bluuebunny/crossref_metadata_2025 huggingface dataset:
Presynaptic kainate receptors at hippocampal mossy fiber synapses
Authors: Schmitz Dietmar , Mellor Jack , Frerking Matthew , Nicoll Roger A.
Published: July 2002

Hippocampal mossy fibers, which are the axons of dentate
granule cells, form powerful excitatory synapses onto the proximal
dendrites of CA3 pyramidal cells. It has long been known that
high-affinity binding sites for kainate, a glutamate receptor agonist,
are present on mossy fibers. Here we summarize recent experiments on
the role of these presynaptic kainate receptors (KARs). Application of
kainate has a direct effect on the amplitude of the extracellularly
recorded fiber volley, with an enhancement by low concentrations and a
depression by high concentrations. These effects are mediated by KARs,
because they persist in the presence of the
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor-selective antagonist GYKI 53655, but are blocked by the
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/KAR antagonist
6-cyano-7-nitroquinoxaline-2,3-dione and the KAR antagonist SYM2081.
The effects on the fiber volley are most likely caused by a
depolarization of the fibers via the known ionotropic actions of
KARs, because application of potassium mimics the effects. In addition
to these effects on fiber excitability, low concentrations of kainate
enhance transmitter release, whereas high concentrations depress
transmitter release. Importantly, the synaptic release of glutamate
from mossy fibers also activates these presynaptic KARs, causing an
enhancement of the fiber volley and a facilitation of release that
lasts for many seconds. This positive feedback contributes to the
dramatic frequency facilitation that is characteristic of mossy fiber
synapses. It will be interesting to determine how widespread
facilitatory presynaptic KARs are at other synapses in the central
nervous system.
Calcium regulation of neuronal gene expression
Authors: West Anne E. , Chen Wen G. , Dalva Matthew B. , Dolmetsch Ricardo E. , Kornhauser Jon M. , Shaywitz Adam J. , Takasu Mari A. , Tao Xu , Greenberg Michael E.
Published: July 2002

Plasticity is a remarkable feature of the brain, allowing neuronal
structure and function to accommodate to patterns of electrical
activity. One component of these long-term changes is the
activity-driven induction of new gene expression, which is required for
both the long-lasting long-term potentiation of synaptic transmission
associated with learning and memory, and the activitydependent
survival events that help to shape and wire the brain during
development. We have characterized molecular mechanisms by which
neuronal membrane depolarization and subsequent calcium influx into the
cytoplasm lead to the induction of new gene transcription. We have
identified three points within this cascade of events where the
specificity of genes induced by different types of stimuli can be
regulated. By using the induction of the gene that encodes
brain-derived neurotrophic factor (BDNF) as a model, we have found that
the ability of a calcium influx to induce transcription of this gene is
regulated by the route of calcium entry into the cell, by the pattern
of phosphorylation induced on the transcription factor cAMP-response
element (CRE) binding protein (CREB), and by the complement of active
transcription factors recruited to the
BDNF
promoter.
These results refine and expand the working model of activity-induced
gene induction in the brain, and help to explain how different types of
neuronal stimuli can activate distinct transcriptional responses.
Chemosensitivity prediction by transcriptional profiling
Authors: Staunton Jane E. , Slonim Donna K. , Coller Hilary A. , Tamayo Pablo , Angelo Michael J. , Park Johnny , Scherf Uwe , Lee Jae K. , Reinhold William O. , Weinstein John N. , Mesirov Jill P. , Lander Eric S. , Golub Todd R.
Published: July 2002

In an effort to develop a genomics-based approach to the prediction of drug response, we have developed an algorithm for classification of cell line chemosensitivity based on gene expression profiles alone. Using oligonucleotide microarrays, the expression levels of 6,817 genes were measured in a panel of 60 human cancer cell lines (the NCI-60) for which the chemosensitivity profiles of thousands of chemical compounds have been determined. We sought to determine whether the gene expression signatures of untreated cells were sufficient for the prediction of chemosensitivity. Gene expression-based classifiers of sensitivity or resistance for 232 compounds were generated and then evaluated on independent sets of data. The classifiers were designed to be independent of the cells' tissue of origin. The accuracy of chemosensitivity prediction was considerably better than would be expected by chance. Eighty-eight of 232 expression-based classifiers performed accurately (with
P
< 0.05) on an independent test set, whereas only 12 of the 232 would be expected to do so by chance. These results suggest that at least for a subset of compounds genomic approaches to chemosensitivity prediction are feasible.
Divergent effect of bacillus Calmette–Guérin (BCG) vaccination on
Mycobacterium tuberculosis
infection in highly related macaque species: Implications for primate models in tuberculosis vaccine research
Authors: Langermans Jan A. M. , Andersen Peter , van Soolingen Dick , Vervenne Richard A. W. , Frost Patrice A. , van der Laan Tridia , van Pinxteren Laurens A. H. , van den Hombergh Jan , Kroon Saskia , Peekel Inge , Florquin Sandrine , Thomas Alan W.
Published: July 2002

Despite the widespread use of bacillus Calmette–Guérin
vaccination,
Mycobacterium tuberculosis
infection
remains globally the leading cause of death from a single infectious
disease. The complicated and often protracted dynamics of infection and
disease make clinical trials to test new tuberculosis vaccines
extremely complex. Preclinical selection of only the most promising
candidates is therefore essential. Because macaque monkeys develop a
disease very similar to humans, they have potential to provide
important information in addition to small animal models. To assess the
relative merits of rhesus and cynomolgus monkeys as screens for
tuberculosis vaccines, we compared the efficacy of bacillus
Calmette–Guérin vaccination and the course of infection in both
species. Unvaccinated rhesus and cynomolgus monkeys both developed
progressive disease with high levels of C-reactive protein,
M.
tuberculosis
-specific IgG, and extensive pathology including
cavitation and caseous necrosis. Bacillus Calmette–Guérin
vaccination protected cynomolgus almost completely toward the
development of pathology, reflected in a striking 2-log reduction in
viable bacteria in the lungs compared with nonvaccinated animals.
Rhesus, on the other hand, were not protected efficiently by the
bacillus Calmette–Guérin. The vaccinated animals developed
substantial pathology and had negligible reductions of colony-forming
units in the lungs. Comparative studies in these closely related
species are likely to provide insight into mechanisms involved in
protection against tuberculosis.
Monarch larvae sensitivity to
Bacillus thuringiensis
- purified proteins and pollen
Authors: Hellmich Richard L. , Siegfried Blair D. , Sears Mark K. , Stanley-Horn Diane E. , Daniels Michael J. , Mattila Heather R. , Spencer Terrence , Bidne Keith G. , Lewis Leslie C.
Published: July 2002

Laboratory tests were conducted to establish the relative toxicity of
Bacillus thuringiensis
(
Bt
) toxins and pollen from
Bt
corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (
i
) purified toxins incorporated into artificial diet, (
ii
) pollen collected from
Bt
corn hybrids applied directly to milkweed leaf discs, and (
iii
)
Bt
pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified
Bt
toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of
Bt
corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings.
β-Amyloid peptides enhance α-synuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer's disease and Parkinson's disease
Authors: Masliah Eliezer , Rockenstein Edward , Veinbergs Isaac , Sagara Yutaka , Mallory Margaret , Hashimoto Makoto , Mucke Lennart
Published: July 2002

Alzheimer's disease and Parkinson's disease are associated with the cerebral accumulation of β-amyloid and α-synuclein, respectively. Some patients have clinical and pathological features of both diseases, raising the possibility of overlapping pathogenetic pathways. We generated transgenic (tg) mice with neuronal expression of human β-amyloid peptides, α-synuclein, or both. The functional and morphological alterations in doubly tg mice resembled the Lewy-body variant of Alzheimer's disease. These mice had severe deficits in learning and memory, developed motor deficits before α-synuclein singly tg mice, and showed prominent age-dependent degeneration of cholinergic neurons and presynaptic terminals. They also had more α-synuclein-immunoreactive neuronal inclusions than α-synuclein singly tg mice. Ultrastructurally, some of these inclusions were fibrillar in doubly tg mice, whereas all inclusions were amorphous in α-synuclein singly tg mice. β-Amyloid peptides promoted aggregation of α-synuclein in a cell-free system and intraneuronal accumulation of α-synuclein in cell culture. β-Amyloid peptides may contribute to the development of Lewy-body diseases by promoting the aggregation of α-synuclein and exacerbating α-synuclein-dependent neuronal pathologies. Therefore, treatments that block the production or accumulation of β-amyloid peptides could benefit a broader spectrum of disorders than previously anticipated.
Suppressor of cytokine signaling 3 (SOCS-3) protects β-cells against interleukin-1β- and interferon-γ-mediated toxicity
Authors: Karlsen Allan E. , Rønn Sif G. , Lindberg Karen , Johannesen Jesper , Galsgaard Elisabeth D. , Pociot Flemming , Nielsen Jens H. , Mandrup-Poulsen Thomas , Nerup Jørn , Billestrup Nils
Published: July 2002

Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-γ signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IFN-γ. IL-1β and IFN-γ alone, or potentiated by TNF-α, are cytotoxic to the insulin producing pancreatic β-cells and β-cell lines
in vitro
and suggested to contribute to the specific β-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat β-cell line INS-1, we demonstrate that the toxic effect of both IL-1β or IFN-γ at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-γ signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1β signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1β-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1β revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of β-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.
Role of the C-terminal domain in inactivation of brain and cardiac sodium channels
Authors: Mantegazza Massimo , Yu Frank H. , Catterall William A. , Scheuer Todd
Published: July 2002

Inactivation is a fundamental characteristic of Na
+
channels, and small changes cause skeletal muscle paralysis and
myotonia, epilepsy, and cardiac arrhythmia. Brain
Na
v
1.2a channels have faster inactivation than cardiac
Na
v
1.5 channels, but minor differences in inactivation gate
structure are not responsible. We constructed chimeras in which the C
termini beyond the fourth homologous domains of Na
v
1.2a and
Na
v
1.5 were exchanged. Replacing the C-terminal domain (CT)
of Na
v
1.2a with that of Na
v
1.5
(Na
v
1.2/1.5CT) slowed inactivation at +40 mV ≈2-fold,
making it similar to Na
v
1.5. Conversely, replacing the CT
of Na
v
1.5 with that of Na
v
1.2a (Nav1.5/1.2CT)
accelerated inactivation, making it similar to Na
v
1.2a.
Activation properties were unaffected. The voltage dependence of
steady-state inactivation of Na
v
1.5 is 16 mV more negative
than that of Na
v
1.2a. The steady-state inactivation curve
of Na
v
1.2a was shifted +12 mV in
Na
v
1.2/1.5CT, consistent with destabilization of the
inactivated state. Conversely, Na
v
1.5/1.2CT was shifted
−14 mV relative to Na
v
1.5, consistent with stabilization
of the inactivated state. Although these effects of exchanging C
termini were consistent with their effects on inactivation kinetics,
they magnified the differences in the voltage dependence of
inactivation between brain and cardiac channels rather than
transferring them. Thus, other parts of these channels determine the
basal difference in steady-state inactivation. Deletion of the distal
half of either the Na
v
1.2 or Na
v
1.5 CTs
accelerated open-state inactivation and negatively shifted steady-state
inactivation. Thus, the C terminus has a strong influence on kinetics
and voltage dependence of inactivation in brain
Na
v
1.2 and cardiac Na
v
1.5 channels and is
primarily responsible for their differing rates of channel
inactivation.
Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering
Authors: Zhang Jin , Ma Yuliang , Taylor Susan S. , Tsien Roger Y.
Published: July 2002

The complexity and specificity of many forms of signal
transduction are widely suspected to require spatial
microcompartmentation of protein kinase and phosphatase activities, yet
current relevant imaging methods such as phosphorylation-specific
antibodies or fluorescent peptide substrates require fixation or
microinjection and lack temporal or spatial resolution. We
present a genetically encoded fluorescent reporter for protein kinase A
(PKA) consisting of fusions of cyan fluorescent protein, a phosphoamino
acid binding domain (14–3-3τ), a consensus substrate for PKA, and
yellow fluorescent protein. cAMP elevations cause 25–50% changes in
the ratios of yellow to cyan emissions in live cells caused by
phosphorylation-induced changes in fluorescence resonance energy
transfer. The reporter response was accelerated by tethering to PKA
holoenzyme and slowed by localization to the nucleus. We
demonstrate that deliberate redistribution of a substrate or
colocalizing a substrate and PKA can modulate its susceptibility to
phosphorylation by the kinase. The successful design of a fluorescent
reporter of PKA activity and its application for studying
compartmentalized and dynamic modulation of kinases lays a foundation
for studying targeting and compartmentation of PKA and other kinases
and phosphatases.
Auditory representation of the vocal repertoire in a songbird with multiple song types
Authors: Mooney Richard , Hoese William , Nowicki Stephen
Published: July 2002

Neural mechanisms for representing complex communication sounds must solve the problem of encoding multiple and potentially overlapping signals. Birdsong provides an excellent model for such processing, in that many songbird species produce multiple song types. Although auditory song representations in single song type species have been studied, how song is represented in the brains of species that sing multiple song types remains unknown. Here we examine song type representations in swamp sparrows (
Melospiza georgiana
), a multiple song type species, by making
in vivo
intracellular recordings from the telencephalic nucleus HVc, the major auditory-vocal interface in the songbird brain. These recordings show that single HVc relay neurons often generate action potentials to playback of only a single song type, even though synaptic inputs on these cells can be activated by playback of other song types in the bird's repertoire and songs of other birds. These subthreshold response patterns suggest that the song evoked action potential discharge of a single relay neuron is more selective than its presynaptic network. One component of this presynaptic network is likely to be in HVc, because multiple recordings from single birds show that different relay neurons can respond best to different song types, whereas single interneurons can generate action potentials to all song types in the bird's repertoire. These results show that single HVc neurons can generate song type-specific action potential responses, a feature that may facilitate the selective auditory encoding of multiple learned vocalizations in a single brain area.
Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes
Authors: Lange B. Markus , Rujan Tamas , Martin William , Croteau Rodney
Published: July 2002

Isopentenyl diphosphate (IPP) is the central intermediate in the
biosynthesis of isoprenoids, the most ancient and diverse class of
natural products. Two distinct routes of IPP biosynthesis occur in
nature: the mevalonate pathway and the recently discovered
deoxyxylulose 5-phosphate (DXP) pathway. The evolutionary history of
the enzymes involved in both routes and the phylogenetic distribution
of their genes across genomes suggest that the mevalonate pathway is
germane to archaebacteria, that the DXP pathway is germane to
eubacteria, and that eukaryotes have inherited their genes for IPP
biosynthesis from prokaryotes. The occurrence of genes specific to the
DXP pathway is restricted to plastid-bearing eukaryotes, indicating
that these genes were acquired from the cyanobacterial ancestor of
plastids. However, the individual phylogenies of these genes, with only
one exception, do not provide evidence for a specific affinity between
the plant genes and their cyanobacterial homologues. The results
suggest that lateral gene transfer between eubacteria subsequent to the
origin of plastids has played a major role in the evolution of this
pathway.
Evaluation of methods for detecting recombination from DNA sequences: Computer simulations
Authors: Posada David , Crandall Keith A.
Published: July 2002

Recombination is a key evolutionary process that shapes the architecture of genomes and the genetic structure of populations. Although many statistical methods are available for the detection of recombination from DNA sequences, their absolute and relative performance is still unknown. Here we evaluated the performance of 14 different recombination detection algorithms. We used the coalescent with recombination to simulate DNA sequences with different levels of recombination, genetic diversity, and rate variation among sites. Recombination detection methods were applied to these data sets, and whether they detected or not recombination was recorded. Different recombination methods showed distinct performance depending on the amount of recombination, genetic diversity, and rate variation among sites. The model of nucleotide substitution under which the data were generated did not seem to have a significant effect. Most methods increase power with more sequence divergence. In general, recombination detection methods seem to capture the presence of recombination, but they are not very powerful. Methods that use substitution patterns or incompatibility among sites were more powerful than methods based on phylogenetic incongruence. Most methods do not seem to infer more false positives than expected by chance. Especially depending on the amount of diversity in the data, different methods could be used to attain maximum power while minimizing false positives. Results shown here will provide some guidance in the selection of the most appropriate method/s for the analysis of the particular data at hand.
Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2receptor for vasoactive intestinal peptide
Authors: Goetzl Edward J. , Voice Julia K. , Shen Sanbing , Dorsam Glenn , Kong Yvonne , West Katrine M. , Morrison Christine F. , Harmar Anthony J.
Published: July 2002

Vasoactive intestinal peptide (VIP) and its G protein-coupled receptors, VPAC1R and VPAC2R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC1R is expressed constitutively, whereas VPAC2R is induced by immune stimuli. VPAC2R-null (VPAC2R−/−) mice on a C57BL/6 background are shown here to have normal basic immune characteristics, including serum Ig concentrations, blood levels of all leukocytes, and spleen number of total T cells (CD3+) and T cells bearing CD4, CD8, and CD28. Hapten-evoked cutaneous delayed-type hypersensitivity (DTH) was significantly enhanced in VPAC2R-null mice compared with age- and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were ≥70% lower in VPAC2R-null mice than in wild-type controls. Cytokine production by splenic CD4+T cells, stimulated with adherent anti-CD3 plus anti-CD28 antibodies, revealed higher levels of IL-2 (mean = 3-fold) and IFN-γ (mean = 3-fold), and lower levels of IL-4 (mean = one-fifth) in VPAC2R-null mice than wild-type controls. Loss of VIP-VPAC2R maintenance of the normal ratio of Th2/Th1 cytokines thus leads to a state of enhanced DTH and depressed immediate-type hypersensitivity, which may alter both host defense and susceptibility to immune-mediated diseases.
RelE, a global inhibitor of translation, is activated during nutritional stress
Authors: Christensen Susanne K. , Mikkelsen Marie , Pedersen Kim , Gerdes Kenn
Published: July 2002

The stringent response is defined as the physiological changes
elicited by amino acid starvation. Many of these changes depend
on the regulatory nucleotide ppGpp (guanosine tetraphosphate)
synthesized by RelA (ppGpp synthetase I), the
relA
-encoded protein. The second
rel
locus of
Escherichia coli
is called
relBE
and encodes RelE cytotoxin and RelB antitoxin. RelB counteracts the
toxic effect of RelE. In addition, RelB is an autorepressor of
relBE
transcription. Here we reveal a ppGpp-independent
mechanism that reduces the level of translation during amino acid
starvation. Artificial overexpression of RelE severely inhibited
translation. During amino acid starvation, the presence of
relBE
caused a significant reduction in the
poststarvation level of translation. Concomitantly,
relBE
transcription was rapidly and strongly induced.
Induction of transcription occurred independently of
relA
and
spoT
(encoding ppGpp synthetase
II), but instead depended on Lon protease. Consistently, Lon was
required for degradation of RelB. Replacement of the
relBE
promoter with a LacI-regulated promoter indicated
that strong and ongoing transcription of
relBE
is
required to maintain a proper RelB:RelE ratio during starvation. Thus
relBE
may be regarded as a previously
uncharacterized type of stress-response element that reduces the
global level of translation during nutritional stress.
NMR structure reveals intramolecular regulation mechanism for pheromone binding and release
Authors: Horst Reto , Damberger Fred , Luginbühl Peter , Güntert Peter , Peng Guihong , Nikonova Larisa , Leal Walter S. , Wüthrich Kurt
Published: July 2002

Odorants are transmitted by small hydrophobic molecules that cross
the aqueous sensillar lymph surrounding the dendrites of the olfactory
neurons to stimulate the olfactory receptors. In insects, the
transport of pheromones, which are a special class of odorants, is
mediated by pheromone-binding proteins (PBPs), which occur at high
concentrations in the sensillar lymph. The PBP from the silk moth
Bombyx mori
(BmPBP) undergoes a pH-dependent
conformational transition between the forms BmPBP
A
present
at pH 4.5 and BmPBP
B
present at pH 6.5. Here, we describe
the NMR structure of BmPBP
A
, which consists of a tightly
packed arrangement of seven α-helices linked by well defined peptide
segments and knitted together by three disulfide bridges. A scaffold of
four α-helices that forms the ligand binding site in the crystal
structure of a BmPBP–pheromone complex is preserved in
BmPBP
A
. The C-terminal dodecapeptide segment, which is in
an extended conformation and located on the protein surface in the
pheromone complex, forms a regular helix, α
7
, which is
located in the pheromone-binding site in the core of the unliganded
BmPBP
A
. Because investigations by others indicate that the
pH value near the membrane surface is reduced with respect to the bulk
sensillar lymph, the pH-dependent conformational transition of BmPBP
suggests a novel physiological mechanism of intramolecular regulation
of protein function, with the formation of α
7
triggering
the release of the pheromone from BmPBP to the membrane-standing
receptor.
Chlorophyllin intervention reduces aflatoxin–DNA adducts in individuals at high risk for liver cancer
Authors: Egner Patricia A. , Wang Jin-Bing , Zhu Yuan-Rong , Zhang Bao-Chu , Wu Yan , Zhang Qi-Nan , Qian Geng-Sun , Kuang Shuang-Yuan , Gange Stephen J. , Jacobson Lisa P. , Helzlsouer Kathy J. , Bailey George S. , Groopman John D. , Kensler Thomas W.
Published: July 2002

Residents of Qidong, People's Republic of China, are at high
risk for development of hepatocellular carcinoma, in part from
consumption of foods contaminated with aflatoxins. Chlorophyllin, a
mixture of semisynthetic, water-soluble derivatives of chlorophyll that
is used as a food colorant and over-the-counter medicine, has been
shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in
animal models by blocking carcinogen bioavailability. In a randomized,
double-blind, placebo-controlled chemoprevention trial, we tested
whether chlorophyllin could alter the disposition of aflatoxin. One
hundred and eighty healthy adults from Qidong were randomly assigned to
ingest 100 mg of chlorophyllin or a placebo three times a day for 4
months. The primary endpoint was modulation of levels of
aflatoxin-N
7
-guanine adducts in urine samples collected 3
months into the intervention measured by using sequential
immunoaffinity chromatography and liquid chromatography–electrospray
mass spectrometry. This aflatoxin–DNA adduct excretion product serves
as a biomarker of the biologically effective dose of aflatoxin, and
elevated levels are associated with increased risk of liver cancer.
Adherence to the study protocol was outstanding, and no adverse events
were reported. Aflatoxin-N
7
-guanine could be detected in
105 of 169 available samples. Chlorophyllin consumption at each meal
led to an overall 55% reduction (
P
= 0.036) in
median urinary levels of this aflatoxin biomarker compared with those
taking placebo. Thus, prophylactic interventions with chlorophyllin or
supplementation of diets with foods rich in chlorophylls may represent
practical means to prevent the development of hepatocellular carcinoma
or other environmentally induced cancers.
Structural basis of transcription: α-Amanitin–RNA polymerase II cocrystal at 2.8 Å resolution
Authors: Bushnell David A. , Cramer Patrick , Kornberg Roger D.
Published: July 2002

The structure of RNA polymerase II in a complex with the inhibitor α-amanitin has been determined by x-ray crystallography. The structure of the complex indicates the likely basis of inhibition and gives unexpected insight into the transcription mechanism.
Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.
Authors: Tiruppathi C , Finnegan A , Malik A B
Published: July 2002

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process involving transcytosis. In the present study, we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells. The endothelial cell membranes were isolated from cultured cells by differential centrifugation and solubilized with sodium cholate and urea. The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.
Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.
Authors: Xu J , Lapham J , Crothers D M
Published: July 2002

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.
A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture.
Authors: López-García J C , Arancio O , Kandel E R , Baranes D
Published: July 2002

The complex circuitry of the CA3 region and the abundance of collateral connections has made it difficult to study the mossy fiber pathway in hippocampal slices and therefore to establish the site of expression of long-term potentiation at these synapses. Using a novel cell culture system, we have produced long-term potentiation of the elementary synaptic connections on single CA3 pyramidal neurons following tetanic stimulation of individual dentate gyrus granule cells. As is the case for the hippocampal slice, this potentiation was independent of N-methyl-D-aspartate receptor activation, was simulated by application of forskolin, and its induction did not require any modulatory input. The increase in synaptic strength was accompanied by a reduction in the number of failures of transmission and by an increase in the coefficient of variation of the responses and was prevented by presynaptic injection of an inhibitor of protein kinase A. These findings show that mossy fiber long-term potentiation has a presynaptic locus and that its expression is dependent on protein kinase A.
Selective expression of m2 muscarinic receptor in the parvocellular channel of the primate visual cortex.
Authors: Mrzljak L , Levey A I , Rakic P
Published: July 2002

Visual information in primates is relayed from the dorsal lateral geniculate nucleus to the cerebral cortex by three parallel neuronal channels designated the parvocellular, magnocellular, and interlaminar pathways. Here we report that m2 muscarinic acetylcholine receptor in the macaque monkey visual cortex is selectively associated with synaptic circuits subserving the function of only one of these channels. The m2 receptor protein is enriched both in layer IV axons originating from parvocellular layers of the dorsal lateral geniculate nucleus and in cytochrome oxidase poor interblob compartments in layers II and III, which are linked with the parvocellular pathway. In these compartments, m2 receptors appear to be heteroreceptors, i.e., they are associated predominantly with asymmetric, noncholinergic synapses, suggesting a selective role in the modulation of excitatory neurotransmission through the parvocellular visual channel.
Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.
Authors: Xu W , Ellington A D
Published: July 2002

In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of human immunodeficiency virus type I Rev, were selected from a random sequence RNA pool. Several of the selected RNAs could bind the free peptide more tightly than a natural RNA ligand, the Rev-binding element. In accord with the hypothesis that protein and nucleic acid binding cusps are functionally similar, interactions between aptamers and the peptide target could be disrupted by sequence substitutions. Moreover, the aptamers appeared to be able to bind peptides with different solution conformations, implying an induced fit mechanism for binding. Just as anti-peptide antibodies can sometimes recognize the corresponding epitope when presented in a protein, the anti-peptide aptamers were found to specifically bind to Rev.
Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD8+ cell-mediated immunity against tumors located in the "immunologically privileged" central nervous system.
Authors: Sampson J H , Archer G E , Ashley D M , Fuchs H E , Hale L P , Dranoff G , Bigner D D
Published: July 2002

Vaccination with cytokine-producing tumor cells generates potent immune responses against tumors outside the central nervous system (CNS). The CNS, however, is a barrier to allograft and xenograft rejection, and established tumors within the CNS have failed to respond to other forms of systemic immunotherapy. To determine what barriers the "immunologically privileged" CNS would pose to cytokine-assisted tumor vaccines and what cytokines would be most efficacious against tumors within the CNS, we irradiated B16 murine melanoma cells producing murine interleukin 2 (IL-2), IL-3, IL-4, IL-6, gamma-interferon, or granulocyte-macrophage colony stimulating factor (GM-CSF) and used these cells as subcutaneous vaccines against tumors within the brain. Under conditions where untransfected B16 cells had no effect, cells producing IL-3, IL-6, or GM-CSF increased the survival of mice challenged with viable B16 cells in the brain. Vaccination with B16 cells producing IL-4 or gamma-interferon had no effect, and vaccination with B16 cells producing IL-2 decreased survival time. GM-CSF-producing vaccines were also able to increase survival in mice with pre-established tumors. The response elicited by GM-CSF-producing vaccines was found to be specific to tumor type and to be abrogated by depletion of CD8+ cells. Unlike the immunity generated against subcutaneous tumors by GM-CSF, however, the effector responses generated against tumors in the CNS were not dependent on CD4+ cells. These data suggest that cytokine-producing tumor cells are very potent stimulators of immunity against tumors within the CNS, but effector responses in the CNS may be different from those obtained against subcutaneous tumors.
Potassium homeostasis in vacuolate plant cells.
Authors: Walker D J , Leigh R A , Miller A J
Published: July 2002

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understanding the way this nutrient affects plant growth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (aK) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measurements revealed that vacuolar aK declined linearly with decreases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data have been used in thermodynamic calculations to predict the need for, and likely mechanisms of, active K+ transport into the vacuole and cytosol. The direction of active K+ transport at the vacuolar membrane changes with tissue K+ status.
Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells.
Authors: Sheng S , Carey J , Seftor E A , Dias L , Hendrix M J , Sager R
Published: July 2002

Maspin, a novel serine protease inhibitor (serpin), inhibits tumor invasion and metastasis of mammary carcinoma. We show here that recombinant maspin protein blocks the motility of these carcinoma cells in culture over 12 h, as demonstrated by time-lapse video microscopy. Lamellopodia are withdrawn but ruffling continues. Both exogenous recombinant maspin and maspin expressed by tumor transfectants exhibit inhibitory effects on cell motility and cell invasion as shown in modified Boyden chamber assays. In addition, three prostatic cancer cell lines treated with recombinant maspin exhibited similar inhibition of both invasion and motility, suggesting a similar mode of maspin action in these two glandular epithelial cancers. When mammary carcinoma cells were treated with recombinant maspin, the protein was shown by immunostaining to bind specifically to the cell surface, suggesting that maspin activity is membrane associated. When pretreated with antimaspin antibody, maspin loses its inhibitory effects on both invasion and motility. However, when maspin is added to these cells preceding antibody treatment, the activity of maspin is no longer inhibited by subsequent addition of the antibody. It is concluded therefore that the inhibition of invasion and motility by maspin is initially localized to the cell surface.
Heteromultimeric CLC chloride channels with novel properties
Authors: Lorenz Claudius , Pusch Michael , Jentsch Thomas J.
Published: July 2002

The skeletal muscle chloride channel CLC-1 and the ubiquitous
volume-activated chloride channel CLC-2 belong to a large gene family
whose members often show overlapping expression patterns. CLC-1 and
CLC-2 are coexpressed in skeletal and smooth muscle and in the heart.
By coexpressing CLC-1 and CLC-2 in
Xenopus
oocytes, we
now show the formation of novel CLC-1/CLC-2 heterooligomers that
yield time-independent linear chloride currents with a chloride →
bromide → iodide selectivity sequence. Formation of heterooligomeric
CLC channels increases the number and possible functions of chloride
channels.
Relationships of dehydroepiandrosterone sulfate in the elderly with functional, psychological, and mental status, and short-term mortality: A French community-based study
Authors: Berr Claudine , Lafont Sylviane , Debuire Brigitte , Dartigues Jean-François , Baulieu Etienne-Emile
Published: July 2002

In human beings of both sexes, dehydroepiandrosterone sulfate
(DHEAS) circulating in blood is mostly an adrenally secreted steroid
whose serum concentration (in the micromolar range and 30–50% higher
in men than in women) decreases with age, toward ≈20–10% of its
value in young adults during the 8th and 9th decades. The mechanism of
action of DHEA and DHEAS is poorly known and may include partial
transformation into sex steroids, increase of bioavailable insulin-like
growth factor I, and effects on neurotransmitter receptors. Whether
there is a cause-to-effect relationship between the decreasing levels
of DHEAS with age and physiological and pathological manifestations of
aging is still undecided, but this is of obvious theoretical and
practical interest in view of the easy restoration by DHEA
administration. Here we report on 622 subjects over 65 years of age,
studied for the 4 years since DHEAS baseline values had been obtained,
in the frame of the PAQUID program, analyzing the functional,
psychological, and mental status of a community-based population in the
south-west of France. We confirm the continuing decrease of DHEAS serum
concentration with age, more in men than in women, even if men retain
higher levels. Significantly lower values of baseline DHEAS were
recorded in women in cases of functional limitation (Instrumental
Activities of Daily Living), confinement, dyspnea, depressive
symptomatology, poor subjective perception of health and life
satisfaction, and usage of various medications. In men, there was a
trend for the same correlations, even though not statistically
significant in most categories. No differences in DHEAS levels were
found in cases of incident dementia in the following 4 years. In men
(but not in women), lower DHEAS was significantly associated with
increased short-term mortality at 2 and 4 years after baseline
measurement. These results, statistically established by taking into
account corrections for age, sex, and health indicators, suggest the
need for further careful trials of the administration of replacement
doses of DHEA in aging humans. Indeed, the first noted results of such
“treatment” are consistent with correlations observed here
between functional and psychological status and endogenous
steroid serum concentrations.
Involvement of the amygdala in memory storage: Interaction with other brain systems
Authors: McGaugh James L. , Cahill Larry , Roozendaal Benno
Published: July 2002

There is extensive evidence that the amygdala is involved in
affectively influenced memory. The central hypothesis guiding the
research reviewed in this paper is that emotional arousal activates the
amygdala and that such activation results in the modulation of memory
storage occurring in other brain regions. Several lines of evidence
support this view. First, the effects of stress-related hormones
(epinephrine and glucocorticoids) are mediated by influences involving
the amygdala. In rats, lesions of the amygdala and the stria terminalis
block the effects of posttraining administration of epinephrine and
glucocorticoids on memory. Furthermore, memory is enhanced by
posttraining intra-amygdala infusions of drugs that activate
β-adrenergic and glucocorticoid receptors. Additionally, infusion of
β-adrenergic blockers into the amygdala blocks the memory-modulating
effects of epinephrine and glucocorticoids, as well as those of drugs
affecting opiate and GABAergic systems. Second, an intact amygdala is
not required for expression of retention. Inactivation of the amygdala
prior to retention testing (by posttraining lesions or drug infusions)
does not block retention performance. Third, findings of studies using
human subjects are consistent with those of animal experiments.
β-Blockers and amygdala lesions attenuate the effects of
emotional arousal on memory. Additionally, 3-week recall of emotional
material is highly correlated with positron-emission tomography
activation (cerebral glucose metabolism) of the right amygdala during
encoding. These findings provide strong evidence supporting the
hypothesis that the amygdala is involved in modulating long-term memory
storage.
Presence of a mitochondrial-type 70-kDa heat shock protein inTrichomonas vaginalissuggests a very early mitochondrial endosymbiosis in eukaryotes
Authors: Germot Agnès , Philippe Hervé , Le Guyader Hervé
Published: July 2002

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryoteEntamoeba histolytica. We have now isolated fromTrichomonas vaginalisa gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, theT. vaginalisprotein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.