buckner Randy L. , Dale Anders M.
Published: July 2002

The past two decades have seen an enormous growth in the field of
human brain mapping. Investigators have extensively exploited
techniques such as positron emission tomography and MRI to map patterns
of brain activity based on changes in cerebral hemodynamics. However,
until recently, most studies have investigated equilibrium changes in
blood flow measured over time periods upward of 1 min. The advent of
high-speed MRI methods, capable of imaging the entire brain with a
temporal resolution of a few seconds, allows for brain mapping based on
more transient aspects of the hemodynamic response. Today it is now
possible to map changes in cerebrovascular parameters essentially in
real time, conferring the ability to observe changes in brain state
that occur over time periods of seconds. Furthermore, because robust
hemodynamic alterations are detectable after neuronal stimuli lasting
only a few tens of milliseconds, a new class of task paradigms designed
to measure regional responses to single sensory or cognitive events can
now be studied. Such “event related” functional MRI should
provide for fundamentally new ways to interrogate brain function, and
allow for the direct comparison and ultimately integration of data
acquired by using more traditional behavioral and
electrophysiological methods.