bluuebunny/crossref_metadata_2025 huggingface dataset:
The pair of bacteriophytochromes from
Agrobacterium tumefaciens
are histidine kinases with opposing photobiological properties
Authors: Karniol Baruch , Vierstra Richard D.
Published: March 2003

Bacteriophytochrome photoreceptors (BphPs) are a family of phytochrome-like sensor kinases that help a wide variety of bacteria respond to their light environment. In
Agrobacterium tumefaciens
, a unique pair of BphPs with potentially opposing roles in light sensing are present. Both
At
BphPs contain an N-terminal chromophore-binding domain that covalently attaches a biliverdin chromophore. Whereas
At
BphP1 assumes a Pr ground state,
At
BphP2 is unusual in that it assumes a Pfr ground state that is produced nonphotochemically after biliverdin binding through a transient Pr-like intermediate. Photoconversion of
At
BphP2 with far-red light then generates Pr but this Pr is also unstable and rapidly reverts nonphotochemically to Pfr.
At
BphP1 contains a typical two-component histidine kinase domain at its C terminus whose activity is repressed after photoconversion to Pfr.
At
BphP2 also functions as a histidine kinase but instead uses a distinct two-component kinase motif that is repressed after photoconversion to Pr. We identified sequences related to this domain in numerous predicted sensing proteins in
A. tumefaciens
and other bacteria, indicating that
At
BphP2 might represent the founding member of a family of histidine phosphorelay proteins that is widely used in environmental signaling. By using these mutually opposing BphPs,
A. tumefaciens
presumably has the capacity to simultaneously sense red light-rich and far-red light-rich environments through deactivation of their associated kinase cascades.
Gravity-regulated differential auxin transport from columella to lateral root cap cells
Authors: Ottenschläger Iris , Wolff Patricia , Wolverton Chris , Bhalerao Rishikesh P. , Sandberg Göran , Ishikawa Hideo , Evans Mike , Palme Klaus
Published: March 2003

Gravity-induced root curvature has long been considered to be regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients, and the transport mechanisms involved, remain to be identified. Here, we describe a GFP-based auxin biosensor to monitor auxin during
Arabidopsis
root gravitropism at cellular resolution. We identify elevated auxin levels at the root apex in columella cells, the site of gravity perception, and an asymmetric auxin flux from these cells to the lateral root cap (LRC) and toward the elongation zone after gravistimulation. We differentiate between an efflux-dependent lateral auxin transport from columella to LRC cells, and an efflux- and influx-dependent basipetal transport from the LRC to the elongation zone. We further demonstrate that endogenous gravitropic auxin gradients develop even in the presence of an exogenous source of auxin. Live-cell auxin imaging provides unprecedented insights into gravity-regulated auxin flux at cellular resolution, and strongly suggests that this flux is a prerequisite for root gravitropism.
IL-1 is required for tumor invasiveness and angiogenesis
Authors: Voronov Elena , Shouval Dror S. , Krelin Yakov , Cagnano Emanuela , Benharroch Daniel , Iwakura Yoichiro , Dinarello Charles A. , Apte Ron N.
Published: March 2003

Here, we describe that microenvironmental IL-1β and, to a lesser extent, IL-1α are required forin vivoangiogenesis and invasiveness of different tumor cells. In IL-1β knockout (KO) mice, local tumor or lung metastases of B16 melanoma cells were not observed compared with WT mice. Angiogenesis was assessed by the recruitment of blood vessel networks into Matrigel plugs containing B16 melanoma cells; vascularization of the plugs was present in WT mice, but was absent in IL-1β KO mice. The addition of exogenous IL-1 into B16-containing Matrigel plugs in IL-1β KO mice partially restored the angiogenic response. Moreover, the incorporation of IL-1 receptor antagonist to B16-containing plugs in WT mice inhibited the ingrowth of blood vessel networks into Matrigel plugs. In IL-1α KO mice, local tumor development and induction of an angiogenic response in Matrigel plugs was less pronounced than in WT mice, but significantly higher than in IL-1β KO mice. These effects of host-derived IL-1α and IL-1β were not restricted to the melanoma model, but were also observed in DA/3 mammary and prostate cancer cell models. In addition to thein vivofindings, IL-1 contributed to the production of vascular endothelial cell growth factor and tumor necrosis factor in cocultures of peritoneal macrophages and tumor cells. Host-derived IL-1 seems to control tumor angiogenesis and invasiveness. Furthermore, the anti-angiogenic effects of IL-1 receptor antagonist, shown here, suggest a possible therapeutic role in cancer, in addition to its current use in rheumatoid arthritis.
Controlling market power and price spikes in electricity networks: Demand-side bidding
Authors: Rassenti Stephen J. , Smith Vernon L. , Wilson Bart J.
Published: March 2003

In this article we report an experiment that examines how demand-side bidding can discipline generators in a market for electric power. First we develop a treatment without demand-side bidding; two large firms are allocated baseload and intermediate cost generators such that either firm might unilaterally withhold the capacity of its intermediate cost generators from the market to benefit from the supracompetitive prices that would result from only selling its baseload units. In a converse treatment, ownership of some of the intermediate cost generators is transferred from each of these firms to two other firms such that no one firm could unilaterally restrict output to spawn supracompetitive prices. Having established a well controlled data set with price spikes paralleling those observed in the naturally occurring economy, we also extend the design to include demand-side bidding. We find that demand-side bidding completely neutralizes the exercise of market power and eliminates price spikes even in the presence of structural market power.
Phase locking between human primary and secondary somatosensory cortices
Authors: Simões Cristina , Jensen Ole , Parkkonen Lauri , Hari Riitta
Published: March 2003

Unilateral stimulation of human peripheral nerves activates the primary somatosensory cortex (SI) contralaterally and the secondary somatosensory cortex (SII) bilaterally. We aimed at characterizing phase locking between SI and SII in response to electric stimuli applied once every 3 s to the right median nerve at the wrist; phase locking between brain regions has been proposed to either reflect joined processing or information exchange. Ongoing neuromagnetic activity of healthy volunteers was recorded with 204 planar gradiometers covering the whole scalp. After selecting a sensor maximally sensitive to activity in the left (contralateral) SI, phase locking between this sensor and the other 203 sensors was examined from single trial data. Statistically significant phase locking was found at ≈20 Hz, 80–90 ms after the stimuli between the left SI and the right SII in 9 of 10 subjects. Sensors with high phase-locking values over the left SI and right SII were separated by sensors with no phase-locked activity over the scalp midline, indicating that the phase locking was not caused by the sensors seeing activity from the same sources. The observed SI–SII phase locking would not be reflected in the evoked responses because a considerable part of it was not time-locked to the stimuli. Thus, our finding reveals a unique interaction in the sensorimotor system.
Cdc42-interacting protein 4 binds to huntingtin: Neuropathologic and biological evidence for a role in Huntington's disease
Authors: Holbert Sébastien , Dedeoglu Alpaslan , Humbert Sandrine , Saudou Frédéric , Ferrante Robert J. , Néri Christian
Published: March 2003

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Pathogenesis in HD seems to involve the formation of neuronal intranuclear inclusions and the abnormal regulation of transcription and signal transduction. To identify previously uncharacterized htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a
Caenorhabditis elegans
activation domain library. We found a predicted SH3 domain protein (K08E3.3b) that interacts with N-terminal htt in two-hybrid tests. A human homolog of K08E3.3b is the Cdc42-interacting protein 4 (CIP4), a protein involved in Cdc42 and Wiskott–Aldrich syndrome protein-dependent signal transduction. CIP4 interacted
in vitro
with full-length htt from lymphoblastoid cells. Neuronal CIP4 immunoreactivity increased with neuropathological severity in the neostriatum of HD patients and partially colocalized to ubiquitin-positive aggregates. Marked CIP4 overexpression also was observed in Western blot from human HD brain striatum. The overexpression of CIP4 induced the death of striatal neurons. Our data suggest that CIP4 accumulation and cellular toxicity may have a role in HD pathogenesis.
Transcriptional analysis of the B cell germinal center reaction
Authors: Klein Ulf , Tu Yuhai , Stolovitzky Gustavo A. , Keller Jeffrey L. , Haddad Joseph , Miljkovic Vladan , Cattoretti Giorgio , Califano Andrea , Dalla-Favera Riccardo
Published: March 2003

The germinal center (GC) reaction is crucial for T cell-dependent immune responses and is targeted by B cell lymphomagenesis. Here we analyzed the transcriptional changes that occur in B cells during GC transit (naïve B cells → centroblasts → centrocytes → memory B cells) by gene expression profiling. Naïve B cells, characterized by the expression of cell cycle-inhibitory and antiapoptotic genes, become centroblasts by inducing an atypical proliferation program lacking c-Myc expression, switching to a proapoptotic program, and down-regulating cytokine, chemokine, and adhesion receptors. The transition from GC to memory cells is characterized by a return to a phenotype similar to that of naïve cells except for an apoptotic program primed for both death and survival and for changes in the expression of cell surface receptors including IL-2 receptor β. These results provide insights into the dynamics of the GC reaction and represent the basis for the analysis of B cell malignancies.
Insulin resistance is a poor predictor of type 2 diabetes in individuals with no family history of disease
Authors: Goldfine Allison B. , Bouche Clara , Parker Robert A. , Kim Caroline , Kerivan Amy , Soeldner J. Stuart , Martin Blaise C. , Warram James H. , Kahn C. Ronald
Published: March 2003

In normoglycemic offspring of two type 2 diabetic parents, low insulin sensitivity (S
I
) and low insulin-independent glucose effectiveness (S
G
) predict the development of diabetes one to two decades later. To determine whether low S
I
, low S
G,
or low acute insulin response to glucose are predictive of diabetes in a population at low genetic risk for disease, 181 normoglycemic individuals with no family history of diabetes (FH−) and 150 normoglycemic offspring of two type 2 diabetic parents (FH+) underwent i.v. glucose tolerance testing (IVGTT) between the years 1964–82. During 25 ± 6 years follow-up, comprising 2,758 person years, the FH− cohort (54 ± 9 years) had an age-adjusted incidence rate of type 2 diabetes of 1.8 per 1,000 person years, similar to that in other population-based studies, but significantly lower than 16.7 for the FH+ cohort. Even when the two study populations were subdivided by initial values of S
I
and S
G
derived from IVGTT's performed at study entry, there was a 10- to 20-fold difference in age-adjusted incidence rates for diabetes in the FH− vs. FH+ individuals with low S
I
and low S
G
. The acute insulin response to glucose was not predictive of the development of diabetes when considered independently or when assessed as a function of S
I
, i.e., the glucose disposition index. These data demonstrate that low glucose disposal rates are robustly associated with the development of diabetes in the FH+ individuals, but insulin resistance per se is not sufficient for the development of diabetes in individuals without family history of disease and strongly suggest a familial factor, not detectable in our current measures of the dynamic responses of glucose or insulin to an IVGTT is an important risk factor for type 2 diabetes. Low S
I
and low S
G
, both measures of glucose disposal, interact with this putative familial factor to result in a high risk of type 2 diabetes in the FH+ individuals, but not in the FH− individuals.
Development of murine ischemic cardiomyopathy is associated with a transient inflammatory reaction and depends on reactive oxygen species
Authors: Dewald Oliver , Frangogiannis Nikolaos G. , Zoerlein Martin , Duerr Georg D. , Klemm Christina , Knuefermann Pascal , Taffet George , Michael Lloyd H. , Crapo James D. , Welz Armin , Entman Mark L.
Published: March 2003

We examined the effects of daily repetitive brief (15 min) myocardial ischemia and reperfusion (I/R) in WT C57/BL6 and extracellular superoxide dismutase (EC-SOD)-overexpressing mice. In the absence of myocardial necrosis, I/R resulted in persistent fibrosis in ischemic areas of C57/BL6 mice associated with persistent global and segmental anterior wall dysfunction. The I/R protocol induced chemokines (peak 3 days) followed sequentially by infiltration of macrophages and myofibroblasts (5 days). Fibrosis peaked at 7 days and was stable at 28 days despite regression of the chemokine and cellular response. Discontinuation of I/R at 7 or 28 days led to regression of fibrosis and ventricular dysfunction. In contrast, the EC-SOD mice developed markedly less chemokine induction, cell response, and fibrosis, with no ventricular dysfunction. Reversible fibrosis and ventricular dysfunction are features of human hibernating myocardium. The reduction of the cellular and functional response in EC-SOD mice suggests a role for reactive O
2
in the pathogenesis of ischemic cardiomyopathy.
Mechanism of estrogen-mediated neuroprotection: Regulation of mitochondrial calcium and Bcl-2 expression
Authors: Nilsen Jon , Brinton Roberta Diaz
Published: March 2003

Estrogens are neuroprotective against glutamate excitotoxicity caused by an excessive rise in intracellular calcium ([Ca
2+
]
i
). In this study, we demonstrate that 17β-estradiol (E
2
) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in bulk-free [Ca
2+
]
i
despite potentiating the influx of Ca
2+
induced by glutamate. E
2
-induced attenuation of bulk-free [Ca
2+
]
i
depends on mitochondrial sequestration of Ca
2+
, which is blocked in the presence of the combination of rotenone and oligomycin or in the presence of antimycin, which collapse the mitochondrial membrane potential, thereby preventing mitochondrial Ca
2+
transport. Release of mitochondrial Ca
2+
by carbonyl cyanide
p
-trifluoromethoxyphenylhydrazone (FCCP) after excitotoxic glutamate treatment resulted in a greater [Ca
2+
]
i
in E
2
-treated cells, indicating an E
2
-induced increase in the mitochondrial calcium ([Ca
2+
]
m
) load. The increased [Ca
2+
]
m
load was accompanied by increased expression of Bcl-2, which can promote mitochondrial Ca
2+
load tolerance. These findings provide a mechanism of E
2
-induced neuronal survival by attenuation of excitotoxic glutamate [Ca
2+
]
i
rise via increased mitochondrial sequestration of cytosolic Ca
2+
coupled with an increase in Bcl-2 expression to sustain mitochondrial Ca
2+
load tolerance and function.
Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes
Authors: Panyi G. , Bagdány M. , Bodnár A. , Vámosi G. , Szentesi G. , Jenei A. , Mátyus L. , Varga S. , Waldmann T. A. , Gáspár R. , Damjanovich S.
Published: March 2003

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C= 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E= 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.
Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand
Authors: Luo Bing-Hao , Springer Timothy A. , Takagi Junichi
Published: March 2003

The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligand-binding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aVβ3 show two different conformations. The open conformation of the headpiece is present when a ligand mimetic peptide is bound and differs from the closed conformation in the presence of an obtuse angle between the β3 subunit hybrid and I-like domains. We tested the hypothesis that opening of the hybrid-I-like domain interface increases ligand-binding affinity by mutationally introducing an N-glycosylation site into it. Both β3 and β1 integrin glycan wedge mutants exhibit constitutively high affinity for physiological ligands. The data uniquely support one model of integrin activation and suggest that movement at the interface with the hybrid domain pulls down the C-terminal helix of the I-like domain and activates its metal ion-dependent adhesion site, analogously to activation of the integrin I domain.
Five components of the ethylene-response pathway identified in a screen for
weak ethylene-insensitive
mutants in
Arabidopsis
Authors: Alonso Jose M. , Stepanova Anna N. , Solano Roberto , Wisman Ellen , Ferrari Simone , Ausubel Frederick M. , Ecker Joseph R.
Published: March 2003

Five ethylene-insensitive loci (
wei1
–
wei5
) were identified by using a low-dose screen for “weak” ethylene-insensitive mutants.
wei1
,
wei2
, and
wei3
seedlings showed hormone insensitivity only in roots, whereas
wei4
and
wei5
displayed insensitivity in both roots and hypocotyls. The genes corresponding to
wei1
,
wei4
, and
wei5
were isolated using a positional cloning approach. The
wei1
mutant harbored a recessive mutation in
TIR1
, which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response.
wei4
, a dominant mutant, resulted from a mutation in the ethylene receptor
ERS
, whereas
wei5
, a semidominant mutant, was caused by a mutation in the
EIN3
-related transcription factor gene
EIL1
. The simultaneous loss of functional
WEI5
/
EIL1
and
EIN3
nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen
Botrytis cinerea
. Moreover,
wei5
/
eil1 ein3
double mutants were able to fully suppress constitutive signaling caused by
ctr1
, suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants,
wei2
and
wei3
were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that
wei2
and
wei3
correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.
The integrity of a cholesterol-binding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels
Authors: Ko Dennis C. , Binkley Jonathan , Sidow Arend , Scott Matthew P.
Published: March 2003

The neurodegenerative disease Niemann–Pick Type C2 (NPC2) results from mutations in the
NPC2
(
HE1
) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (
K
d
= 30–50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.
Hypoxic neuronal necrosis: Protein synthesis-independent activation of a cell death program
Authors: Niquet Jerome , Baldwin Roger A. , Allen Suni G. , Fujikawa Denson G. , Wasterlain Claude G.
Published: March 2003

Hypoxic necrosis of dentate gyrus neurons in primary culture required the activation of an orderly cell death program independent of protein synthesis. Early mitochondrial swelling and loss of the mitochondrial membrane potential were accompanied by release of cytochrome
c
and followed by caspase-9-dependent activation of caspase-3. Caspase-3 and -9 inhibitors reduced neuronal necrosis. Calcium directly induced cytochrome
c
release from isolated mitochondria. Hypoxic neuronal necrosis may be an active process in which the direct effect of hypoxia on mitochondria may lead to the final common pathway of caspase-3-mediated neuronal death.
Photoexcited breathers in conjugated polyenes: An excited-state molecular dynamics study
Authors: Tretiak S. , Saxena A. , Martin R. L. , Bishop A. R.
Published: March 2003

π-conjugated polymers have become an important class of materials for electronic devices. Design of these devices requires understanding such processes as photochemical reactions, spatial dynamics of photoexcitations, and energy and charge transport, which in turn involve complex coupled electron-vibrational dynamics. Here we study nonlinear photoexcitation dynamics in the polyene oligomers by using a quantum-chemical method suitable for the simulation of excited-state molecular dynamics in extended molecular systems with sizes up to hundreds of atoms. The method is based on the adiabatic propagation of the ground-state and transition single-electron density matrices along the trajectory. The simulations reveal formation of a self-localized vibronic excitation (“breather” or multiquanta bound state) with a typical period of 34 fs and allows us to identify specific slow and fast nuclear motions strongly coupled to the electronic degrees of freedom. The effect of chain imperfections and chemical defects on the dynamics is also investigated. A complementary two-dimensional analysis of corresponding transition density matrices provides an efficient way to monitor time-dependent real-space localization of the photoexcitation by identifying the underlying changes in charge densities and bond orders. Possible correlated electronic and vibrational spectroscopic signatures of photoexcited breathers are predicted, and generalizations to energy localization in complex macromolecules are discussed.
Launching the yeast 23S RNA Narnavirus shows 5′ and 3′ cis-acting signals for replication
Authors: Esteban Rosa , Fujimura Tsutomu
Published: March 2003

Narnavirus 23S RNA is a persistent positive-stranded RNA virus found in yeast
Saccharomyces cerevisiae
. The viral genome (2.9 kb) only encodes its RNA-dependent RNA polymerase, p104. Here we report the generation of 23S RNA virus, with high frequency, from a vector containing the entire viral cDNA sequence. When the conserved GDD (Gly-Asp-Asp) motif of RNA-dependent RNA polymerase was modified, the vector failed to generate the virus, indicating that an active p104 is essential for replication. Successful launching required transcripts having the proper viral 3′ terminus generated
in vivo
. This was accomplished through
in vivo
processing of the primary transcripts by the hepatitis delta virus antigenomic ribozyme directly fused to the 3′ terminus of the 23S RNA genome. Although the primary transcripts also contained extra nucleotides at their 5′ ends derived from the vector, the launched virus possessed the authentic 5′ terminus of the viral genome without these extra nucleotides. Modifications of the genome sequence at the 5′ and 3′ termini abolished viral generation, indicating that the viral genome has cis-acting signals for replication at both termini. The great ease to generate the virus will facilitate the identification of these cis-acting signals. Furthermore, the virus, once generated, can be transmitted to daughter cells indefinitely without the vector or any selection, which makes the 23S RNA virus-launching system particularly useful for investigating the basis for RNA virus persistence.
Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia
Authors: Marijt W. A. Erik , Heemskerk Mirjam H. M. , Kloosterboer Freke M. , Goulmy Els , Kester Michel G. D. , van der Hoorn Menno A. W. G. , van Luxemburg-Heys Simone A. P. , Hoogeboom Manja , Mutis Tuna , Drijfhout Jan Wouter , van Rood Jon J. , Willemze Roel , Falkenburg J. H. Frederik
Published: March 2003

Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2/HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8+T cells in the blood of the recipients 5–7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cellsin vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies thatin vitrogenerated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignances relapsing after alloSCT.
Cholinergic plasticity in the hippocampus
Authors: Colgin Laura Lee , Kubota Don , Lynch Gary
Published: March 2003

Tests were made for use-dependent plasticity in the cholinergic projections to hippocampus. Transient infusion of the cholinergic agonist carbachol into hippocampal slices induced rhythmic activity that persisted for hours after washout. Comparable effects were obtained with physostigmine, a drug that blocks acetylcholine breakdown and thereby enhances cholinergic transmission. It thus seems that activation of cholinergic synapses induces lasting changes in hippocampal physiology. Two lines of evidence indicated that cholinergic synapses are also the sites at which the plasticity is expressed. First, the induction and expression of the rhythms were not blocked by the
N
-methyl-
d
-aspartate receptor antagonist
d
-2-amino-5-phosphonovaleric acid, indicating that a long-term potentiation effect between pyramidal cells was not involved. Second, a muscarinic antagonist (atropine) completely abolished stable rhythmic activity after agonist washout. This result indicates that endogenous cholinergic activity is responsible for the persistence of rhythmic oscillations. These experiments suggest that short periods of intense cholinergic activity induce lasting changes in cholinergic synapses and thus extend such forms of plasticity to beyond the glutamatergic system.
Molecular classification of familial non-
BRCA1/BRCA2
breast cancer
Authors: Hedenfalk Ingrid , Ringnér Markus , Ben-Dor Amir , Yakhini Zohar , Chen Yidong , Chebil Gunilla , Ach Robert , Loman Niklas , Olsson Håkan , Meltzer Paul , Borg Åke , Trent Jeffrey
Published: March 2003

In the decade since their discovery, the two major breast cancer susceptibility genes
BRCA1
and
BRCA2
, have been shown conclusively to be involved in a significant fraction of families segregating breast and ovarian cancer. However, it has become equally clear that a large proportion of families segregating breast cancer alone are not caused by mutations in
BRCA1
or
BRCA2.
Unfortunately, despite intensive effort, the identification of additional breast cancer predisposition genes has so far been unsuccessful, presumably because of genetic heterogeneity, low penetrance, or recessive/polygenic mechanisms. These non-
BRCA1/2
breast cancer families (termed
BRCAx
families) comprise a histopathologically heterogeneous group, further supporting their origin from multiple genetic events. Accordingly, the identification of a method to successfully subdivide
BRCAx
families into recognizable groups could be of considerable value to further genetic analysis. We have previously shown that global gene expression analysis can identify unique and distinct expression profiles in breast tumors from
BRCA1
and
BRCA2
mutation carriers. Here we show that gene expression profiling can discover novel classes among
BRCAx
tumors, and differentiate them from
BRCA1
and
BRCA2
tumors. Moreover, microarray-based comparative genomic hybridization (CGH) to cDNA arrays revealed specific somatic genetic alterations within the
BRCAx
subgroups. These findings illustrate that, when gene expression-based classifications are used,
BRCAx
families can be grouped into homogeneous subsets, thereby potentially increasing the power of conventional genetic analysis.
Abundance not linked to survival across the end-Cretaceous mass extinction: Patterns in North American bivalves
Authors: Lockwood Rowan
Published: March 2003

Ecological studies suggest that rare taxa are more likely to go extinct than abundant ones, but the influence of abundance on survivorship in the fossil record has received little attention. An analysis of Late Maastrichtian bivalve subgenera from the North American Coastal Plain found no evidence that survivorship is tied to abundance across the end-Cretaceous mass extinction (65 million years ago), regardless of abundance metric or spatial scale examined. The fact that abundance does not promote survivorship in end-Cretaceous bivalves suggests that the factors influencing survivorship during mass extinctions in the fossil record may differ from those operating during intervals of background extinction.
An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion
Authors: Krsmanovic Lazar Z. , Mores Nadia , Navarro Carlos E. , Arora Krishan K. , Catt Kevin J.
Published: March 2003

The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from normal and immortalized hypothalamic GnRH neurons is highly calcium-dependent and is stimulated by cAMP. It is also influenced by agonist activation of the endogenous GnRH receptor (GnRH-R), which couples to G
q/11
as indicated by release of membrane-bound α
q/11
subunits and increased inositol phosphate/Ca
2+
signaling. Conversely, GnRH antagonists increase membrane-associated α
q/11
subunits and abolish pulsatile GnRH secretion. GnRH also stimulates cAMP production but at high concentrations has a pertussis toxin-sensitive inhibitory effect, indicative of receptor coupling to G
i
. Coupling of the agonist-activated GnRH-R to both G
s
and G
i
proteins was demonstrated by the ability of nanomolar GnRH concentrations to reduce membrane-associated α
s
and α
i3
levels and of higher concentrations to diminish α
i3
levels. Conversely, α
i3
was increased during GnRH antagonist and pertussis toxin treatment, with concomitant loss of pulsatile GnRH secretion. In cholera toxin-treated GnRH neurons, decreases in α
s
immunoreactivity and increases in cAMP production paralleled the responses to nanomolar GnRH concentrations. Treatment with cholera toxin and 8-bromo-cAMP amplified episodic GnRH pulses but did not affect their frequency. These findings suggest that an agonist concentration-dependent switch in coupling of the GnRH-R between specific G proteins modulates neuronal Ca
2+
signaling via G
s
-cAMP stimulatory and G
i
-cAMP inhibitory mechanisms. Activation of G
i
may also inhibit GnRH neuronal function and episodic secretion by regulating membrane ion currents. This autocrine mechanism could serve as a timer to determine the frequency of pulsatile GnRH release by regulating Ca
2+
- and cAMP-dependent signaling and GnRH neuronal firing.
Separable features of visual cortical plasticity revealed by
N
-methyl-
d
-aspartate receptor 2A signaling
Authors: Fagiolini Michela , Katagiri Hiroyuki , Miyamoto Hiroyuki , Mori Hisashi , Grant Seth G. N. , Mishina Masayoshi , Hensch Takao K.
Published: March 2003

How individual receptive field properties are formed in the maturing sensory neocortex remains largely unknown. The shortening of
N
-methyl-
d
-aspartate (NMDA) receptor currents by 2A subunit (NR2A) insertion has been proposed to delimit the critical period for experience-dependent refinement of circuits in visual cortex. In mice engineered to maintain prolonged NMDA responses by targeted deletion of NR2A, the sensitivity to monocular deprivation was surprisingly weakened but restricted to the typical critical period and delayed normally by dark rearing from birth. Orientation preference instead failed to mature, occluding further effects of dark rearing. Interestingly, a full ocular dominance plasticity (but not orientation bias) was selectively restored by enhanced inhibition, reflecting an imbalanced excitation in the absence of NR2A. Many of the downstream pathways involved in NMDA signaling are coupled to the receptor through a variety of protein–protein interactions and adaptor molecules. To further investigate a mechanistic dissociation of receptive field properties in the developing visual system, mice carrying a targeted disruption of the NR2A-associated 95-kDa postsynaptic density (PSD95) scaffolding protein were analyzed. Although the development and plasticity of ocular dominance was unaffected, orientation preference again failed to mature in these mice. Taken together, our results demonstrate that the cellular basis generating individual sensory response properties is separable in the developing neocortex.
Nigrostriatal α-synucleinopathy induced by viral vector-mediated overexpression of human α-synuclein: A new primate model of Parkinson's disease
Authors: Kirik Deniz , Annett Lucy E. , Burger Corinna , Muzyczka Nicholas , Mandel Ronald J. , Björklund Anders
Published: March 2003

We used a high-titer recombinant adeno-associated virus (rAAV) vector to express WT or mutant human α-synuclein in the substantia nigra of adult marmosets. The α-synuclein protein was expressed in 90–95% of all nigral dopamine neurons and distributed by anterograde transport throughout their axonal and dendritic projections. The transduced neurons developed severe neuronal pathology, including α-synuclein-positive cytoplasmic inclusions and granular deposits; swollen, dystrophic, and fragmented neuritis; and shrunken and pyknotic, densely α-synuclein-positive perikarya. By 16 wk posttransduction, 30–60% of the tyrosine hydroxylase-positive neurons were lost, and the tyrosine hydroxylase-positive innervation of the caudate nucleus and putamen was reduced to a similar extent. The rAAV-α-synuclein-treated monkeys developed a type of motor impairment, i.e., head position bias, compatible with this magnitude of nigrostriatal damage. rAAV vector-mediated α-synuclein gene transfer provides a transgenic primate model of nigrostriatal α-synucleinopathy that is of particular interest because it develops slowly over time, like human Parkinson's disease (PD), and expresses neuropathological features (α-synuclein-positive inclusions and dystrophic neurites, in particular) that are similar to those seen in idiopathic PD. This model offers new opportunities for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human PD.
Long-term depression of climbing fiber-evoked calcium transients in Purkinje cell dendrites
Authors: Weber John T. , De Zeeuw Chris I. , Linden David J. , Hansel Christian
Published: March 2003

In recent years much has been learned about the molecular requirements for inducing long-term synaptic depression (LTD) in various brain regions. However, very little is known about the consequences of LTD induction for subsequent signaling events in postsynaptic neurons. We have addressed this issue by examining homosynaptic LTD at the cerebellar climbing fiber (CF)–Purkinje cell (PC) synapse. This synapse is built for reliable and massive excitation: Activation of a single axon produces an unusually large α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated synaptic current, the depolarization of which drives a regenerative complex spike producing a large, widespread Ca
2+
transient in PC dendrites. Here we test whether CF LTD has an impact on dendritic, complex spike-evoked Ca
2+
signals by simultaneously performing long-term recordings of complex spikes and microfluorimetric Ca
2+
measurements in PC dendrites in rat cerebellar slices. Our data show that LTD of the CF excitatory postsynaptic current produces a reduction in both slow components of the complex spike waveform and complex spike-evoked dendritic Ca
2+
transients. This LTD of dendritic Ca
2+
signals may provide a neuroprotective mechanism and/or constitute “heterosynaptic metaplasticity” by reducing the probability for subsequent induction of those forms of use-dependent plasticity, which require CF-evoked Ca
2+
signals such as parallel fiber–PC LTD and interneuron–PC LTP.
Nuclear and cytosolic calcium are regulated independently
Authors: Leite M. F. , Thrower E. C. , Echevarria W. , Koulen P. , Hirata K. , Bennett A. M. , Ehrlich B. E. , Nathanson M. H.
Published: March 2003

Nuclear calcium (Ca
2+
) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca
2+
signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca
2+
release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP
3
) receptor (InsP
3
R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP
3
R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca
2+
signals induced by low InsP
3
concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca
2+
stores for InsP
3
. Nuclear InsP
3
R channels were active at lower InsP
3
concentrations than InsP
3
R from cytosol. Enriched expression of type II InsP
3
R in the nucleus results in greater sensitivity of the nucleus to InsP
3
, thus providing a mechanism for independent regulation of Ca
2+
-dependent processes in this cellular compartment.
The ATP hydrolyzing transcription activator phage shock protein F of
Escherichia coli
: Identifying a surface that binds σ
54
Authors: Bordes Patricia , Wigneshweraraj Siva R. , Schumacher Jörg , Zhang Xiaodong , Chaney Matthew , Buck Martin
Published: March 2003

Members of the protein family called ATPases associated with various cellular activities (AAA
+
) play a crucial role in transforming chemical energy into biological events. AAA
+
proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The
Escherichia coli
transcription activator phage shock protein F (PspF) is an AAA
+
mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the σ
54
-RNA polymerase. Closed promoter complexes formed by the σ
54
-RNA polymerase are substrates for the action of PspF. By using a protein fragmentation approach, we identify here at least one σ
54
-binding surface in the PspF AAA
+
domain. Results suggest that ATP hydrolysis by PspF is coupled to the exposure of at least one σ
54
-binding surface. This nucleotide hydrolysis-dependent presentation of a substrate binding surface can explain why complexes that form between σ
54
and PspF are transient and could be part of a mechanism used generally by other AAA
+
proteins to regulate activity.
Lipolysis of triglyceride-rich lipoproteins generates PPAR ligands: Evidence for an antiinflammatory role for lipoprotein lipase
Authors: Ziouzenkova Ouliana , Perrey Stephane , Asatryan Liana , Hwang Juliana , MacNaul Karen L. , Moller David E. , Rader Daniel J. , Sevanian Alex , Zechner Rudolf , Hoefler Gerald , Plutzky Jorge
Published: March 2003

Increased levels of triglyceride-rich lipoproteins provoke lipid accumulation in the artery wall, triggering early inflammatory responses central to atherosclerosis like endothelial adhesion molecule expression. The endogenous mechanisms limiting such reactions remain poorly defined. Lipoprotein lipase (LPL) plays a central role in lipid metabolism by hydrolyzing triglyceride rich lipoproteins and releasing fatty acids. We found that LPL treatment reversed tumor necrosis factor α and very low-density lipoprotein (VLDL)-stimulated endothelial vascular cell adhesion molecule 1 (VCAM1) induction and VCAM1 promoter responses, thus recapitulating effects reported with synthetic peroxisome proliferator-activated receptor (PPAR) agonists. In fact, these LPL effects on VCAM1 were absent in endothelial cells isolated from PPARα-deficient mice. This finding suggests a novel antiinflammatory role for LPL. Further studies reveal specificity for PPAR activation through lipolysis in regards to lipoprotein substrate (VLDL ≫ LDL > HDL), PPAR isoform (PPARα ≫ PPARδ > PPARγ), and among fatty acid-releasing lipases. These PPAR responses required intact LPL catalytic activity.
In vivo
, transgenic mice overexpressing LPL had increased peroxisome proliferation, but not in the genetic absence of PPARα. Although human plasma possesses minimal PPARα activation despite containing abundant free fatty acids, marked PPARα activation is seen with human plasma after LPL is added
in vitro
or systemically released
in vivo
. These data suggest a previously uncharacterized pathway in which the key lipolytic enzyme LPL can act on circulating lipoproteins to generate PPARα ligands, providing a potentially important link between lipoprotein metabolism and distal PPARα transcriptional effects.
Selective killing of cancer cells by β-lapachone: Direct checkpoint activation as a strategy against cancer
Authors: Li Youzhi , Sun Xiangao , LaMont J. Thomas , Pardee Arthur B. , Li Chiang J.
Published: March 2003

Most chemotherapeutic drugs kill cancer cells by indirectly activating checkpoint-mediated apoptosis after creating nonselective damage to DNA or microtubules, which accounts for their toxicity toward normal cells. We seek to target cancer cells by directly activating checkpoint regulators without creating such damage. Here, we show that β-lapachone selectively induces apoptosis in cancer cells without causing the death of nontransformed cells in culture. This unusual selectivity against cancer cells is preceded by activation of S-phase checkpoint and selective induction of E2F1, a regulator of checkpoint-mediated apoptosis. This study suggests direct checkpoint activation as a strategy against cancer.
Bacillus thuringiensiscrystal proteins that target nematodes
Authors: Wei Jun-Zhi , Hale Kristina , Carta Lynn , Platzer Edward , Wong Cynthie , Fang Su-Chiung , Aroian Raffi V.
Published: March 2003

Bacillus thuringiensis(Bt) crystal proteins are pore-forming toxins used as insecticides around the world. Previously, the extent to which these proteins might also target the invertebrate phylum Nematoda has been mostly ignored. We have expressed seven different crystal toxin proteins from two largely unstudiedBtcrystal protein subfamilies. By assaying their toxicity on diverse free-living nematode species, we demonstrate that four of these crystal proteins are active against multiple nematode species and that each nematode species tested is susceptible to at least one toxin. We also demonstrate that a rat intestinal nematode is susceptible to some of the nematicidal crystal proteins, indicating these may hold promise in controlling vertebrate-parasitic nematodes. Toxicity in nematodes correlates with damage to the intestine, consistent with the mechanism of crystal toxin action in insects. Structure–function analyses indicate that one novel nematicidal crystal protein can be engineered to a small 43-kDa active core. These data demonstrate that at least twoBtcrystal protein subfamilies contain nematicidal toxins.
Crystal structure of
Mycobacterium tuberculosis
SecA, a preprotein translocating ATPase
Authors: Sharma Vivek , Arockiasamy Arulandu , Ronning Donald R. , Savva Christos G. , Holzenburg Andreas , Braunstein Miriam , Jacobs William R. , Sacchettini James C.
Published: March 2003

In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation. Here we report the three-dimensional structure of the SecA protein of
Mycobacterium tuberculosis
. Each subunit of the homodimer contains a “motor” domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process.
RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells
Authors: Wilson Joyce A. , Jayasena Sumedha , Khvorova Anastasia , Sabatinos Sarah , Rodrigue-Gervais Ian Gaël , Arya Sudha , Sarangi Farida , Harris-Brandts Marees , Beaulieu Sylvie , Richardson Christopher D.
Published: March 2003

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.