bluuebunny/crossref_metadata_2025 huggingface dataset:
The
SPANX
gene family of cancer/testis-specific antigens: Rapid evolution and amplification in African great apes and hominids
Authors: Kouprina Natalay , Mullokandov Michael , Rogozin Igor B. , Collins N. Keith , Solomon Greg , Otstot John , Risinger John I. , Koonin Eugene V. , Barrett J. Carl , Larionov Vladimir
Published: March 2004

Human sperm protein associated with the nucleus on the X chromosome (
SPANX
) genes comprise a gene family with five known members (
SPANX
-
A1
, -
A2
, -
B
, -
C
, and -
D
), encoding cancer/testis-specific antigens that are potential targets for cancer immunotherapy. These highly similar paralogous genes cluster on the X chromosome at Xq27. We isolated and sequenced primate genomic clones homologous to human
SPANX
. Analysis of these clones and search of the human genome sequence revealed an uncharacterized group of genes,
SPANX
-
N
, which are present in all primates as well as in mouse and rat. In humans, four
SPANX
-
N
genes comprise a series of tandem duplicates at Xq27; a fifth member of this subfamily is located at Xp11. Similarly to
SPANX
-
A
/
D
, human
SPANX
-
N
genes are expressed in normal testis and some melanoma cell lines; testis-specific expression of
SPANX
is also conserved in mouse. Analysis of the taxonomic distribution of the long and short forms of the intron indicates that
SPANX
-
N
is the ancestral form, from which the
SPANX
-
A
/
D
subfamily evolved in the common ancestor of the hominoid lineage. Strikingly, the coding sequences of the
SPANX
genes evolved much faster than the intron and the 5′ untranslated region. There is a strong correlation between the rates of evolution of synonymous and nonsynonymous codon positions, both of which are accelerated 2-fold or more compared to the noncoding sequences. Thus, evolution of the
SPANX
family appears to have involved positive selection that affected not only the protein sequence but also the synonymous sites in the coding sequence.
The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA
in vitro
Authors: Kahmann Jan D. , Wecking Diana A. , Putter Vera , Lowenhaupt Ky , Kim Yang-Gyun , Schmieder Peter , Oschkinat Hartmut , Rich Alexander , Schade Markus
Published: March 2004

The N-terminal domain of the vaccinia virus protein E3L (Zα
E3L
) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Zα
ADAR1
and Zα
DLM1
. Here, we report the solution structure of Zα
E3L
and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Zα
E3L
are very similar to the high-affinity Z-DNA-binding domains Zα
ADAR1
and Zα
DLM1
. However, the key Z-DNA contacting residue Y48 of Zα
E3L
adopts a different side chain conformation in unbound Zα
E3L
, which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower
in vitro
affinity of Zα
E3L
to Z-DNA compared with its homologues.
Activation of the Wnt signaling pathway in chronic lymphocytic leukemia
Authors: Lu Desheng , Zhao Yandong , Tawatao Rommel , Cottam Howard B. , Sen Malini , Leoni Lorenzo M. , Kipps Thomas J. , Corr Maripat , Carson Dennis A.
Published: March 2004

B cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, functionally incompetent B cells. Wnts are a large family of secreted glycoproteins involved in cell proliferation, differentiation, and oncogenesis. The classical Wnt signaling cascade inhibits the activity of the enzyme glycogen synthase kinase-3β, augmenting β-catenin translocation to the nucleus, and the transcription of target genes. Little is known about the potential roles of Wnt signaling in CLL. In this study, we quantified the gene expression profiles of the Wnt family, and their cognate frizzled (Fzd) receptors in primary CLL cells, and determined the role of Wnt signaling in promoting CLL cell survival. Wnt3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16, as well as the Wnt receptor Fzd3, were highly expressed in CLL, compared with normal B cells. Three lines of evidence suggested that the Wnt signaling pathway was active in CLL. First, the Wnt/β-catenin-regulated transcription factor lymphoid-enhancing factor-1, and its downstream target cyclin D1, were overexpressed in CLL. Second, a pharmacological inhibitor of glycogen synthase kinase-3β, SB-216763, activated β-catenin-mediated transcription, and enhanced the survival of CLL lymphocytes. Third, Wnt/β-catenin signaling was diminished by an analog of a nonsteroidal antiinflammatory drug (R-etodolac), at concentrations that increased apoptosis of CLL cells. Taken together, these results indicate that Wnt signaling genes are overexpressed and are active in CLL. Uncontrolled Wnt signaling may contribute to the defect in apoptosis that characterizes this malignancy.
Revealing modularity and organization in the yeast molecular network by integrated analysis of highly heterogeneous genomewide data
Authors: Tanay Amos , Sharan Roded , Kupiec Martin , Shamir Ron
Published: March 2004

The dissection of complex biological systems is a challenging task, made difficult by the size of the underlying molecular network and the heterogeneous nature of the control mechanisms involved. Novel high-throughput techniques are generating massive data sets on various aspects of such systems. Here, we perform analysis of a highly diverse collection of genomewide data sets, including gene expression, protein interactions, growth phenotype data, and transcription factor binding, to reveal the modular organization of the yeast system. By integrating experimental data of heterogeneous sources and types, we are able to perform analysis on a much broader scope than previous studies. At the core of our methodology is the ability to identify modules, namely, groups of genes with statistically significant correlated behavior across diverse data sources. Numerous biological processes are revealed through these modules, which also obey global hierarchical organization. We use the identified modules to study the yeast transcriptional network and predict the function of >800 uncharacterized genes. Our analysis framework,samba(Statistical-Algorithmic Method for Bicluster Analysis), enables the processing of current and future sources of biological information and is readily extendable to experimental techniques and higher organisms.
Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit
Authors: Martínez-Torres Ataúlfo , Miledi Ricardo
Published: March 2004

γ-Aminobutyric acid A (GABA
A
) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABA
A
receptor, injected alone into
Xenopus
oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl
-
ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABA
A
receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in
Xenopus
oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABA
A
receptors
in situ
require further clarification.
Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients
Authors: Wang Tian-Li , Diaz Luis A. , Romans Katharine , Bardelli Alberto , Saha Saurabh , Galizia Gennaro , Choti Michael , Donehower Ross , Parmigiani Giovanni , Shih Ie-Ming , Iacobuzio-Donahue Christine , Kinzler Kenneth W. , Vogelstein Bert , Lengauer Christoph , Velculescu Victor E.
Published: March 2004

Resistance to chemotherapy is a major cause of mortality in advanced cancer patients. In this study, digital karyotyping was used to search for genomic alterations in liver metastases that were clinically resistant to 5-fluorouracil (5-FU). In two of four patients, we identified amplification of an ≈100-kb region on 18p11.32 that was of particular interest because it contained the gene encoding thymidylate synthase (TYMS), a molecular target of 5-FU. Analysis of TYMS by fluorescence
in situ
hybridization identified TYMS gene amplification in 23% of 31 5-FU-treated cancers, whereas no amplification was observed in metastases of patients that had not been treated with 5-FU. Patients with metastases containing TYMS amplification had a substantially shorter median survival (329 days) than those without amplification (1,021 days,
P
<0.01). These data suggest that genetic amplification of TYMS is a major mechanism of 5-FU resistance
in vivo
and have important implications for the management of colorectal cancer patients with recurrent disease.
A small molecular activator of cardiac hypertrophy uncovered in a chemical screen for modifiers of the calcineurin signaling pathway
Authors: Bush Erik , Fielitz Jens , Melvin Lawrence , Martinez-Arnold Michael , McKinsey Timothy A. , Plichta Ryan , Olson Eric N.
Published: March 2004

The calcium, calmodulin-dependent phosphatase calcineurin, regulates growth and gene expression of striated muscles. The activity of calcineurin is modulated by a family of cofactors, referred to as modulatory calcineurin-interacting proteins (MCIPs). In the heart, the
MCIP1
gene is activated by calcineurin and has been proposed to fulfill a negative feedback loop that restrains potentially pathological calcineurin signaling, which would otherwise lead to abnormal cardiac growth. In a high-throughput screen for small molecules capable of regulating MCIP1 expression in muscle cells, we identified a unique 4-aminopyridine derivative exhibiting an embedded partial structural motif of serotonin (5-hydroxytryptamine, 5-HT). This molecule, referred to as pyridine activator of myocyte hypertrophy, acts as a selective agonist for 5-HT
2A/2B
receptors and induces hypertrophy of cardiac muscle cells through a signaling pathway involving calcineurin and a kinase-dependent mechanism that inactivates class II histone deacetylases, which act as repressors of cardiac growth. These findings identify MCIP1 as a downstream target of 5-HT
2A/2B
receptor signaling in cardiac muscle cells and suggest possible uses for 5-HT
2A/2B
agonists and antagonists as modulators of cardiac growth and gene expression.
Role for neuronal insulin resistance in neurodegenerative diseases
Authors: Schubert Markus , Gautam Dinesh , Surjo David , Ueki Kojihiko , Baudler Stephanie , Schubert Dominic , Kondo Tatsuya , Alber Jens , Galldiks Norbert , Küstermann Eckehardt , Arndt Saskia , Jacobs Andreas H. , Krone Wilhelm , Kahn C. Ronald , Brüning Jens C.
Published: March 2004

Impairment of insulin signaling in the brain has been linked to neurodegenerative diseases. To test the hypothesis that neuronal insulin resistance contributes to defects in neuronal function, we have performed a detailed analysis of brain/neuron-specific insulin receptor knockout (NIRKO) mice. We find that NIRKO mice exhibit a complete loss of insulin-mediated activation of phosphatidylinositol 3-kinase and inhibition of neuronal apoptosis. In intact animals, this loss results in markedly reduced phosphorylation of Akt and GSK3β, leading to substantially increased phosphorylation of the microtubule-associated protein Tau, a hallmark of neurodegenerative diseases. Nevertheless, these animals exhibit no alteration in neuronal proliferation/survival, memory, or basal brain glucose metabolism. Thus, lack of insulin signaling in the brain may lead to changes in Akt and GSK3β activity and Tau hyperphosphorylation but must interact with other mechanisms for development of Alzheimer's disease.
RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells
Authors: Maxwell Michele M. , Pasinelli Piera , Kazantsev Aleksey G. , Brown Robert H.
Published: March 2004

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder resulting from selective death of motor neurons in the brain and spinal cord. In ≈25% of familial ALS cases, the disease is caused by dominantly acting point mutations in the gene encoding cytosolic Cu,Zn superoxide dismutase (SOD1). In cell culture and in rodent models of ALS, mutant SOD1 proteins exhibit dose-dependent toxicity; thus, agents that reduce mutant protein expression would be powerful therapeutic tools. A wealth of recent evidence has demonstrated that the mechanism of RNA-mediated interference (RNAi) can be exploited to achieve potent and specific gene silencing
in vitro
and
in vivo
. We have evaluated the utility of RNAi for selective silencing of mutant SOD1 expression in cultured cells and have identified small interfering RNAs capable of specifically inhibiting expression of ALS-linked mutant, but not wild-type, SOD1. We have investigated the functional effects of RNAi-mediated silencing of mutant SOD1 in cultured murine neuroblastoma cells. In this model, stable expression of mutant, but not wild-type, human SOD1 sensitizes cells to cytotoxic stimuli. We find that silencing of mutant SOD1 protects these cells against cyclosporin A-induced cell death. These results demonstrate a positive physiological effect caused by RNAi-mediated silencing of a dominant disease allele. The present study further supports the therapeutic potential of RNAi-based methods for the treatment of inherited human diseases, including ALS.
The X3LYP extended density functional for accurate descriptions of nonbond interactions, spin states, and thermochemical properties
Authors: Xu Xin , Goddard William A.
Published: March 2004

We derive the form for an exact exchange energy density for a density decaying with Gaussian-like behavior at long range. Based on this, we develop the X3LYP (extended hybrid functional combined with Lee–Yang–Parr correlation functional) extended functional for density functional theory to significantly improve the accuracy for hydrogen-bonded and van der Waals complexes while also improving the accuracy in heats of formation, ionization potentials, electron affinities, and total atomic energies [over the most popular and accurate method, B3LYP (Becke three-parameter hybrid functional combined with Lee–Yang–Parr correlation functional)]. X3LYP also leads to a good description of dipole moments, polarizabilities, and accurate excitation energies from s to d orbitals for transition metal atoms and ions. We suggest that X3LYP will be useful for predicting ligand binding in proteins and DNA.
Fluorescence-activated cell sorting (FACS) of
Drosophila
hemocytes reveals important functional similarities to mammalian leukocytes
Authors: Tirouvanziam Rabindra , Davidson Colin J. , Lipsick Joseph S. , Herzenberg Leonard A.
Published: March 2004

Drosophila
is a powerful model for molecular studies of hematopoiesis and innate immunity. However, its use for functional cellular studies remains hampered by the lack of single-cell assays for hemocytes (blood cells). Here we introduce a generic method combining fluorescence-activated cell sorting and nonantibody probes that enables the selective gating of live
Drosophila
hemocytes from the lymph glands (larval hematopoietic organ) or hemolymph (blood equivalent). Gated live hemocytes are analyzed and sorted at will based on precise quantitation of fluorescence levels originating from metabolic indicators, lectins, reporters (GFP and β-galactosidase) and antibodies. With this approach, we discriminate and sort plasmatocytes, the major hemocyte subset, from lamellocytes, an activated subset present in gain-of-function mutants of the Janus kinase and Toll pathways. We also illustrate how important, evolutionarily conserved, blood-cell-regulatory molecules, such as calcium and glutathione, can be studied functionally within hemocytes. Finally, we report an
in vivo
transfer of sorted live hemocytes and their successful reanalysis on retrieval from single hosts. This generic and versatile fluorescence-activated cell sorting approach for hemocyte detection, analysis, and sorting, which is efficient down to one animal, should critically enhance
in vivo
and
ex vivo
hemocyte studies in
Drosophila
and other species, notably mosquitoes.
Impetus for sowing and the beginning of agriculture: Ground collecting of wild cereals
Authors: Kislev Mordechai E. , Weiss Ehud , Hartmann Anat
Published: March 2004

The Agricultural Revolution in Western Asia, which took place some 11,000 years ago, was a turning point in human history [Childe, V. G. (1952)
New Light on the Most Ancient East
(Routledge & Kegan Paul, London)]. In investigating the cultural processes that could have led from gathering to intentional cultivation, various authors have discussed and tested wild cereal harvesting techniques. Some argue that Near Eastern foragers gathered grains by means of sickle harvesting, uprooting, plucking (hand stripping), or beating into baskets [Hillman, G. C. & Davies, M. S. (1999) in
Prehistory of Agriculture: New Experimental and Ethnographic Approaches
, ed. Anderson, P. (The Institute of Archaeology, University of California, Los Angeles), pp. 70–102]. During systematic experiments, we found that archaeobotanical data from regional Neolithic sites support ground collection of grains by early hunter-gatherers. Ground collecting suits the natural shattering of wild species that ripen and drop grains at the beginning of summer. We show that continual collection off the ground from May to October would have provided surplus grains for deliberate sowing in more desirable fields, and facilitate the transition to intentional cultivation. Because ground gathering enabled collectors to observe that fallen seeds are responsible for the growth of new plants in late fall, they became aware of the profitability of sowing their surplus seeds for next year's food. Ground collecting of wild barley and wild wheat may comprise the missing link between seed collecting by hunter-gatherers and cereal harvesting by early farmers.
K channel gating by an affinity-switching selectivity filter
Authors: VanDongen Antonius M. J.
Published: March 2004

A universal property of ion channels is their ability to alternate stochastically between two permeation states, open and closed. This behavior is thought to be controlled by a steric “gate”, a structure that physically impedes ion flow in the closed state and moves out of the way during channel opening. Experiments employing macroscopic currents in the
Shaker
K channel have suggested a cytoplasmic localization for the gate. Crystallographic structures of the KcsA K channel indeed reveal a cytoplasmic constriction, implying that the gate and selectivity filter are localized to opposite ends of the permeation pathway. However, analysis of K channel subconductance behavior has suggested a strict coupling between channel opening (gating) and permeation. The idea that the selectivity filter is the gate was therefore investigated by using Monte Carlo simulations. Gating is accomplished by allowing the filter to alternate stochastically between two conformations: a high-affinity state, which selectively binds K ions (but not Na ions) and traps them, and a completely nonselective, low-affinity state, which allows both Na and K ions to permeate. The results of these simulations indicate that affinity switching not only endows the selectivity filter with gating abilities, it also allows efficient permeation without jeopardizing ion selectivity. In this model, permeation and gating result from the same process.
Global analysis of
Escherichia coli
RNA degradosome function using DNA microarrays
Authors: Bernstein Jonathan A. , Lin Pei-Hsun , Cohen Stanley N. , Lin-Chao Sue
Published: March 2004

RNase E, an essential endoribonuclease of
Escherichia coli
, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289
E. coli
mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some
E. coli
mRNAs
in vivo
depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes.
Predicted 3D structure for the human β2 adrenergic receptor and its binding site for agonists and antagonists
Authors: Freddolino Peter L. , Kalani M. Yashar S. , Vaidehi Nagarajan , Floriano Wely B. , Hall Spencer E. , Trabanino Rene J. , Kam Victor Wai Tak , Goddard William A.
Published: March 2004

We report the 3D structure of human β2 adrenergic receptor (AR) predicted by using the MembStruk first principles method. To validate this structure, we use the HierDock first principles method to predict the ligand-binding sites for epinephrine and norepinephrine and for eight other ligands, including agonists and antagonists to β2 AR and ligands not observed to bind to β2 AR. The binding sites agree well with available mutagenesis data, and the calculated relative binding energies correlate reasonably with measured binding affinities. In addition, we find characteristic differences in the predicted binding sites of known agonists and antagonists that allow us to infer the likely activity of other ligands. The predicted ligand-binding properties validate the methods used to predict the 3D structure and function. This validation is a successful step toward applying these procedures to predict the 3D structures and function of the other eight subtypes of ARs, which should enable the development of subtype-specific antagonists and agonists with reduced side effects.
Defining and identifying communities in networks
Authors: Radicchi Filippo , Castellano Claudio , Cecconi Federico , Loreto Vittorio , Parisi Domenico
Published: March 2004

The investigation of community structures in networks is an important issue in many domains and disciplines. This problem is relevant for social tasks (objective analysis of relationships on the web), biological inquiries (functional studies in metabolic and protein networks), or technological problems (optimization of large infrastructures). Several types of algorithms exist for revealing the community structure in networks, but a general and quantitative definition of community is not implemented in the algorithms, leading to an intrinsic difficulty in the interpretation of the results without any additional nontopological information. In this article we deal with this problem by showing how quantitative definitions of community are implemented in practice in the existing algorithms. In this way the algorithms for the identification of the community structure become fully self-contained. Furthermore, we propose a local algorithm to detect communities which outperforms the existing algorithms with respect to computational cost, keeping the same level of reliability. The algorithm is tested on artificial and real-world graphs. In particular, we show how the algorithm applies to a network of scientific collaborations, which, for its size, cannot be attacked with the usual methods. This type of local algorithm could open the way to applications to large-scale technological and biological systems.
Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture
Authors: Reixach Natàlia , Deechongkit Songpon , Jiang Xin , Kelly Jeffery W. , Buxbaum Joel N.
Published: March 2004

The transthyretin (TTR) amyloidoses are human diseases in which the misfolded TTR protein aggregates in tissues with subsequent visceral, peripheral, and autonomic nerve dysfunction. Recent reports have stressed the importance of oligomeric intermediates as major cytotoxic species in various forms of amyloidogenesis. We have examined the cytotoxic effects of several quaternary structural states of wild-type and variant TTR proteins on cells of neural lineage. TTR amyloid fibrils and soluble aggregates >100 kDa were not toxic. Incubation of TTR under the conditions of the cell assay and analysis by size-exclusion chromatography and SDS/PAGE reveal that monomeric TTR or relatively small, rapidly formed aggregates of a maximum size of six subunits were the major cytotoxic species. Small molecules that stabilize the native tetrameric state were shown to prevent toxicity. The studies are consistent with a model in which the misfolded TTR monomer rapidly aggregates to form transient low molecular mass assemblies (<100 kDa) that are highly cytotoxic in tissue culture.
Intrachromosomal excision of a hybrid
Ds
element induces large genomic deletions in
Arabidopsis
Authors: Page Damian R. , Köhler Claudia , da Costa-Nunes José A. , Baroux Célia , Moore James M. , Grossniklaus Ueli
Published: March 2004

Transposon activity is known to cause chromosome rearrangements in the host genome. Surprisingly, extremely little is known about
Dissociation
(
Ds
)-induced chromosome rearrangements in
Arabidopsis
, where
Ds
is intensively used for insertional mutagenesis. Here, we describe three
Arabidopsis
mutants with reduced fertility and propose that excision of a hybrid
Ds
element induced a large genomic deletion flanking
Ds
. In the mutants
anat
and
haumea
, the deletion mechanism consists of a local
Ds
transposition from replicated into unreplicated DNA followed by
Ds
excision, where one end of the newly transposed element and one end of the
Ds
transposon at the donor site served as substrate for transposase. Excision of this hybrid element reminiscent of a macrotransposon leads to loss of the chromosomal piece located between the two ends, including one full
Ds
element and the flanking genomic sequence. This mechanism was found to be responsible for several other deletions and occurs at a genetically trackable frequency. Thus, it could be applied to efficiently generate deletions of various sizes in the vicinity of any existing
Ds
element present in the genome. In the mutant
tons missing
, a mechanism that involves endogenous repetitive sequences caused a large flanking deletion at a position unlinked to the starter locus. Our study of
Ds
transposition in
Arabidopsis
revealed previously undescribed mechanisms that lead to large genomic deletions flanking
Ds
elements, which may contribute to genome dynamics and evolution.
Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate
Authors: Zhang Zhiquan , Rajagopalan P. T. Ravi , Selzer Tzvia , Benkovic Stephen J. , Hammes Gordon G.
Published: March 2004

The interaction of dihydrofolate (H
2
F) and NADPH with a fluorescent derivative of H
2
F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the binding of both substrates and the hydride transfer reaction. For the binding of H
2
F to DHFR, the simplest mechanism consistent with the data postulates that the enzyme exists as slowly interconverting conformers, with the substrate binding preferentially to one of the conformers. At pH 7.0, the binding reaction has a bimolecular rate constant of 1.8 × 10
7
M
-1
·s
-1
, and the formation of the initial complex is followed by a conformational change. The binding of NADPH to DHFR is more complex and suggests multiple conformers of the enzyme exist. NADPH binds to a different conformer than H
2
F with a bimolecular rate constant of 2.6–5.7 × 10
6
M
-1
·s
-1
, with the former value obtained from single-molecule kinetics and the latter from stopped-flow kinetics. Single-molecule studies of DHFR in equilibrium with substrates and products revealed a reaction with ensemble average rate constants of 170 and 470 s
-1
at pH 8.5. The former rate constant has an isotope effect of >2 when NADPD is substituted for NADPH and probably is associated with hydride transfer. The results from stopped-flow and single-molecule methods are complementary and demonstrate that multiple conformations of both the enzyme and enzyme–substrate complexes exist.
On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites
Authors: Wang Ming-Bo , Bian Xue-Yu , Wu Li-Min , Liu Li-Xia , Smith Neil A. , Isenegger Daniel , Wu Rong-Mei , Masuta Chikara , Vance Vicki B. , Watson John M. , Rezaian Ali , Dennis Elizabeth S. , Waterhouse Peter M.
Published: March 2004

Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.
The archaeal feast/famine regulatory protein: Potential roles of its assembly forms for regulating transcription
Authors: Koike Hideaki , Ishijima Sanae A. , Clowney Lester , Suzuki Masashi
Published: March 2004

The classification feast/famine regulatory proteins (FFRPs) encompasses archaeal DNA-binding proteins with
Escherichia coli
transcription factors, the leucine-responsive regulatory protein and the asparagine synthase
C
gene product. In this paper, we describe two forms of the archaeal FFRP FL11 (pot0434017), both assembled from dimers. When crystallized, a helical cylinder is formed with six dimers per turn. In contrast, in solution, disks are formed, most likely consisting of four dimers each; an observation by cryoelectron microscopy. Whereas each dimer binds a 13-bp sequence, different forms will discriminate between promoters, based on the numbers of repeating 13-bp sequences, and types of linkers inserted between them, which are either of 7-8 or ≈18 bp. The amino acid sequences of these FFRPs are designed to form the same type of 3D structures, and the transition between their assembly forms is regulated by interaction with small molecules. These considerations lead us to propose a possible mechanism for regulating a number of genes by varying assembly forms and by combining different FFRPs into these assemblies, responding to environmental changes.
CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in
Arabidopsis
Authors: Daniel Xavier , Sugano Shoji , Tobin Elaine M.
Published: March 2004

The circadian clock controls numerous physiological and molecular processes in organisms ranging from fungi to human. In plants, these processes include leaf movement, stomata opening, and expression of a large number of genes. At the core of the circadian clock, the central oscillator consists of a negative autoregulatory feedback loop that is coordinated with the daily environmental changes, and that generates the circadian rhythms of the overt processes. Phosphorylation of some of the central oscillator proteins is necessary for the generation of normal circadian rhythms of
Drosophila
, humans, and
Neurospora
, where CK1 and CK2 are emerging as the main protein kinases involved in the phosphorylation of PER and FRQ. We have previously shown that in
Arabidopsis
, the protein kinase CK2 can phosphorylate the clock-associated protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)
in vitro
. The overexpression of one of its regulatory subunits, CKB3, affects the regulation of circadian rhythms. Whether the effects of CK2 on the clock were due to its phosphorylation of a clock component had yet to be proven. By examining the effects of constitutively expressing a mutant form of the
Arabidopsis
clock protein CCA1 that cannot be phosphorylated by CK2, we demonstrate here that CCA1 phosphorylation by CK2 is important for the normal functioning of the central oscillator.
TLR2 is expressed on activated T cells as a costimulatory receptor
Authors: Komai-Koma Mousa , Jones Louise , Ogg Graham S. , Xu Damo , Liew Foo Y.
Published: March 2004

Toll is the founder of a group of pattern recognition receptors that play a critical role in the innate immunity in
Drosophila
. At least 10 distinct Toll-like receptors (TLRs), recognizing pathogen-associated molecular patterns, have now been identified in humans. Most investigations on TLRs have focused on cells of the innate system. We report here that naïve human T cells expressed high levels of cell-surface TLR2 after activation by anti-T cell receptor antibody and IFN-α. Activated cells produced elevated levels of cytokines in response to the TLR2 ligand, bacterial lipopeptide. Furthermore, CD4
+
CD45RO
+
memory T cells from peripheral blood constitutively expressed TLR2 and produced IFN-γ in response to bacterial lipopeptide, which also markedly enhanced the proliferation and IFN-γ production by CD45RO
+
T cells in the presence of IL-2 or IL-15. Thus, TLR2 serves as a costimulatory receptor for antigen-specific T cell development and participates in the maintenance of T cell memory. This suggests that pathogens, via their pathogen-associated molecular patterns, may contribute directly to the perpetuation and activation of long-term T cell memory in both antigen-dependent and independent manner.
Plasma membrane localization signals in the light chain of botulinum neurotoxin
Authors: Fernández-Salas Ester , Steward Lance E. , Ho Helen , Garay Patton E. , Sun Sarah W. , Gilmore Marcella A. , Ordas Joseph V. , Wang Joanne , Francis Joseph , Aoki K. Roger
Published: March 2004

Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT/A last from 3 to 12 months, whereas the effects of BoNT/E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT/A light chain (BoNT/A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25
197
. In contrast, the short-duration BoNT/E serotype is cytoplasmic. Mutations in the BoNT/A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes.
Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a
Drosophila
model of Huntington's disease
Authors: Lee Wyan-Ching Mimi , Yoshihara Motojiro , Littleton J. Troy
Published: March 2004

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, we generated
Drosophila
transgenic strains expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to disruption of axonal transport and accumulation of aggregates at synapses. In contrast,
Drosophila
expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. Our findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease.
Cardiovascular fitness, cortical plasticity, and aging
Authors: Colcombe Stanley J. , Kramer Arthur F. , Erickson Kirk I. , Scalf Paige , McAuley Edward , Cohen Neal J. , Webb Andrew , Jerome Gerry J. , Marquez David X. , Elavsky Steriani
Published: March 2004

Cardiovascular fitness is thought to offset declines in cognitive performance, but little is known about the cortical mechanisms that underlie these changes in humans. Research using animal models shows that aerobic training increases cortical capillary supplies, the number of synaptic connections, and the development of new neurons. The end result is a brain that is more efficient, plastic, and adaptive, which translates into better performance in aging animals. Here, in two separate experiments, we demonstrate for the first time to our knowledge, in humans that increases in cardiovascular fitness results in increased functioning of key aspects of the attentional network of the brain during a cognitively challenging task. Specifically, highly fit (Study 1) or aerobically trained (Study 2) persons show greater task-related activity in regions of the prefrontal and parietal cortices that are involved in spatial selection and inhibitory functioning, when compared with low-fit (Study 1) or nonaerobic control (Study 2) participants. Additionally, in both studies there exist groupwise differences in activation of the anterior cingulate cortex, which is thought to monitor for conflict in the attentional system, and signal the need for adaptation in the attentional network. These data suggest that increased cardiovascular fitness can affect improvements in the plasticity of the aging human brain, and may serve to reduce both biological and cognitive senescence in humans.
Two discriminatory binding sites in theEscherichia colireplication origin are required for DNA strand opening by initiator DnaA-ATP
Authors: McGarry Kevin C. , Ryan Valorie T. , Grimwade Julia E. , Leonard Alan C.
Published: March 2004

Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studiedEscherichia colireplication origin,oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5′TGTGNAT/AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5′TG/TGGATCAG/A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and openoriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation.
Transportin2 functions as importin and mediates nuclear import of HuR
Authors: Güttinger Stephan , Mühlhäusser Petra , Koller-Eichhorn Roland , Brennecke Julius , Kutay Ulrike
Published: March 2004

The RanGTP-binding nuclear transport receptors transportin1 (TRN1) and transportin2 (TRN2) are highly similar in sequence but are reported to function in nuclear import and export, respectively. Here we show that TRN2 possesses properties of a nuclear import receptor. TRN1/2 both interacted with a similar set of RNA-binding proteins in a RanGTP-sensitive manner. TRN2 bound RanGTP with high affinity, a feature of nuclear import receptors. As expected of an import complex, RanGTP also disrupted the interaction between TRN2 and HuR, an RNA-binding protein previously described as a TRN2 export substrate. The HuR nucleocytoplasmic shuttling signal, a sequence resembling the M9 nuclear import signal of hnRNP A1, was necessary and sufficient for TRN-mediated nuclear import of HuR
in vitro
. Finally, crosscompetition experiments demonstrated that HuR nucleocytoplasmic shuttling signal and M9 are imported along redundant pathways involving TRN1/2, substantiating the function of TRN2 in nuclear import.
Correspondence of presaccadic activity in the monkey primary visual cortex with saccadic eye movements
Authors: Supèr Hans , van der Togt Chris , Spekreijse Henk , Lamme Victor A. F.
Published: March 2004

We continuously scan the visual world via rapid or saccadic eye movements. Such eye movements are guided by visual information, and thus the oculomotor structures that determine when and where to look need visual information to control the eye movements. To know whether visual areas contain activity that may contribute to the control of eye movements, we recorded neural responses in the visual cortex of monkeys engaged in a delayed figure-ground detection task and analyzed the activity during the period of oculomotor preparation. We show that ≈100 ms before the onset of visually and memory-guided saccades neural activity in V1 becomes stronger where the strongest presaccadic responses are found at the location of the saccade target. In addition, in memory-guided saccades the strength of presaccadic activity shows a correlation with the onset of the saccade. These findings indicate that the primary visual cortex contains saccade-related responses and participates in visually guided oculomotor behavior.
Interspecific competition between the nematode-trapping fungus, Duddingtonia flagrans, and selected microorganisms and the effect of spore concentration on the efficacy of nematode trapping
Authors: Grønvold J. , Wolstrup J. , Larsen M. , Gillespie A. , Giacomazzi F.
Published: February 2004

The fungus, Duddingtonia flagrans, is able to trap and kill free-living nematode larvae of the cattle parasite Cooperia oncophora when chlamydospores are mixed in cattle faeces. Isolates of Bacillus subtilis (two isolates), Pseudomonas spp. (three isolates) and single isolates of the fungal genera Alternaria, Cladosporium, Fusarium, Trichoderma and Verticillium were isolated from cattle faeces and shown to reduce D. flagrans growth on agar plates. When these isolates were added to cattle faeces containing D. flagrans and nematode larvae of C. oncophora, developing from eggs, none of the isolates reduced nematode mortality attributed to D. flagrans. Similarly, the coprophilic fungus Pilobolus kleinii, which cannot be cultivated on agar, also failed to suppress the ability of D. flagrans to trap and kill developing larvae of C. oncophora. Increasing chlamydospore doses of D. flagrans in faecal cultures resulted in higher nematode mortality. Thus, no evidence of interspecific or intraspecific competition was observed. The consequences of these findings are discussed.
Pallisentis rexus from the Chiang Mai Basin, Thailand: ultrastructural studies on egg envelope development and the mechanism of egg expansion
Authors: Wongkham W. , Whitfield P.J.
Published: February 2004

Pallisentis rexus Wongkham & Whitfield, 1999 (Eoacanthocephala: Quadrigyridae) infects the freshwater snakehead fish, Channa striata, in the Chiang Mai Basin, Thailand. All stages of egg development within the body cavity of the female parasite were observed, using transmission electron microscopy. Changes in mature eggs after contact with water were also investigated. The mature egg has five egg envelopes separated from each other by four gaps. The fertilization membrane, which is formed first, is pushed centrifugally by other, subsequently formed, envelopes and gaps, which produces a final total shell thickness of 8–36 μm around the acanthor. The disappearance of the outermost layer and the unpleating of an adjacent inner layer causes the expansion of eggs on contact with water. The volume of an expanded egg is approximately 27 times that of an unexpanded one, but the density of eggs is reduced from a value greater than water to one almost equal to water. This is believed to aid the dispersion of eggs.
Metacercarial utilization of a naturally infected single species (Lymnaea peregra) snail community by Echinoparyphium recurvatum
Authors: Morley N.J. , Lewis J.W. , Adam M.E.
Published: February 2004

Utilization of a single-species molluscan community of Lymnaea peregra by metacercariae of Echinoparyphium recurvatum over a summer (July–September) period in south-east England showed an increase in the mean number of cysts per host with host size and time of exposure. Aggregation resulting from host and habitat-related factors increased with host size and time of exposure. Encystment within the host was restricted to the peripheral organs in smaller juvenile snails but as snails increased in size, metacercariae were distributed throughout the tissues.
Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni
Authors: Long X.-C. , Bahgat M. , Chlichlia K. , Ruppel A. , Li Y.-L.
Published: February 2004

Schistosoma japonicum and S. mansoni were tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental in S. japonicum and parenchymal in S. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme of S. japonicum had an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite–host interrelation remains to be clarified.