bluuebunny/crossref_metadata_2025 huggingface dataset:
Anopheles gambiae
salivary gland proteins as putative targets for blocking transmission of malaria parasites
Authors: Brennan Justin D. G. , Kent Melissa , Dhar Ravi , Fujioka Hisashi , Kumar Nirbhay
Published: July 2002

Anopheles gambiae
is the primary vector of human
malaria in sub-Saharan Africa. Invasion of
Anopheles
salivary glands by
Plasmodium
sporozoites is a necessary
step in the transmission of malaria and is likely to be mediated by
specific receptor–ligand interactions. We are interested in
identifying putative an
A. gambiae
salivary gland
receptor or receptors for sporozoite invasion as a possible target for
blocking malaria transmission. By using monoclonal antibodies against
female-specific
A. gambiae
salivary gland proteins, two
molecules, one of 29 kDa and one of 100 kDa, were identified and
characterized with respect to the age and blood-feeding process of
mosquitoes. In an
in vivo
bioassay, the monoclonal
antibody against the 100-kDa protein inhibited
Plasmodium
yoelii
sporozoite invasion of
salivary glands ≥75%. These results show that
A.
gambiae
salivary gland proteins are accessible to monoclonal
antibodies that inhibit sporozoite invasion of the salivary glands and
suggest alternate targets for blocking the transmission of malaria by
this most competent of malaria vectors.
PAS-A domain of phosphorelay sensor kinase A: A catalytic ATP-binding domain involved in the initiation of development in
Bacillus subtilis
Authors: Stephenson Keith , Hoch James A.
Published: July 2002

The major sensor kinase controlling the initiation of
development in
Bacillus subtilis
, KinA, functions
by activating the phosphorelay signal-transduction system in response
to as yet unknown signal ligands. KinA contains, within its
amino-terminal signal-sensing region, three PAS domains that, in other
proteins, are known to be involved in sensing changes in oxygen
concentration and redox potential among other functions. The most
amino-terminal PAS domain, PAS-A, was found to bind ATP and catalyze
exchange of phosphate between ATP and nucleoside diphosphates. A
cysteine-to-alanine mutation in PAS-A increased the affinity for ATP
5-fold, decreased the exchange reaction 2-fold, and stimulated
KinA-dependent sporulation. A model for the role of ATP and the
exchange reaction in the PAS domain in sensor kinase signal
transduction is presented in which the free energy of nucleotide
hydrolysis drives the conformational changes that activate or
deactivate the sensor kinase in response to signal ligand binding.
High frequency mitotic gene conversion in genetic hybrids of the oomycete
Phytophthora
sojae
Authors: Chamnanpunt Jureerat , Shan Wei-xing , Tyler Brett M.
Published: July 2002

Microbial populations depend on genetic variation to
respond to novel environmental challenges. Plant pathogens are
notorious for their ability to overcome pesticides and host resistance
genes as a result of genetic changes. We report here that in particular
hybrid strains of
Phytophthora sojae
, an oomycete
pathogen of soybean, high frequency mitotic gene conversion rapidly
converts heterozygous loci to homozygosity, resulting in heterokaryons
containing highly diverse populations of diploid nuclei. In hybrids
involving strain P7076, conversion rates of up to 3 ×
10
−2
per locus per nucleus per generation were observed.
In other hybrids, rates were of the order of 5 ×
10
−5
. Independent gene conversion was observed within a
selected linkage group including loci as close as 0.7 kb apart and in
unlinked markers throughout the genome. Gene conversions continued
throughout vegetative growth and were stimulated by further sexual
reproduction. At many loci, conversion showed extreme disparity, with
one allele always being lost, suggesting that conversion was initiated
by allele-specific double-stranded breaks. Pedigree analysis indicated
that individual loci undergo multiple independent conversions within
the nuclei of a vegetative clone and that conversion may be preceded by
a heritable “activation” state.
Algebraic orbifold conformal field theories
Authors: Xu Feng
Published: July 2002

The unitary rational orbifold conformal field theories in the
algebraic quantum field theory and subfactor theory framework are
formulated. Under general conditions, it is shown that the orbifold of
a given unitary rational conformal field theory generates a unitary
modular category. Many new unitary modular categories are obtained. It
is also shown that the irreducible representations of orbifolds of rank
one lattice vertex operator algebras give rise to unitary modular
categories and determine the corresponding modular matrices, which has
been conjectured for some time.
Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation
Authors: Center Rob J. , Schuck Peter , Leapman Richard D. , Arthur Larry O. , Earl Patricia L. , Moss Bernard , Lebowitz Jacob
Published: July 2002

The envelope proteins (env) of simian immunodeficiency virus (SIV)
and HIV type 1 assemble to form noncovalently associated oligomers in
the endoplasmic reticulum. After cleavage in a Golgi
compartment, oligomeric env complexes are transported to the surface of
infected cells, where incorporation into budding virions can occur.
Difficulties in obtaining adequate quantities of virions retaining env,
as well as the unstable nature and hydrophobicity of the oligomer, may
account for the absence of previous biophysical studies to determine
the oligomeric valency of membrane-associated env. The aim of this
study was to evaluate the oligomeric state of SIV env before
membrane-fusion activation. Virion-associated env, obtained by
crosslinking and detergent extraction, and non-crosslinked secreted env
ectodomain (recombinant gp140) were purified by lentil-lectin
chromatography and gel filtration as single predominant species.
Sedimentation equilibrium-derived mass values for both forms of SIV env
were close to those predicted for trimeric assemblies. Determination of
the mass of individual molecules by scanning transmission electron
microscopy confirmed that SIV virion-associated env and gp140 formed
largely homogeneous populations of trimers. Furthermore, a triangular
or tri-lobed morphology was clearly visualized in a subset of the
trimers.
Drainage basins and channel incision on Mars
Authors: Aharonson Oded , Zuber Maria T. , Rothman Daniel H. , Schorghofer Norbert , Whipple Kelin X.
Published: July 2002

Measurements acquired by the Mars Orbiter Laser Altimeter on board the Mars Global Surveyor indicate that large drainage systems on Mars have geomorphic characteristics inconsistent with prolonged erosion by surface runoff. We find the topography has not evolved to an expected equilibrium terrain form, even in areas where runoff incision has been previously interpreted. By analogy with terrestrial examples, groundwater sapping may have played an important role in the incision. Longitudinally flat floor segments may provide a direct indication of lithologic layers in the bedrock, altering subsurface hydrology. However, it is unlikely that floor levels are entirely due to inherited structures due to their planar cross-cutting relations. These conclusions are based on previously unavailable observations, including extensive piece-wise linear longitudinal profiles, frequent knickpoints, hanging valleys, and small basin concavity exponents.
The lpf fimbrial operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches.
Authors: Bäumler A J , Tsolis R M , Heffron F
Published: July 2002

We investigated the role of the Salmonella typhimurium fimbrial operon formed by the genes lpfABCDE in infection of mice. A mutant in lpfC, the gene encoding the fimbrial outer membrane usher, had an approximately 5-fold increased 50% lethal dose when administered orally to mice. When mice were infected with a mixture of the lpfC mutant and isogenic wild-type S. typhimurium, the lpfC mutant was recovered in lower numbers from Peyer's patches, mesenteric lymph nodes, liver, and spleen. In an organ culture model using murine intestinal loops, lpfC mutants were shown to be associated in lower numbers than wild-type bacteria with Peyer's patches but not with villous intestine. The defect of the lpfC mutant in adhesion to Peyer's patches could be complemented by introducing lpfABCDE on a cosmid. Similarly, heterologous expression of the Salmonella lpf operon in Escherichia coli resulted in an increased adhesion to histological thin sections of Peyer's patch lymph follicles. Electron microscopic analysis of histological sections taken from Peyer's patches after intragastric infection of mice showed that, in contrast to the S. typhimurium wild type, the isogenic lpfC mutant did not destroy M cells of the follicle-associated epithelium. These data show that the Salmonella lpf operon is involved in adhesion to murine Peyer's patches.
How optimization of potential functions affects protein folding.
Authors: Hao M H , Scheraga H A
Published: July 2002

The relationship between the optimization of the potential function and the foldability of theoretical protein models is studied based on investigations of a 27-mer cubic-lattice protein model and a more realistic lattice model for the protein crambin. In both the simple and the more complicated systems, optimization of the energy parameters achieves significant improvements in the statistical-mechanical characteristics of the systems and leads to foldable protein models in simulation experiments. The foldability of the protein models is characterized by their statistical-mechanical properties--e.g., by the density of states and by Monte Carlo folding simulations of the models. With optimized energy parameters, a high level of consistency exists among different interactions in the native structures of the protein models, as revealed by a correlation function between the optimized energy parameters and the native structure of the model proteins. The results of this work are relevant to the design of a general potential function for folding proteins by theoretical simulations.
Heat-shock protein 104 expression is sufficient for thermotolerance in yeast.
Authors: Lindquist S , Kim G
Published: July 2002

In all organisms, mild heat pretreatments induce tolerance to high temperatures. In the yeast Saccharomyces cerevisiae, such pretreatments strongly induce heat-shock protein (Hsp) 104, and hsp104 mutations greatly reduce high-temperature survival, indicating Hsp1O4 plays a critical role in induced thermotolerance. Surprisingly, however, a heat-shock transcription factor mutation (hsf1-m3) that blocks the induction of Hsps does not block induced thermotolerance. To resolve these apparent contradictions, we reexamined Hsp expression in hsf1-m3 cells. HsplO4 was expressed at a higher basal level in this strain than in other S. cerevisiae strains. Moreover, whereas the hsf1-m3 mutation completely blocked the induction of Hsp26 by heat, it did not block the induction of Hsp1O4. HSP104 could not be deleted in hsf1-m3 cells because the expression of heat-shock factor (and the viability of the strain) requires nonsense suppression mediated by the yeast prion [PSI+], which in turn depends upon Hsp1O4. To determine whether the level of Hsp1O4 expressed in hsf1-m3 cells is sufficient for thermotolerance, we used heterologous promoters to regulate Hsp1O4 expression in other strains. In the presence of other inducible factors (with a conditioning pretreatment), low levels of Hsp1O4 are sufficient to provide full thermotolerance. More remarkably, in the absence of other inducible factors (without a pretreatment), high levels of Hsp1O4 are sufficient. We conclude that Hsp1O4 plays a central role in ameliorating heat toxicity. Because Hsp1O4 is nontoxic and highly conserved, manipulating the expression of Hsp1OO proteins provides an excellent prospect for manipulating thermotolerance in other species.
Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex.
Authors: Fox K , Schlaggar B L , Glazewski S , O'Leary D D
Published: July 2002

The segregation of thalamocortical inputs into eye-specific stripes in the developing cat or monkey visual cortex is prevented by manipulations that perturb or abolish neural activity in the visual pathway. Such findings show that proper development of the functional organization of visual cortex is dependent on normal patterns of neural activity. The generalisation of this conclusion to other sensory cortices has been questioned by findings that the segregation of thalamocortical afferents into a somatotopic barrel pattern in developing rodent primary somatosensory cortex (S1) is not prevented by activity blockade. We show that a temporary block of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat S1 during the critical period for barrel development disrupts the topographic refinement of thalamocortical connectivity and columnar organization. These effects are evident well after the blockade is ineffective and thus may be permanent. Our findings show that neural activity and specifically the activation of postsynaptic cortical neurons has a prominent role in establishing the primary sensory map in S1, as well as the topographic organization of higher order synaptic connections.
Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.
Authors: Lichtarge O , Bourne H R , Cohen F E
Published: July 2002

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.
Secretory granule targeting of atrial natriuretic peptide correlates with its calcium-mediated aggregation.
Authors: Canaff L , Brechler V , Reudelhuber T L , Thibault G
Published: July 2002

Atrial natriuretic peptide (ANP) is a 28-aa peptide hormone secreted predominantly from atrial cardiocytes. ANP is first synthesized in the form of a 126-aa precursor (proANP) which is targeted to dense core granules of the regulated secretory pathway. ProANP is stored until the cell receives a signal that triggers the processing and release of the mature peptide (regulated secretion). Various models have been proposed to explain the targeting of selected proteins to the regulated secretory pathway, including specific "sorting receptors" and calcium-mediated aggregation. As potential calcium binding regions had previously been reported in the profragment of ANP, the current study was undertaken in an effort to determine the relationship between the ability of ANP to enter the regulated secretory pathway and its calcium-mediated aggregation. Deletion and site-directed mutagenesis of selected regions of the prosegment demonstrates that acidic amino acids at positions 23 and 24 are critical for both regulated secretion of proANP from transfected AtT-20 cells and calcium-mediated aggregation of purified recombinant proANP in vitro. These results demonstrate that the ability of certain proteins to enter secretory granules is directly linked to their calcium-mediated aggregation.
The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.
Authors: Mazin A V , Kowalczykowski S C
Published: July 2002

The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound to the RecA protein primary site. We conclude that the specificity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNa molecules within nucleoprotein filament--i.e., self-recognition. The secondary DNA binding site displays a higher affinity for ssDNA than for double-stranded DNA, and the binding of ssDNA to the secondary site strongly inhibits DNA strand exchange. We suggest that the secondary binding site has a dual role in DNA strand exchange. During the homology search, it binds double-stranded DNA weakly; upon finding local homology, this site binds, with higher affinity, the ssDNA strand that is displaced during DNA strand exchange. These characteristics facilitate homologous pairing, promote stabilization of the newly formed heteroduplex DNA, and contribute to the directionality of DNA strand exchange.
Ligand-activated site-specific recombination in mice.
Authors: Feil R , Brocard J , Mascrez B , LeMeur M , Metzger D , Chambon P
Published: July 2002

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.
Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma.
Authors: Bonini S , Lambiase A , Bonini S , Angelucci F , Magrini L , Manni L , Aloe L
Published: July 2002

Nerve growth factor (NGF) serum levels were measured in 49 patients with asthma and/or rhinoconjunctivitis and/or urticaria-angioedema. Clinical and biochemical parameters, such as bronchial reactivity, total and specific serum IgE levels, and circulating eosinophil cationic protein levels, were evaluated in relation to NGF values in asthma patients. NGF was significantly increased in the 42 allergic (skin-test- or radioallergosorbent-test-positive) subjects (49.7 +/- 28.8 pg/ml) versus the 18 matched controls (3.8 +/- 1.7 pg/ml; P < 0.001). NGF levels in allergic patients with asthma, rhinoconjunctivitis, and urticaria-angioedema were 132.1 +/- 90.8, 17.6 +/- 6.1, and 7.6 +/- 1.8 pg/ml (P < 0.001, P < 0.002, and P < 0.05 versus controls), respectively. Patients with more than one allergic disease had higher NGF serum values than those with a single disease. When asthma patients were considered as a group, NGF serum values (87.6 +/- 59.8 pg/ml) were still significantly higher than those of control groups (P < 0.001), but allergic asthma patients had elevated NGF serum levels compared with nonallergic asthma patients (132.1 +/- 90.8 versus 4.9 +/- 2.9 pg/ml; P < 0.001). NGF serum levels correlate to total IgE serum values (rho = 0.43; P < 0.02). The highest NGF values were found in patients with severe allergic asthma, a high degree of bronchial hyperreactivity, and high total IgE and eosinophil cationic protein serum levels. This study represents the first observation (that we know of) that NGF is increased in human allergic inflammatory diseases and asthma.
Microtubules mediate mitochondrial distribution in fission yeast.
Authors: Yaffe M P , Harata D , Verde F , Eddison M , Toda T , Nurse P
Published: July 2002

The Schizosaccharomyces pombe mutant, ban5-4, displays aberrant mitochondrial distribution. Incubation of this conditional-lethal mutant at the nonpermissive temperature led to aggregated mitochondria that were distributed asymmetrically within the cell. Development of this mitochondrial asymmetry but not mitochondrial aggregation required progression through the cell division cycle. Genetic analysis revealed that ban5-4 is an allele of atb2 encoding alpha 2-tubulin. Consistent with this finding, cells with the cold-sensitive nda3 mutation in beta-tubulin displayed aggregated and asymmetrically distributed mitochondria after incubation at lowered temperatures. These results indicate that microtubules mediate mitochondrial distribution in fission yeast and provide the first genetic evidence for the role of microtubules in mitochondrial movement.
Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins.
Authors: Boye E , Stokke T , Kleckner N , Skarstad K
Published: July 2002

We describe here the development of a new approach to the analysis of Escherichia coli replication control. Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with G1, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division. Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass. The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9%. Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation. In dnaA (Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that for wild-type cells. In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony. The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.
The Pto kinase mediates a signaling pathway leading to the oxidative burst in tomato
Authors: Chandra Sreeganga , Martin Gregory B. , Low Philip S.
Published: July 2002

The
Pto
gene encodes a serine/threonine kinase
that confers resistance in tomato to
Pseudomonas
syringae
pv.
tomato
strains that express the
avirulence gene
avrPto
. Partial characterization of the
Pto
signal transduction pathway and the availability of
transgenic tomato lines (±
Pto
) make this an ideal
system for exploring the molecular basis of disease resistance. In this
paper, we test two transgenic tomato cell suspension cultures
(±
Pto
) for production of H
2
O
2
following independent challenge with two strains of
P.
syringae
pv.
tomato
(±
avrPto
).
Only when
Pto
and
avrPto
are present in
the corresponding organisms are two distinct phases of the oxidative
burst seen, a rapid first burst followed by a slower and more prolonged
second burst. In the remaining three plant–pathogen interactions, we
observe either no burst or only a first burst, indicating that the
second burst is correlated with disease resistance. Further support for
this observation comes from the finding that both resistant and
susceptible tomato lines produce the critical second oxidative burst
when challenged with
P. syringae
pv.
tabaci
, a nonhost pathogen that elicits a hypersensitive
response on both tomato lines. The
Pto
kinase is not
required, however, for the oxidative burst initiated by non-specific
elicitors such as oligogalacturonides or osmotic stress. A model
describing a possible role for the
Pto
kinase in the
overall scheme of oxidative burst signaling is proposed.
Ensemble codes involving hippocampal neurons are at risk during delayed performance tests
Authors: Hampson Robert E. , Deadwyler Sam A.
Published: July 2002

Multielectrode recording techniques were used to record ensemble
activity from 10 to 16 simultaneously active CA1 and CA3 neurons in the
rat hippocampus during performance of a spatial
delayed-nonmatch-to-sample task. Extracted sources of variance were
used to assess the nature of two different types of errors that
accounted for 30% of total trials. The two types of errors included
ensemble “miscodes” of sample phase information and errors
associated with delay-dependent corruption or disappearance of sample
information at the time of the nonmatch response. Statistical
assessment of trial sequences and associated “strength” of
hippocampal ensemble codes revealed that miscoded error trials always
followed delay-dependent error trials in which encoding was
“weak,” indicating that the two types of errors were
“linked.” It was determined that the occurrence of weakly
encoded, delay-dependent error trials initiated an ensemble encoding
“strategy” that increased the chances of being correct on the
next trial and avoided the occurrence of further delay-dependent
errors. Unexpectedly, the strategy involved “strongly” encoding
response position information from the prior (delay-dependent) error
trial and carrying it forward to the sample phase of the next trial.
This produced a miscode type error on trials in which the “carried
over” information obliterated encoding of the sample phase response
on the next trial. Application of this strategy, irrespective of
outcome, was sufficient to reorient the animal to the proper between
trial sequence of response contingencies (nonmatch-to-sample) and boost
performance to 73% correct on subsequent trials. The capacity for
ensemble analyses of strength of information encoding combined with
statistical assessment of trial sequences therefore provided unique
insight into the “dynamic” nature of the role hippocampus plays
in delay type memory tasks.
A conserved repetitive DNA element located in the centromeres of cereal chromosomes
Authors: Jiang Jiming , Nasuda Shuhei , Dong Fenggao , Scherrer Christopher W. , Woo Sung-Sick , Wing Rod A. , Gill Bikram S. , Ward David C.
Published: July 2002

Repetitive DNA sequences have been demonstrated to play an
important role for centromere function of eukaryotic chromosomes,
including those from fission yeast,
Drosophila
melanogaster,
and humans. Here we report on the isolation of a
repetitive DNA element located in the centromeric regions of cereal
chromosomes. A 745-bp repetitive DNA clone, pSau3A9, was isolated from
sorghum (
Sorghum bicolor
). This DNA element is located
in the centromeric regions of all sorghum chromosomes, as demonstrated
by fluorescence
in situ
hybridization. Repetitive DNA
sequences homologous to pSau3A9 also are present in the centromeric
regions of chromosomes from other cereal species, including rice,
maize, wheat, barley, rye, and oats. Probe pSau3A9 also hybridized to
the centromeric region of B chromosomes from rye and maize. The
repetitive nature and its conservation in distantly related plant
species indicate that the pSau3A9 family may be associated with
centromere function of cereal chromosomes. The absence of DNA sequences
homologous to pSau3A9 in dicot species suggests a faster divergence of
centromere-related sequences compared with the telomere-related
sequences in plants.
A novel host factor for integration of mycobacteriophage L5
Authors: Pedulla Marisa L. , Lee Mong Hong , Lever Dawn C. , Hatfull Graham F.
Published: July 2002

Bacterial integration host factors (IHFs) play central roles in the
cellular processes of recombination, DNA replication, transcription,
and bacterial pathogenesis. We describe here a novel mycobacterial IHF
(mIHF) of
Mycobacterium smegmatis
and
Mycobacterium tuberculosis
that stimulates integration
of mycobacteriophage L5. mIHF is the product of a single gene and is
unrelated at the sequence level to other integration host factors. By
itself, mIHF does not bind preferentially to
attP
DNA,
although it significantly alters the pattern of integrase (Int)
binding, promoting the formation of specific
integrase–mIHF–
attP
intasome complexes.
Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.
Authors: Hughes S R , Goyal S , Sun J E , Gonzalez-DeWhitt P , Fortes M A , Riedel N G , Sahasrabudhe S R
Published: July 2002

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.
Recombinant human uteroglobin suppresses cellular invasiveness via a novel class of high-affinity cell surface binding site.
Authors: Kundu G C , Mantile G , Miele L , Cordella-Miele E , Mukherjee A B
Published: July 2002

The mechanism(s) that regulates invasion of trophoblasts through the uterine epithelium during embryo implantation and nidation in hemochorial placental mammals is poorly understood. While limited trophoblast invasion is essential for the establishment of normal pregnancy, dysregulation of this process may contribute to the pathogenesis of choriocarcinoma, a highly invasive and lethal form of cancer arising from the trophoblasts. We have previously demonstrated that rabbit uteroglobin (UG), a cytokine-like, antiinflammatory protein, produced by the endometrial epithelium during pregnancy, has a potent antichemotactic effect on neutrophils and monocytes in vitro. Here, we report that recombinant human UG (hUG) dramatically suppresses invasion of human trophoblasts and NIH 3T3 cells through an artificial basement membrane (Matrigel) in vitro but has no effect on that of human choriocarcinoma cells. We identified a previously unreported high-affinity, high molecular weight (approximately 190 kDa), nonglycosylated hUG-binding protein, readily detectable on human trophoblasts and NIH 3T3 cells but totally lacking on choriocarcinoma cells. Taken together, these results raise the possibility that (i) hUG plays a critical role in regulating cellular invasiveness, at least in part, via its previously unrecognized cell surface binding site, and (ii) some of the numerous biological activities of proteins of the UG family, reported so far, may be mediated via this binding site.
Localization of the central rhythm generator involved in spontaneous consummatory licking in rats: functional ablation and electrical brain stimulation studies.
Authors: Brozek G , Zhuravin I A , Megirian D , Bures J
Published: July 2002

Localization of the central rhythm generator (CRG) of spontaneous consummatory licking was studied in freely moving rats by microinjection of tetrodotoxin (TTX) into the pontine reticular formation. Maximum suppression of spontaneous water consumption was elicited by TTX (1 ng) blockade of the oral part of the nucleus reticularis gigantocellularis (NRG), whereas TTX injections into more caudal or rostral locations caused significantly weaker disruption of drinking. To verify the assumption that TTX blocked the proper CRG of licking rather than some relay in its output, spontaneously drinking thirsty rats were intracranially stimulated via electrodes chronically implanted into the oral part of the NRG. Lick-synchronized stimulation (a 100-ms train of 0.1-ms-wide rectangular pulses at 100 Hz and 25-150 microA) applied during continuous licking (after eight regular consecutive licks) caused a phase shift of licks emitted after stimulus delivery. The results suggest that the stimulation has reset the CRG of licking without changing its frequency. The reset-inducing threshold current was lowest during the tongue retraction and highest during the tongue protrusion period of the lick cycle. It is concluded that the CRG of licking is located in the oral part of NRG.
Characterization of mouse angiogenin-related protein: implications for functional studies on angiogenin.
Authors: Nobile V , Vallee B L , Shapiro R
Published: July 2002

Angiogenin-related protein (Angrp), the putative product of a recently discovered mouse gene, shares 78% sequence identity with mouse angiogenin (Ang). In the present study, the relationship of Angrp to Ang has been investigated by producing both proteins in bacteria and comparing their functional properties. We find that mouse Ang is potently angiogenic, but Angrp is not, even when assayed at relatively high doses. A deficiency in catalytic capacity, which is essential for the biological activity of Ang, does not appear to underlie Angrp's lack of angiogenicity. In fact, Angrp has somewhat greater ribonucleolytic activity toward tRNA and dinucleotide substrates than does Ang. Instead, an inability to bind cellular receptors is implicated since Angrp does not inhibit Ang-induced angiogenesis. Poor conservation of the Ang receptor recognition sequence 58-69 in Angrp most likely contributes to this defect. However, other substitutions must also influence receptor binding since an Angrp quadruple mutant that is identical to Ang in this segment still lacks both angiogenic activity and the capacity to inhibit Ang. The functional differences between Ang and Angrp, together with evidence presented herein that Angrp is regulated differently than Ang, suggest that the roles of the two proteins in vivo may be quite distinct.
The bi-loop, a new general four-stranded DNA motif
Authors: Salisbury Stephen A. , Wilson Sarah E. , Powell Harold R. , Kennard Olga , Lubini Paolo , Sheldrick George M. , Escaja Nuria , Alazzouzi ElMostafa , Grandas Anna , Pedroso Enrique
Published: July 2002

The crystal structure of the cyclic octanucleotide d contains two independent molecules that form a novel quadruplex by means of intermolecular Watson–Crick A⋅T pairs and base stacking. A virtually identical quadruplex composed of G⋅C pairs was found by earlier x-ray analysis of the linear heptamer d(GCATGCT), when the DNA was looped in the crystal. The close correspondence between these two structures of markedly dissimilar oligonucleotides suggests that they are both examples of a previously unrecognized motif. Their nucleotide sequences have little in common except for two separated 5′-purine-pyrimidine dinucleotides forming the quadruplex, and by implication these so-called “bi-loops” could occur widely in natural DNA. Such structures provide a mechanism for noncovalent linking of polynucleotides
in vivo
. Their capacity to associate by base stacking, demonstrated in the crystal structure of d(GCATGCT), creates a compact molecular framework made up of four DNA chains within which strand exchange could take place.
The T cell receptor repertoire of intestinal intraepithelial γδ T lymphocytes is influenced by genes linked to the major histocompatibility complex and to the T cell receptor loci
Authors: Pereira Pablo , Lafaille Juan J. , Gerber David , Tonegawa Susumu
Published: July 2002

Most of the γδ T cells in the intestinal epithelium of normal mice use the Vγ1 or the Vγ7 gene segments. However, the relative proportions of γδ intraepithelial lymphocytes expressing either the Vγ1 or the Vγ7 chain vary among different strains of mice whereas they are quite constant between different individuals of the same strain, suggesting that genetic factors, rather than environmental factors, are responsible for the observed differences. To analyze the genetic factors influencing the representation of different γδ T cell subsets in the intestinal epithelium, we used available anti-T cell antigen receptor (TCR) V region-specific mAbs against Vγ1, Vγ4, Vγ7, and Vδ4 to examine the TCR repertoire of intraepithelial γδ lymphocytes in a set of (C57BL/6 × DBA/2) recombinant inbred strains. Our results show that the representation of different Vγ and Vδ gene products among γδ intestinal intraepithelial lymphocytes is under a complex genetic control with a marked influence by genes closely linked to the TCRγ, TCRδ, and major histocompatibility complex loci.
A critical role for neutralizing-antibody-producing B cells, CD4+T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: Implications for adoptive immunotherapy of virus carriers
Authors: Planz Oliver , Ehl Stephan , Furrer Esther , Horvath Edit , Bründler Marie-Anne , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

This study demonstrates that neutralizing-antibody-producing B cells, CD4+T cells, and interferons (IFNs) are of key importance in virus control both in adoptive immunotherapy of persistent infection and in the late phase of acute infection with the WE strain of lymphocytic choriomeningitis virus (LCMV). We report the following results. (i) Clearance of LCMV-WE from C57BL/6 carrier mice by adoptive transfer of memory spleen cells requires B cells and CD4+T cells but not necessarily CD8+T cells. (ii) At the doses examined, CD8+T cells contribute to the initial reduction of viral titers but are alone not sufficient to clear the virus because they are exhausted. (iii) In the presence of functional IFN-γ, virus clearance correlates well with the generation of neutralizing antibodies in the treated carrier mice. (iv) In the absence of receptors for IFN-γ, virus clearance is not achieved. (v) Adoptive immunotherapy of mice persistently infected with a distinct virus isolate, LCMV-Armstrong, revealed only low levels of neutralizing antibodies; in this case, CD8+T cells were needed for virus clearance in addition to B and CD4+T cells. (vi) After low dose infection of C57BL/6 mice with LCMV-WE, virus is eliminated below detectable levels by CD8+T cells, but long-term (>2 months) virus control is usually not achieved in the absence of B cells or CD4+T cells; reappearance of the virus is paralleled either by exhaustion of virus-specific cytotoxic T lymphocytes or lethal immunopathology. These findings are of importance for adoptive immunotherapy strategies against persistent virus infections in humans.
The interferon-inducible murine p48 (ISGF3γ) gene is regulated by protooncogene c-
myc
Authors: Weihua Xiao , Lindner Daniel J. , Kalvakolanu Dhananjaya V.
Published: July 2002

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-γ-regulated element. In addition to the IFN-regulated element, a myc–max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor.
Distal enhancer regulation by promoter derepression in topologically constrained DNA
in vitro
Authors: Barton Michelle Craig , Madani Navid , Emerson Beverly M.
Published: July 2002

Long-range promoter–enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken β
A
-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved
in vitro
at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein–protein interactions
in vitro
may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.
The roles of the two proton input channels in cytochrome
c
oxidase from
Rhodobacter sphaeroides
probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer
Authors: Konstantinov Alexander A. , Siletsky Sergey , Mitchell David , Kaulen Andrey , Gennis Robert B.
Published: July 2002

The crystal structures of cytochrome
c
oxidase from both bovine and
Paracoccus denitrificans
reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome
c
oxidase from
Rhodobacter sphaeroides
. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a
3
(Fe
4+
= O
2−
) to the oxidized form (Fe
3+
OH
−
). This redox step requires the delivery of a “chemical” H
+
to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995)
Nature (London)
376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a
3
in the K362M and E286Q mutants under aerobic steady-state turnover conditions.
Listeria monocytogenes
infection of P388D
1
macrophages results in a biphasic NF-κB (RelA/p50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by IκBα and IκBβ degradation
Authors: Hauf Nadja , Goebel Werner , Fiedler Franz , Sokolovic Zeljka , Kuhn Michael
Published: July 2002

As previously reported,
Listeria monocytogenes
infection of P388D
1
macrophages results in a rapid induction of NF-κB DNA-binding activity. Here we show that this induction of NF-κB activity occurs in a biphasic mode: first, a transient, IκBα degradation-dependent phase of activity, also induced by the nonvirulent species
Listeria innocua
, which is mediated by binding of the bacteria to the macrophage, or by adding
Listeria
-derived lipoteichoic acid to the macrophage; the second persistent phase of activation is only markedly induced when the bacteria enter the cytoplasm of the host cell and express the virulence genes
plcA
and
plcB,
encoding two phospholipases. We suggest that products of the enzymatic activity of phospholipases directly interfere with host cell signal transduction pathways, thus leading to persistent NF-κB activation via persistent IκBβ degradation.
UV endonuclease of
Micrococcus luteus
, a cyclobutane pyrimidine dimer–DNA glycosylase/abasic lyase: Cloning and characterization of the gene
Authors: Shiota Susumu , Nakayama Hiroaki
Published: July 2002

The gene of
Micrococcus luteus
UV endonuclease (cyclobutane pyrimidine dimer–DNA glycosylase/abasic lyase) was cloned and characterized. The cloned gene, whose product had a predicted molecular mass of 17,120 Da, was found to be capable of complementing the
Escherichia coli uvrA6
mutation
in vivo
with respect to resistance to acetone-mediated molecular photosensitization, a treatment producing exclusively cyclobutane pyrimidine dimers in DNA. It also generated a nicking activity specific for photosensitization-treated DNA by
in vitro
transcription/translation. When expressed in
E. coli
cells, the gene produced a protein structurally identical with UV endonuclease and possessing an activity consistent with cyclobutane pyrimidine dimer–DNA glycosylase/abasic lyase with respect to the effect of inhibitors and the site of the DNA backbone scission. Furthermore, the UV endonuclease-deficient mutant DB7 was shown to regain the enzyme through transformation with the cloned gene. The deduced amino acid sequence of the gene product was at best 27% identical with that of endonuclease V of phage T4, an enzyme strikingly similar to UV endonuclease in molecular and catalytic properties. Despite this marginal overall similarity in amino acid sequence, four of the seven amino acid residues reported to be functionally important in the T4 enzyme were found to be conserved in the
M. luteus
enzyme. We propose that the gene be called
uveA
.