bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamical hierarchy in transition states: Why and how does a system climb over the mountain?
Authors: Komatsuzaki Tamiki , Berry R. Stephen
Published: July 2002

How a reacting system climbs through a transition state during the course of a reaction has been an intriguing subject for decades. Here we present and quantify a technique to identify and characterize local invariances about the transition state of anN-particle Hamiltonian system, using Lie canonical perturbation theory combined with microcanonical molecular dynamics simulation. We show that at least three distinct energy regimes of dynamical behavior occur in the region of the transition state, distinguished by the extent of their local dynamical invariance and regularity. Isomerization of a six-atom Lennard–Jones cluster illustrates this: up to energies high enough to make the system manifestly chaotic, approximate invariants of motion associated with a reaction coordinate in phase space imply a many-body dividing hypersurface in phase space that is free of recrossings even in a sea of chaos. The method makes it possible to visualize the stable and unstable invariant manifolds leading to and from the transition state, i.e., the reaction path in phase space, and how this regularity turns to chaos with increasing total energy of the system. This, in turn, illuminates a new type of phase space bottleneck in the region of a transition state that emerges as the total energy and mode coupling increase, which keeps a reacting system increasingly trapped in that region.
A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme
Authors: Kuchin Sergei , Treich Isabelle , Carlson Marian
Published: July 2002

RNA polymerase II holoenzymes respond to activators and repressors that are regulated by signaling pathways. Here we present evidence for a “shortcut” mechanism in which the Snf1 protein kinase of the glucose signaling pathway directly regulates transcription by the yeast holoenzyme. In response to glucose limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been artificially recruited to a reporter by a LexA fusion to a holoenzyme component. We show that Snf1 interacts physically with the Srb/mediator proteins of the holoenzyme in both two-hybrid and coimmunoprecipitation assays. We also show that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion protein, activates transcription in a glucose-regulated manner; moreover, this activation depends on the integrity of the Srb/mediator complex. These results suggest that direct regulatory interactions between signal transduction pathways and RNA polymerase II holoenzyme provide a mechanism for transcriptional control in response to important signals.
Break-induced replication: A review and an example in budding yeast
Authors: Kraus Eliyahu , Leung Wai-Ying , Haber James E.
Published: July 2002

Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in
Saccharomyces cerevisiae
where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5′ end of the
LEU2
gene at one end was transformed into a strain in which the 5′ end of
LEU2
was replaced by
ADE1
, preventing simple homologous gene replacement to become Leu2
+
. Ade1
+
Leu2
+
transformants were recovered in which the entire
LEU2
gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an
ARS
and homologous to only 50 bp of
ADE2
added to the ends of a
URA3
gene. Autonomously replicating circles were recovered, containing
URA3
and as much as 8 kb of
ADE2
-adjacent sequences, including a nearby
ARS
, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.
Evolutionary self-organization of cell-free genetic coding
Authors: Füchslin Rudolf M. , McCaskill John S.
Published: July 2002

Genetic encoding provides a generic construction scheme for biomolecular functions. This paper addresses the key problem of coevolution and exploitation of the multiple components necessary to implement a replicable genetic encoding scheme. Extending earlier results on multicomponent replication, the necessity of spatial structure for the evolutionary stabilization of the genetic coding system is established. An individual-based stochastic model of interacting molecules in three-dimensional space is presented that allows the evolution of genetic coding to be analyzed explicitly. A massively parallel configurable computer (NGEN) is used to implement the model, on the time scale of millions of generations, directly in electronic hardware. The spatial correlations between components of the genetic coding system are analyzed and found to be essential for evolutionary stability.
Transgene analysis proves mRNA trans-splicing at the complex
mod(mdg4)
locus in
Drosophila
Authors: Dorn Rainer , Reuter Gunter , Loewendorf Andrea
Published: July 2002

The
Drosophila
BTB domain containing gene
mod(mdg4)
produces a large number of protein isoforms
combining a common N-terminal region of 402 aa with different C
termini. We have deduced the genomic structure of this complex locus
and found that at least seven of the
mod(mdg4)
isoforms
are encoded on both of its antiparallel DNA strands, suggesting the
generation of mature mRNAs by trans-splicing. In transgenic assays, we
demonstrate the ability of
Drosophila
to produce
mod(mdg4)
mRNAs by trans-splicing of pre-mRNAs generated
from transgenes inserted at distant chromosomal positions. Furthermore,
evidence is presented for occurring of trans-splicing of
mod(mdg4)
-specific exons encoded by the parallel DNA
strand. The
mod(mdg4)
locus represents a new type of
comlpex gene structure in which genetic complexity is resolved by
extensive trans-splicing, giving important implications for genome
sequencing projects. Demonstration of naturally occurring
trans-splicing in the model organism
Drosophila
opens
new experimental approaches toward an analysis of the underlying
mechanisms.
Polymorphism in glutathione
S
-transferase P1 is associated with susceptibility to chemotherapy-induced leukemia
Authors: Allan James M. , Wild Christopher P. , Rollinson Sara , Willett Eleanor V. , Moorman Anthony V. , Dovey Gareth J. , Roddam Philippa L. , Roman Eve , Cartwright Raymond A. , Morgan Gareth J.
Published: July 2002

Glutathione
S
-transferases (GSTs) detoxify
potentially mutagenic and toxic DNA-reactive electrophiles, including
metabolites of several chemotherapeutic agents, some of which are
suspected human carcinogens. Functional polymorphisms exist in at least
three genes that encode GSTs, including
GSTM1,
GSTT1,
and
GSTP1.
We hypothesize,
therefore, that polymorphisms in genes that encode GSTs alter
susceptibility to chemotherapy-induced carcinogenesis, specifically to
therapy-related acute myeloid leukemia (t-AML), a devastating
complication of long-term cancer survival. Elucidation of genetic
determinants may help to identify individuals at increased risk of
developing t-AML. To this end, we have examined 89 cases of t-AML, 420
cases of
de novo
AML, and 1,022 controls for
polymorphisms in
GSTM1,
GSTT1,
and
GSTP1
. Gene deletion of
GSTM1
or
GSTT1
was not specifically associated with
susceptibility to t-AML. Individuals with at least one
GSTP1
codon 105 Val allele were significantly
over-represented in t-AML cases compared with
de novo
AML cases [odds ratio (OR), 1.81; 95% confidence interval (CI),
1.11–2.94]. Moreover, relative to
de novo
AML, the
GSTP1
codon 105 Val allele occurred more often among
t-AML patients with prior exposure to chemotherapy (OR, 2.66; 95% CI,
1.39–5.09), particularly among those with prior exposure to known
GSTP1 substrates (OR, 4.34; 95% CI, 1.43–13.20), and not among those
t-AML patients with prior exposure to radiotherapy alone (OR,1.01; 95%
CI, 0.50–2.07). These data suggest that inheritance of at least one
Val allele at
GSTP1
codon 105 confers a significantly
increased risk of developing t-AML after cytotoxic chemotherapy, but
not after radiotherapy.
Sparing of neuronal function postseizure with gene therapy
Authors: McLaughlin John , Roozendaal Benno , Dumas Theodore , Gupta Anurag , Ajilore Olusola , Hsieh Joseph , Ho Dora , Lawrence Matthew , McGaugh James L. , Sapolsky Robert
Published: July 2002

Numerous studies have demonstrated that gene therapy interventions
can protect neurons from death after neurological insults. In nearly
all such studies, however, “protection” consists of reduced
neurotoxicity, with no demonstrated preservation of neuronal function.
We used a herpes simplex virus-1 system to overexpress either the
Glut-1 glucose transporter (GT) (to buffer energetics), or the
apoptosis inhibitor Bcl-2. Both decreased hippocampal neuron
loss to similar extents during excitotoxic insults
in
vitro
and
in vivo
. However, the mediating
mechanisms and consequences of the two interventions
differed. GT overexpression attenuated early, energy-dependent facets
of cell death, blocking oxygen radical accumulation. Bcl-2 expression,
in contrast, blocked components of death downstream from the energetic
and oxidative facets. Most importantly, GT- but not Bcl-2-mediated
protection preserved hippocampal function as assessed spatial maze
performance. Thus, gene therapeutic sparing of neurons from
insult-induced death does not necessarily translate into sparing of
function.
Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia
Authors: Kozyraki Renata , Fyfe John , Verroust Pierre J. , Jacobsen Christian , Dautry-Varsat Alice , Gburek Jakub , Willnow Thomas E. , Christensen Erik Ilsø , Moestrup Søren K.
Published: July 2002

Cubilin is a 460-kDa protein functioning as an endocytic receptor for intrinsic factor vitamin B
12
complex in the intestine and as a receptor for apolipoprotein A1 and albumin reabsorption in the kidney proximal tubules and the yolk sac. In the present study, we report the identification of cubilin as a novel transferrin (Tf) receptor involved in catabolism of Tf. Consistent with a cubilin-mediated endocytosis of Tf in the kidney, lysosomes of human, dog, and mouse renal proximal tubules strongly accumulate Tf, whereas no Tf is detectable in the endocytic apparatus of the renal tubule epithelium of dogs with deficient surface expression of cubilin. As a consequence, these dogs excrete increased amounts of Tf in the urine. Mice with deficient synthesis of megalin, the putative coreceptor colocalizing with cubilin, also excrete high amounts of Tf and fail to internalize Tf in their proximal tubules. However, in contrast to the dogs with the defective cubilin expression, the megalin-deficient mice accumulate Tf on the luminal cubilin-expressing surface of the proximal tubule epithelium. This observation indicates that megalin deficiency causes failure in internalization of the cubilin–ligand complex. The megalin-dependent, cubilin-mediated endocytosis of Tf and the potential of the receptors thereby to facilitate iron uptake were further confirmed by analyzing the uptake of
125
I- and
59
Fe-labeled Tf in cultured yolk sac cells.
Diversity of gene expression in adenocarcinoma of the lung
Authors: Garber Mitchell E. , Troyanskaya Olga G. , Schluens Karsten , Petersen Simone , Thaesler Zsuzsanna , Pacyna-Gengelbach Manuela , van de Rijn Matt , Rosen Glenn D. , Perou Charles M. , Whyte Richard I. , Altman Russ B. , Brown Patrick O. , Botstein David , Petersen Iver
Published: July 2002

The global gene expression profiles for 67 human lung tumors representing 56 patients were examined by using 24,000-element cDNA microarrays. Subdivision of the tumors based on gene expression patterns faithfully recapitulated morphological classification of the tumors into squamous, large cell, small cell, and adenocarcinoma. The gene expression patterns made possible the subclassification of adenocarcinoma into subgroups that correlated with the degree of tumor differentiation as well as patient survival. Gene expression analysis thus promises to extend and refine standard pathologic analysis.
Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.
Authors: Lee W J , Lee J D , Kravchenko V V , Ulevitch R J , Brey P T
Published: July 2002

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.
Signal termination in bacterial chemotaxis: CheZ mediates dephosphorylation of free rather than switch-bound CheY.
Authors: Bren A , Welch M , Blat Y , Eisenbach M
Published: July 2002

Chemotaxis in bacteria is controlled by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein CheY. When phosphorylated, CheY binds to FliM, which is one of the proteins that constitute the "gear box" (or "switch") of the flagellar motor. Consequently, the motor shifts from the default direction of rotation, counterclockwise, to clockwise rotation. This biased rotation is terminated when CheY is dephosphorylated either spontaneously or, faster, by a specific phosphatase, CheZ. Logically, one might expect CheZ to act directly on FliM-bound CheY. However, here we provide direct biochemical evidence that, in contrast to this expectation, phosphorylated CheY (CheY approximately P), bound to FliM, is protected from dephosphorylation by CheZ. The complex between CheY approximately P and FliM was trapped by cross-linking with dimethylsuberimidate, and its susceptibility to CheZ was measured. CheY approximately P complexed with FliM, unlike free CheY approximately P, was not dephosphorylated by CheZ. However, it did undergo spontaneous dephosphorylation. Nonspecific cross-linked CheY dimers, measured as a control, were dephosphorylated by CheZ. No significant binding between CheZ and any of the switch proteins was detected. It is concluded that, in the termination mechanism of signal transduction in bacterial chemotaxis, CheZ acts only on free CheY approximately P. We suggest that CheZ affects switch-bound CheY approximately P by shifting the equilibrium between bound and free CheY approximately P.
Synthetic transcripts of double-stranded Birnavirus genome are infectious.
Authors: Mundt E , Vakharia V N
Published: July 2002

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.
Characterization of human cardiac Na
+
channel mutations in the congenital long QT syndrome
Authors: Wang Dao W. , Yazawa Kazuto , George Alfred L. , Bennett Paul B.
Published: July 2002

The congenital long QT syndrome (LQTS) is an inherited disorder
characterized by a prolonged cardiac action potential. This delay in
cellular repolarization can lead to potentially fatal arrhythmias. One
form of LQTS (LQT3) has been linked to the human cardiac voltage-gated
sodium channel gene (
SCN5A
). Three distinct mutations
have been identified in the sodium channel gene. The biophysical and
functional characteristics of each of these mutant channels were
determined by heterologous expression of a recombinant human heart
sodium channel in a mammalian cell line. Each mutation caused a
sustained, non-inactivating sodium current amounting to a few percent
of the peak inward sodium current, observable during long (>50 msec)
depolarizations. The voltage dependence and rate of inactivation were
altered, and the rate of recovery from inactivation was changed
compared with wild-type channels. These mutations in diverse regions of
the ion channel protein, all produced a common defect in channel gating
that can cause the long QT phenotype. The sustained inward current
caused by these mutations will prolong the action potential.
Furthermore, they may create conditions that promote arrhythmias due to
prolonged depolarization and the altered recovery from inactivation.
These results provide insights for successful intervention in the
disease.
The essential yeast RNA binding protein Npl3p is methylated
Authors: Siebel Christian W. , Guthrie Christine
Published: July 2002

Arginine methylation is a prevalent modification found in many RNA
binding proteins, yet little is known about its functional
consequences. Using a monoclonal antibody, 1E4, we have shown that the
yeast
NPL3
gene product Npl3p, an essential RNA binding
protein with repeated RGG motifs, is arginine-methylated
in
vivo
. The 1E4 epitope can be generated by incubating
recombinant Npl3p with partially purified bovine arginine
methyltransferase, and peptides that specifically inhibit arginine
methyltransferases block this reaction. Npl3p methylation requires
S
-adenosyl-
l
-methionine and also occurs in
yeast extracts. An Npl3p deletion mutant lacking the RGG domain is not
a substrate for methylation, suggesting that the methylation sites lie
within the RGG motifs. The discovery of arginine methylation in a
genetically tractable organism provides a powerful entrée to
understanding the function of this modification, particularly in view
of the many roles postulated for Npl3p in RNA processing and transport.
The recent discovery of phosphorylated serine residues within the RGG
domain suggests a hypothesis in which a molecular switch governed by
methylation and phosphorylation regulates the biochemical properties of
the Npl3p RGG domain.
Calmodulin activates nuclear protein import: A link between signal transduction and nuclear transport
Authors: Sweitzer Thomas D. , Hanover John A.
Published: July 2002

In addition to the well-characterized GTP-dependent nuclear
transport observed in permeabilized cells, we detected a mode of
nuclear transport that was GTP-independent at elevated cytoplasmic
calcium concentrations. Nuclear transport under these conditions was
blocked by calmodulin inhibitors. Recombinant
calmodulin restored ATP-dependent nuclear transport in the
absence of cytosol. Calmodulin-dependent transport was
inhibited by wheat germ agglutinin consistent with transport proceeding
through nuclear pores. We propose that release of intracellular calcium
stores upon cell activation inhibits GTP-dependent nuclear transport;
the elevated cytosolic calcium then acts through calmodulin
to stimulate the novel GTP-independent mode of import.
Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the
ech42
(endochitinase-encoding) gene in
Trichoderma harzianum
Authors: Lorito Matteo , Mach Robert L. , Sposato Paola , Strauss Joseph , Peterbauer Clemens K. , Kubicek Christian P.
Published: July 2002

The fungus
Trichoderma harzianum
is a potent
mycoparasite of various plant pathogenic fungi. We have studied the
molecular regulation of mycoparasitism in the host/mycoparasite
system
Botrytis cinerea
/
T. harzianum
.
Protein extracts, prepared from various stages of mycoparasitism, were
used in electrophoretic mobility-shift assays (EMSAs) with two promoter
fragments of the
ech-42
(42-kDa endochitinase-encoding)
gene of
T. harzianum
. This gene was chosen as a model
because its expression is triggered during mycoparasitic interaction
[Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. &
Herrera-Estrella, A. (1994)
Proc. Natl. Acad. Sci. USA
91, 10903–10907]. All cell-free extracts formed
high-molecular weight protein–DNA complexes, but those obtained from
mycelia activated for mycoparasitic attack formed a complex with
greater mobility. Competition experiments, using oligonucleotides
containing functional and nonfunctional consensus sites for binding of
the carbon catabolite repressor Cre1, provided evidence that the
complex from nonmycoparasitic mycelia involves the binding of Cre1 to
both fragments of the
ech-42
promoter. The presence of
two and three consensus sites for binding of Cre1 in the two
ech-42
promoter fragments used is consistent with these
findings. In contrast, the formation of the protein–DNA complex from
mycoparasitic mycelia is unaffected by the addition of the competing
oligonucleotides and hence does not involve Cre1. Addition of equal
amounts of protein of cell-free extracts from nonmycoparasitic mycelia
converted the mycoparasitic DNA–protein complex into the
nonmycoparasitic complex. The addition of the purified
Cre1::glutathione
S
-transferase protein to
mycoparasitic cell-free extracts produced the same effect. These
findings suggest that
ech-42
expression in
T.
harzianum
is regulated by (
i
) binding of Cre1 to
two single sites in the
ech-42
promoter,
(
ii
) binding of a “mycoparasitic” protein–protein
complex to the
ech-42
promoter in vicinity of the Cre1
binding sites, and (
iii
) functional inactivation of Cre1
upon mycoparasitic interaction to enable the formation of the
mycoparasitic protein–DNA complex.
A complexity measure for selective matching of signals by the brain.
Authors: Tononi G , Sporns O , Edelman G M
Published: July 2002

We have previously derived a theoretical measure of neural complexity (CN) in an attempt to characterize functional connectivity in the brain. CN measures the amount and heterogeneity of statistical correlations within a neural system in terms of the mutual information between subsets of its units. CN was initially used to characterize the functional connectivity of a neural system isolated from the environment. In the present paper, we introduce a related statistical measure, matching complexity (CM), which reflects the change in CN that occurs after a neural system receives signals from the environment. CM measures how well the ensemble of intrinsic correlations within a neural system fits the statistical structure of the sensory input. We show that CM is low when the intrinsic connectivity of a simulated cortical area is randomly organized. Conversely, CM is high when the intrinsic connectivity is modified so as to differentially amplify those intrinsic correlations that happen to be enhanced by sensory input. When the input is represented by an individual stimulus, a positive value of CM indicates that the limited mutual information between sensory sheets sampling the stimulus and the rest of the brain triggers a large increase in the mutual information between many functionally specialized subsets within the brain. In this way, a complex brain can deal with context and go "beyond the information given."
ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation.
Authors: Lechner C , Zahalka M A , Giot J F , Møller N P , Ullrich A
Published: July 2002

ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.
Interferon γ-induced transcription of the murine ISGF3γ (p48) gene is mediated by novel factors
Authors: Weihua Xiao , Kolla Venkatadri , Kalvakolanu Dhananjaya V.
Published: July 2002

In this investigation, we show that the gene encoding p48, a subunit of transcription factor ISGF3, is transcriptionally induced by interferon γ (IFN-γ). We have identified a novel IFN-γ-activated response element in the p48 gene promoter. This motif, notated as
g
amma-
a
ctivated
t
ranscriptional
e
lement (GATE), has no significant resemblance to either pIRE (palindromic IFN-response element) or GAS (the IFN-γ-activated sequence) but has partial homology to ISRE (IFN-stimulated response element). When fused to a neutral promoter, GATE, a 24-bp element, induced the expression of reporter genes following IFN-γ treatment. In murine RAW cells, two IFN-
g
amma-
i
nducible
f
actors (GIF) bind to GATE. Binding of these factors to GATE is inhibited by cycloheximide and staurosporine. Although p48 gene induction is dependent on STAT1 and JAK1, activated STAT1 does not bind to GATE. Thus, GIFs appear to be novel trans-acting factors in the IFN-signaling pathway.
Crystal structure of glutamate-1-semialdehyde aminomutase: An α
2
-dimeric vitamin B
6
-dependent enzyme with asymmetry in structure and active site reactivity
Authors: Hennig Michael , Grimm Bernhard , Contestabile Roberto , John Robert A. , Jansonius Johan N.
Published: July 2002

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC
5.4.3.8
), an α
2
-dimeric enzyme from
Synechococcus,
has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-Å resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme’s unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B
6
enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel α-carboxylic acids, thereby suggesting a basis for the enzyme’s reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153–181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153–181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153–181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153–181 remains.
Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression
Authors: Schnorr Jens-Jörg , Xanthakos Stavra , Keikavoussi Petra , Kämpgen Eckhart , ter Meulen Volker , Schneider-Schaulies Sibylle
Published: July 2002

As well as inducing a protective immune response against reinfection, acute measles is associated with a marked suppression of immune functions against superinfecting agents and recall antigens, and this association is the major cause of the current high morbidity and mortality rate associated with measles virus (MV) infections. Dendritic cells (DCs) are antigen-presenting cells crucially involved in the initiation of primary and secondary immune responses, so we set out to define the interaction of MV with these cells. We found that both mature and precursor human DCs generated from peripheral blood monocytic cells express the major MV protein receptor CD46 and are highly susceptible to infection with both MV vaccine (ED) and wild-type (WTF) strains, albeit with different kinetics. Except for the down-regulation of CD46, the expression pattern of functionally important surface antigens on mature DCs was not markedly altered after MV infection. However, precursor DCs up-regulated HLA-DR, CD83, and CD86 within 24 h of WTF infection and 72 h after ED infection, indicating their functional maturation. In addition, interleukin 12 synthesis was markedly enhanced after both ED and WTF infection in DCs. On the other hand, MV-infected DCs strongly interfered with mitogen-dependent proliferation of freshly isolated peripheral blood lymphocytesin vitro. These data indicate that the differentiation of effector functions of DCs is not impaired but rather is stimulated by MV infection. Yet, mature, activated DCs expressing MV surface antigens do give a negative signal to inhibit lymphocyte proliferation and thus contribute to MV-induced immunosuppression.
Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation
Authors: Zhou Yan-Ting , Shimabukuro Michio , Koyama Kazunori , Lee Young , Wang May-Yun , Trieu Falguni , Newgard Christopher B. , Unger Roger H.
Published: July 2002

We have studied mechanisms by which leptin overexpression, which reduces body weight via anorexic and thermogenic actions, induces triglyceride depletion in adipocytes and nonadipocytes. Here we show that leptin alters in pancreatic islets the mRNA of the genes encoding enzymes of free fatty acid metabolism and uncoupling protein-2 (UCP-2). In animals infused with a recombinant adenovirus containing the leptin cDNA, the levels of mRNAs encoding enzymes of mitochondrial and peroxisomal oxidation rose 2- to 3-fold, whereas mRNA encoding an enzyme of esterification declined in islets from hyperleptinemic rats. Islet UCP-2 mRNA rose 6-fold. All
in vivo
changes occurred
in vitro
in normal islets cultured with recombinant leptin, indicating direct extraneural effects. Leptin overexpression increased UCP-2 mRNA by more than 10-fold in epididymal, retroperitoneal, and subcutaneous fat tissue of normal, but not of leptin–receptor-defective obese rats. By directly regulating the expression of enzymes of free fatty acid metabolism and of UCP-2, leptin controls intracellular triglyceride content of certain nonadipocytes, as well as adipocytes.
Phylogenetics and the origin of species
Authors: Avise John C. , Wollenberg Kurt
Published: July 2002

A recent criticism that the biological species concept (BSC) unduly neglects phylogeny is examined under a novel modification of coalescent theory that considers multiple, sex-defined genealogical pathways through sexual organismal pedigrees. A competing phylogenetic species concept (PSC) also is evaluated from this vantage. Two analytical approaches are employed to capture the composite phylogenetic information contained within the braided assemblages of hereditary pathways of a pedigree: (i) consensus phylogenetic trees across allelic transmission routes and (ii) composite phenograms from quantitative values of organismal coancestry. Outcomes from both approaches demonstrate that the supposed sharp distinction between biological and phylogenetic species concepts is illusory. Historical descent and reproductive ties are related aspects of phylogeny and jointly illuminate biotic discontinuity.
Transmembrane β-barrel of staphylococcal α-toxin forms in sensitive but not in resistant cells
Authors: Valeva Angela , Walev Iwan , Pinkernell Matthias , Walker Barbara , Bayley Hagan , Palmer Michael , Bhakdi Sucharit
Published: July 2002

Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118–140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126–140 entered a nonpolar environment in a striking display of an amphipathic transmembrane β-barrel. In contrast, human granulocytes were found to bind α-toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.
Conversion of cysteine to formylglycine: A protein modification in the endoplasmic reticulum
Authors: Dierks Thomas , Schmidt Bernhard , von Figura Kurt
Published: July 2002

In sulfatases a C
α
-formylglycine residue is found at a position where their cDNA sequences predict a cysteine residue. In multiple sulfatase deficiency, an inherited lysosomal storage disorder, catalytically inactive sulfatases are synthesized which retain the cysteine residue, indicating that the C
α
-formylglycine residue is required for sulfatase activity. Using
in vitro
translation in the absence or presence of transport competent microsomes we found that newly synthesized sulfatase polypeptides carry a cysteine residue and that the oxidation of its thiol group to an aldehyde is catalyzed in the endoplasmic reticulum. A linear sequence of 16 residues surrounding the Cys-69 in arylsulfatase A is sufficient to direct the oxidation. This novel protein modification occurs after or at a late stage of cotranslational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure.
MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity
Authors: Li Yong , Gorbea Carlos , Mahaffey David , Rechsteiner Martin , Benezra Robert
Published: July 2002

Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling
Xenopus laevis
egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.
Association of INAD with NORPA is essential for controlled activation and deactivation of
Drosophila
phototransduction
in vivo
Authors: Shieh Bih-Hwa , Zhu Mei-Ying , Lee John K. , Kelly Ingrid M. , Bahiraei Frohar
Published: July 2002

Visual transduction in
Drosophila
is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA–INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD–NORPA association
in vivo
, we generated transgenic flies expressing a modified NORPA, NORPA

C1094S

, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPA

C1094S

, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPA

C1094S

being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA–TRP interactions required for gating of the TRP channel in photoreceptor cells.
Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide
Authors: Thomas Elizabeth A. , Carson Monica J. , Neal Michael J. , Sutcliffe J. Gregor
Published: July 2002

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine
2A
(5HT
2A
) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT
7
receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an ≈65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT
7
receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., γ-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies.
In vitro
-generated neural precursors participate in mammalian brain development
Authors: Brüstle Oliver , Spiro A. Cyril , Karram Khalad , Choudhary Khalid , Okabe Shigeo , McKay Ronald D. G.
Published: July 2002

During embryogenesis, pluripotent stem cells segregate into daughter lineages of progressively restricted developmental potential.
In vitro
, this process has been mimicked by the controlled differentiation of embryonic stem cells into neural precursors. To explore the developmental potential of these cell-culture-derived precursors
in vivo
, we have implanted them into the ventricles of embryonic rats. The transplanted cells formed intraventricular neuroepithelial structures and migrated in large numbers into the brain tissue. Embryonic-stem-cell-derived neurons, astrocytes, and oligodendrocytes incorporated into telencephalic, diencephalic, and mesencephalic regions and assumed phenotypes indistinguishable from neighboring host cells. These observations indicate that entirely
in vitro
-generated neural precursors are able to respond to environmental signals guiding cell migration and differentiation and have the potential to reconstitute neuronal and glial lineages in the central nervous system.
Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in
Nicotiana sylvestris
mitochondrial deletion mutants
Authors: Gutierres Sophie , Sabar Mohammed , Lelandais Christine , Chetrit Philippe , Diolez Philippe , Degand Hervé , Boutry Marc , Vedel Fernand , de Kouchkovsky Yaroslav , De Paepe Rosine
Published: July 2002

We previously have shown that
Nicotiana sylvestris
cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.
On the thermodynamic hypothesis of protein folding
Authors: Govindarajan Sridhar , Goldstein Richard A.
Published: July 2002

The validity of the thermodynamic hypothesis of protein folding was explored by simulating the evolution of protein sequences. Simple models of lattice proteins were allowed to evolve by random point mutations subject to the constraint that they fold into a predetermined native structure with a Monte Carlo folding algorithm. We employed a simple analytical approach to compute the probability of violation of the thermodynamic hypothesis as a function of the size of the protein, the fraction of the total number of possible conformations which are kinetically accessible, and the roughness of the free-energy landscape. It was found that even if the folding is under kinetic control, the sequence will evolve so that the native state is most often the state of minimum free energy.
A Myb homologue, ATR1, activates tryptophan gene expression inArabidopsis
Authors: Bender Judith , Fink Gerald R.
Published: July 2002

InArabidopsis thaliana, tryptophan pathway genes are induced in response to starvation, wounding, and pathogen attack, resulting in increased production of tryptophan and secondary metabolites important for development and defense. TheArabidopsistryptophan pathway therefore provides an ideal system for elucidating how environmental stimuli are transduced into changes in plant gene expression. To characterize the factors that regulate the first gene in the pathway,ASA1, which is the key point of control, we have isolatedalteredtryptophanregulation (atr) mutants with deregulated expression ofASA1. One of these mutants,atr1Dis dominant for increased transcription ofASA1in specific seedling tissues. We have usedatr1Dto clone theATR1gene based on its map position.ATR1encodes a Myb-like transcription factor that modulatesASA1expression. TheATR1transcript also includes a 5′ regulatory region with three short ORFs, one of which is prematurely terminated by theatr1Dmutation. Thus,ATR1defines the first characterized tryptophan gene regulator in plants, and theatr1Dmutation defines a sequence important for ATR1 expression.
Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering
Authors: Stassi D. L. , Kakavas S. J. , Reynolds K. A. , Gunawardana G. , Swanson S. , Zeidner D. , Jackson M. , Liu H. , Buko A. , Katz L.
Published: July 2002

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism
Saccharopolyspora erythraea
. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.