bluuebunny/crossref_metadata_2025 huggingface dataset:
Significance analysis of microarrays applied to the ionizing radiation response
Authors: Tusher Virginia Goss , Tibshirani Robert , Chu Gilbert
Published: July 2002

Microarrays can measure the expression of thousands of genes to identify changes in expression between different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. We describe a method, Significance Analysis of Microarrays (SAM), that assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). When the transcriptional response of human cells to ionizing radiation was measured by microarrays, SAM identified 34 genes that changed at least 1.5-fold with an estimated FDR of 12%, compared with FDRs of 60 and 84% by using conventional methods of analysis. Of the 34 genes, 19 were involved in cell cycle regulation and 3 in apoptosis. Surprisingly, four nucleotide excision repair genes were induced, suggesting that this repair pathway for UV-damaged DNA might play a previously unrecognized role in repairing DNA damaged by ionizing radiation.
The arginine finger of RasGAP helps Gln-61 align the nucleophilic water in GAP-stimulated hydrolysis of GTP
Authors: Resat Haluk , Straatsma T. P. , Dixon David A. , Miller John H.
Published: July 2002

The Ras family of GTPases is a collection of molecular switches
that link receptors on the plasma membrane to signaling pathways that
regulate cell proliferation and differentiation. The accessory
GTPase-activating proteins (GAPs) negatively regulate the cell
signaling by increasing the slow intrinsic GTP to GDP hydrolysis rate
of Ras. Mutants of Ras are found in 25–30% of human tumors. The most
dramatic property of these mutants is their insensitivity to the
negative regulatory action of GAPs. All known oncogenic mutants of Ras
map to a small subset of amino acids. Gln-61 is particularly important
because virtually all mutations of this residue eliminate sensitivity
to GAPs. Despite its obvious importance for carcinogenesis, the role of
Gln-61 in the GAP-stimulated GTPase activity of Ras has remained a
mystery. Our molecular dynamics simulations of the p21ras–p120GAP–GTP
complex suggest that the local structure around the catalytic region
can be different from that revealed by the x-ray crystal structure. We
find that the carbonyl oxygen on the backbone of the arginine finger
supplied in trans by p120GAP (Arg-789) interacts with a water molecule
in the active site that is forming a bridge between the NH
2
group of the Gln-61 and the γ-phosphate of GTP. Thus, Arg-789 may
play a dual role in generating the nucleophile as well as stabilizing
the transition state for P—O bond cleavage.
Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice
Authors: Pruijt Johannes F. M. , Verzaal Perry , van Os Ronald , de Kruijf Evert-Jan F. M. , van Schie Marianke L. J. , Mantovani Alberto , Vecchi Annunciata , Lindley Ivan J. D. , Willemze Roel , Starckx Sofie , Opdenakker Ghislain , Fibbe Willem E.
Published: July 2002

The CXC chemokine interleukin-8 (IL-8/CXCL8) induces rapid mobilization of hematopoietic progenitor cells (HPCs). Previously we showed that mobilization could be prevented completely in mice by pretreatment with neutralizing antibodies against the β2-integrin LFA-1 (CD11a). In addition, murine HPCs do not express LFA-1, indicating that mobilization requires a population of accessory cells. Here we show that polymorphonuclear cells (PMNs) serve as key regulators in IL-8-induced HPC mobilization. The role of PMNs was studied in mice rendered neutropenic by administration of a single injection of antineutrophil antibodies. Absolute neutropenia was observed up to 3–5 days with a rebound neutrophilia at day 7. The IL-8-induced mobilizing capacity was reduced significantly during the neutropenic phase, reappeared with recurrence of the PMNs, and was increased proportionally during the neutrophilic phase. In neutropenic mice, the IL-8-induced mobilizing capacity was restored by the infusion of purified PMNs but not by infusion of mononuclear cells. Circulating metalloproteinase gelatinase B (MMP-9) levels were detectable only in neutropenic animals treated with PMNs in combination with IL-8, showing thatin vivoactivated PMNs are required for the restoration of mobilization. However, IL-8-induced mobilization was not affected in MMP-9-deficient mice, indicating that MMP-9 is not indispensable for mobilization. These data demonstrate that IL-8-induced mobilization of HPCs requires thein vivoactivation of circulating PMNs.
Mnd1p: An evolutionarily conserved protein required for meiotic recombination
Authors: Gerton Jennifer L. , DeRisi Joseph L.
Published: July 2002

We used a functional genomics approach to identify a gene required for meiotic recombination, YGL183c or
MND1. MND1
was spliced in meiotic cells, extending the annotated YGL183c ORF N terminus by 45 aa.
Saccharomyces cerevisiae
mnd1–1
mutants, in which the majority of the
MND1
coding sequence was removed, arrested before the first meiotic division with a phenotype reminiscent of
dmc1
mutants. Physical and genetic analysis showed that these cells initiated recombination, but did not form heteroduplex DNA or double Holliday junctions, suggesting that Mnd1p is involved in strand invasion. Orthologs of
MND1
were identified in protists, several yeasts, plants, and mammals, suggesting that its function has been conserved throughout evolution.
Hd6
, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2
Authors: Takahashi Yuji , Shomura Ayahiko , Sasaki Takuji , Yano Masahiro
Published: July 2002

Hd6
is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the
japonica
variety Nipponbare and the
indica
variety Kasalath. To isolate a gene at
Hd6
, we used a large segregating population for the high-resolution and fine-scale mapping of
Hd6
and constructed genomic clone contigs around the
Hd6
region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited
Hd6
to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of
CK2
α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of
CK2
α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype.
Long-term plasticity in interneurons of the dentate gyrus
Authors: Ross Stephen T. , Soltesz Ivan
Published: July 2002

Single interneurons influence thousands of postsynaptic principal cells, and the control of interneuronal excitability is an important regulator of the computational properties of the hippocampus. However, the mechanisms underlying long-term alterations in the input–output functions of interneurons are not fully understood. We report a mechanism of interneuronal plasticity that leads to the functional enhancement of the gain of glutamatergic inputs in the absence of long-term potentiation of the excitatory synaptic currents. Interneurons in the dentate gyrus exhibit a characteristic, limited (≈8 mV) depolarization of their resting membrane potential after high-frequency stimulation of the perforant path. The depolarization can be observed with either whole-cell or perforated patch electrodes, and it lasts in excess of 3 h. The long-term depolarization is specific to interneurons, because granule cells do not show it. The depolarization requires the activation of Ca
2+
-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the rise of intracellular Ca
2+
, but not
N
-methyl-
d
-aspartate (NMDA) receptor activation. Data on the maintenance of the depolarization point to a major role for a long-term change in the rate of electrogenic Na
+
/K
+
-ATPase pump function in interneurons. As a result of the depolarization, interneurons after the tetanus respond with action potential discharges to previously subthreshold excitatory postsynaptic potentials (EPSPs), even though the EPSPs are not potentiated. These results demonstrate that the plastic nature of the interneuronal resting membrane potential underlies a unique form of long-term regulation of the gain of excitatory inputs to γ-aminobutyric acid (GABA)ergic neurons.
A biosensor assay for studying ligand-membrane receptor interactions: Binding of antibodies and HIV-1 Env to chemokine receptors
Authors: Hoffman Trevor L. , Canziani Gabriela , Jia Li , Rucker Joseph , Doms Robert W.
Published: July 2002

The HIV envelope (Env) protein mediates entry into cells by binding
CD4 and an appropriate coreceptor, which triggers structural changes in
Env that lead to fusion between the viral and cellular membranes. The
major HIV-1 coreceptors are the seven transmembrane domain chemokine
receptors CCR5 and CXCR4. The type of coreceptor used by a virus strain
is an important determinant of viral tropism and pathogenesis, and
virus-receptor interactions can be therapeutic targets. However, Envs
from many virus strains interact with CXCR4 and CCR5 with low affinity
such that direct study of this important interaction is difficult if
not impossible using standard cell-surface binding techniques. We have
developed an approach that makes it possible to study ligand binding to
membrane proteins, including Env–coreceptor interactions, using an
optical biosensor. CCR5, CXCR4, and other membrane proteins were
incorporated into retrovirus particles, which were purified and
attached to the biosensor surface. Binding of conformationally
sensitive antibodies as well as Env to these receptors was readily
detected. The equilibrium dissociation constant for the interaction
between an Env derived from the prototype HIV-1 strain IIIB for CXCR4
was approximately 500 nM, explaining the difficulty in measuring this
interaction using standard equilibrium binding techniques. Retroviral
pseudotypes represent easily produced, stable, homogenous structures
that can be used to present a wide array of single and multiple
membrane-spanning proteins in a native lipid environment for biosensor
studies, thus avoiding the need for detergent solubilization,
purification, and reconstitution. The approach should have general
applicability and can be used to correlate Env–receptor binding
constants to viral tropism and pathogenesis.
Arabidopsis
basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions
Authors: Uno Yuichi , Furihata Takashi , Abe Hiroshi , Yoshida Riichiro , Shinozaki Kazuo , Yamaguchi-Shinozaki Kazuko
Published: July 2002

The induction of the dehydration-responsive
Arabidopsis
gene,
rd29B
, is mediated
mainly by abscisic acid (ABA). Promoter analysis of
rd29B
indicated that two ABA-responsive elements (ABREs)
are required for the dehydration-responsive expression of
rd29B
as cis-acting elements. Three cDNAs encoding basic
leucine zipper (bZIP)-type ABRE-binding proteins were isolated by using
the yeast one-hybrid system and were designated AREB1, AREB2, and AREB3
(ABA-responsive element binding protein). Transcription of the
AREB1
and
AREB2
genes is up-regulated by
drought, NaCl, and ABA treatment in vegetative tissues. In a transient
transactivation experiment using
Arabidopsis
leaf
protoplasts, both the AREB1 and AREB2 proteins activated transcription
of a reporter gene driven by ABRE. AREB1 and AREB2 required ABA for
their activation, because their transactivation activities were
repressed in
aba2
and
abi1
mutants and
enhanced in an
era1
mutant. Activation of AREBs by ABA
was suppressed by protein kinase inhibitors. These results suggest that
both AREB1 and AREB2 function as transcriptional activators in the
ABA-inducible expression of
rd29B
, and further that
ABA-dependent posttranscriptional activation of AREB1 and AREB2,
probably by phosphorylation, is necessary for their maximum activation
by ABA. Using cultured
Arabidopsis
cells, we
demonstrated that a specific ABA-activated protein kinase of 42-kDa
phosphorylated conserved N-terminal regions in the AREB proteins.
Glutathione biosynthesis inArabidopsistrichome cells
Authors: Gutiérrez-Alcalá Gloria , Gotor Cecilia , Meyer Andreas J. , Fricker Mark , Vega José M. , Romero Luis C.
Published: July 2002

InArabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed byin situhybridization the tissue-specific expression of the genesAtcys-3Aand sat5, which encodeO-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well asgsh1andgsh2, which encode γ-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes ofArabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 ± 25, 80 ± 2, and 144 ± 19 μM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes.Atcys-3A,sat5, gsh1, andgsh2mRNA levels show a decrease in transcript abundance, and [GSH]cytis reduced to 47 ± 5 μM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.
Three conformational states of scallop myosin S1
Authors: Houdusse A. , Szent-Györgyi A. G. , Cohen C.
Published: July 2002

We have determined the structure of the intact scallop myosin head,
containing both the motor domain and the lever arm, in the
nucleotide-free state and in the presence of MgADP⋅VO4,
corresponding to the transition state. These two new structures,
together with the previously determined structure of scallop S1
complexed with MgADP (which we interpret as a detached ATP state),
reveal three conformations of an intact S1 obtained from a single
isoform. These studies, together with new crystallization results, show
how the conformation of the motor depends on the nucleotide content of
the active site. The resolution of the two new structures (≈4 Å) is
sufficient to establish the relative positions of the subdomains and
the overall conformation of the joints within the motor domain as well
as the position of the lever arm. Comparison of available crystal
structures from different myosin isoforms and truncated constructs in
either the nucleotide-free or transition states indicates that the
major features within the motor domain are relatively invariant in both
these states. In contrast, the position of the lever arm varies
significantly between different isoforms. These results indicate that
the heavy-chain helix is pliant at the junction between the converter
and the lever arm and that factors other than the precise position of
the converter can influence the position of the lever arm. It is
possible that this pliant junction in the myosin head contributes to
the compliance known to be present in the crossbridge.
Dynamic changes in the localization of thermally unfolded nuclear proteins associated with chaperone-dependent protection
Authors: Nollen Ellen A. A. , Salomons Florian A. , Brunsting Jeanette F. , Want Johannes J. L. van der , Sibon Ody C. M. , Kampinga Harm H.
Published: July 2002

Molecular chaperones are involved in the protection of cells against protein damage through their ability to hold, disaggregate, and refold damaged proteins or their ability to facilitate degradation of damaged proteins. Little is known about how these processes are spatially coordinated in cells. Using a heat-sensitive nuclear model protein luciferase fused to the traceable, heat-stable enhanced green fluorescent protein (N-luc-EGFP), we now show that heat inactivation and insolubilization of luciferase were associated with accumulation of N-luc-EGFP at multiple foci throughout the nucleus. Coexpression of Hsp70, one of the major mammalian chaperones, reduced the formation of these small foci during heat shock. Instead, the heat-unfolded N-luc-EGFP accumulated in large, insoluble foci. Immunofluorescence analysis revealed that these foci colocalized with the nucleoli. Time-lapse analysis demonstrated that protein translocation to the nucleolus, in contrast to the accumulation at small foci, was fully reversible upon return to the normal growth temperature. This reversibility was associated with an increase in the level of active and soluble luciferase. Expression of a carboxyl-terminal deletion mutant of Hsp70(1–543), which lacked chaperone activity, had no effect on the localization of N-luc-EGFP, which suggests that the Hsp70 chaperone activity is required for the translocation events. Our data show that Hsp70 not only is involved in holding and refolding of heat-unfolded nuclear proteins but also drives them to the nucleolus during stress. This might prevent random aggregation of thermolabile proteins within the nucleus, thereby allowing their refolding at the permissive conditions and preventing indirect damage to other nuclear components.
Expression of
bbc3
, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals
Authors: Han Jia-wen , Flemington Cathy , Houghton Anne B. , Gu Zhengming , Zambetti Gerard P. , Lutz Robert J. , Zhu Li , Chittenden Thomas
Published: July 2002

BH3-only proteins function at a proximal point in a conserved
cell death pathway by binding, through their BH3 domains, to other
Bcl-2 family members and triggering mitochondrial events associated
with apoptosis. Here, we describe a strongly
pro-apoptotic BH3-only protein, designated Bbc3, whose
expression increases in response to diverse apoptotic stimuli.
bbc3
mRNA levels were induced by exposure to
DNA-damaging agents and by wild-type p53, which mediates DNA
damage-induced apoptosis. p53 transactivated
bbc3
through consensus p53 binding sites within the
bbc3
promoter region, indicating that
bbc3
is a direct target of p53. Additionally,
bbc3
mRNA was induced by p53-independent
apoptotic stimuli, including dexamethasone treatment of
thymocytes, and serum deprivation of tumor cells. Insulin-like growth
factor-1 and epidermal growth factor, growth factors with broad
anti-apoptotic activity, were each sufficient to suppress Bbc3
expression in serum-starved tumor cells. These results suggest that the
transcriptional regulation of
bbc3
contributes to the
transduction of diverse cell death and survival signals.
Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral,Pocillopora damicornis
Authors: Domart-Coulon Isabelle J. , Elbert David C. , Scully Erik P. , Calimlim Precilia S. , Ostrander Gary K.
Published: July 2002

The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral,Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneousin vitrodissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate thatin vitrocrystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level.
Chemical synthesis and spontaneous folding of a multidomain protein: Anticoagulant microprotein S
Authors: Hackeng Tilman M. , Fernández José A. , Dawson Philip E. , Kent Stephen B. H. , Griffin John H.
Published: July 2002

Because of recent high-yield native ligation techniques, chemical
synthesis of larger multidomain bioactive proteins is rapidly coming
within reach. Here we describe the total chemical synthesis of a
designed “microprotein S,” comprising the γ-carboxyglutamic
acid-rich module, the thrombin-sensitive module, and the first
epidermal growth factor-like module of human plasma protein S (residues
1–116). Synthetic microprotein S expressed anticoagulant cofactor
activity for activated protein C in the down-regulation of blood
coagulation, and the anticoagulant activity of microprotein S was not
neutralized by C4b-binding protein, a natural inhibitor of native
protein S in plasma. The correct folding of this complex multidomain
protein was enhanced compared with individual modules because the
γ-carboxyglutamic acid-rich module and the thrombin-sensitive module
markedly facilitated correct folding of the first epidermal growth
factor-like module compared with folding of the first epidermal growth
factor-like module alone. These results demonstrate that total chemical
synthesis of proteins offers an effective way to generate multidomain
biologically active proteins.
Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.
Authors: Puchta H , Dujon B , Hohn B
Published: July 2002

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.
Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.
Authors: Mattevi A , Vanoni M A , Todone F , Rizzi M , Teplyakov A , Coda A , Bolognesi M , Curti B
Published: July 2002

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.
Cell cycling and patterned cell proliferation in the wing primordium of Drosophila.
Authors: Milán M , Campuzano S , García-Bellido A
Published: July 2002

The pattern of cell proliferation in the Drosophila imaginal wing primordium is spatially and temporally heterogeneous. Direct visualization of cells in S, G2, and mitosis phases of the cell cycle reveals several features invariant throughout development. The fraction of cells in the disc in the different cell cycle stages is constant, the majority remaining in G1. Cells in the different phases of the cell cycle mainly appear in small synchronic clusters that are nonclonally derived but result from changing local cell-cell interactions. Cluster synchronization occurs before S and in the G2/M phases. Rates of cell division are neither constant nor clonal features. Cell cycle progression is linear rather than concentric. Clusters appear throughout the disc but with symmetries related to presumptive wing patterns, compartment boundaries, and vein clonal restrictions.
Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity
Authors: Steinhauer David A. , Martín Javier , Lin Yi Pu , Wharton Stephen A. , Oldstone Michael B. A. , Skehel John J. , Wiley Don C.
Published: July 2002

Amino acid substitutions widely distributed throughout the
influenza hemagglutinin (HA) influence the pH of its membrane fusion
activity. We have combined a number of these substitutions in double
mutants and determined the effects on the pH of fusion and on the pH at
which the refolding of HA required for fusion occurs. By analyzing
combinations of mutations in three regions of the metastable neutral-pH
HA that are rearranged at fusion pH we obtain evidence for both
additive and nonadditive effects and for an apparent order of dominance
in the effects of amino acid substitutions in particular regions on the
pH of fusion. We conclude that there are at least three components in
the structural transition required for membrane fusion activity and
consider possible pathways for the transition in relation to the known
differences between neutral and fusion pH HA structures.
Memory systems in the brain and localization of a memory
Authors: Thompson Richard F. , Kim Jeansok J.
Published: July 2002

It is now clear that there are a number of different forms or
aspects of learning and memory that involve different brain systems.
Broadly, memory phenomena have been categorized as explicit or
implicit. Thus, explicit memories for experience involve the
hippocampus–medial temporal lobe system and implicit basic associative
learning and memory involves the cerebellum, amygdala, and other
systems. Under normal conditions, however, many of these brain–memory
systems are engaged to some degree in learning situations. But each of
these brain systems is learning something different about the
situation. The cerebellum is necessary for classical conditioning of
discrete behavioral responses (eyeblink, limb flexion) under all
conditions; however, in the “trace” procedure where a period of
no stimuli intervenes between the conditioned stimulus and the
unconditioned stimulus the hippocampus plays a critical role. Trace
conditioning appears to provide a simple model of explicit memory where
analysis of brain substrates is feasible. Analysis of the role of the
cerebellum in basic delay conditioning (stimuli overlap) indicates that
the memories are formed and stored in the cerebellum. The phenomenon of
cerebellar long-term depression is considered as a putative mechanism
of memory storage.
beta-Amyloid toxicity in organotypic hippocampal cultures: protection by EUK-8, a synthetic catalytic free radical scavenger.
Authors: Bruce A J , Malfroy B , Baudry M
Published: July 2002

Oxygen free radicals have been proposed to mediate amyloid peptide (beta-AP)-induced neurotoxicity. To test this hypothesis, we evaluated the effects of EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, on neuronal injury produced by beta-AP in organotypic hippocampal slice cultures. Cultures of equivalent postnatal day 35 (defined as mature) and 14 (defined as immature) were exposed to various concentrations of beta-AP (1-42 or 1-40) in the absence or presence of 25 microM EUK-8 for up to 72 hours. Neuronal injury was assessed by lactate dehydrogenase release and semiquantitative analysis of propidium iodide uptake at various times after the initiation of beta-AP exposure. Free radical production was inferred from the relative increase in dichlorofluorescein fluorescence, and the degree of lipid peroxidation was determined by assaying thiobarbituric acid-reactive substances. Treatment of mature cultures with beta-AP (50-250 microg/ml) in serum-free conditions resulted in a reproducible pattern of damage, causing a time-dependent increase in neuronal injury accompanied with formation of reactive oxygen species. However, immature cultures were entirely resistant to beta-AP-induced neurotoxicity and also demonstrated no dichlorofluorescein fluorescence or increased lipid peroxidation after beta-AP treatment. Moreover, mature slices exposed to beta-AP in the presence of 25 microM EUK-8 were significantly protected from beta-AP-induced neurotoxicity. EUK-8 also completely blocked beta-AP-induced free radical accumulation and lipid peroxidation. These results not only support a role for oxygen free radicals in beta-AP toxicity but also highlight the therapeutic potential of synthetic radical scavengers in Alzheimer disease.
The
cluA
−
mutant of
Dictyostelium
identifies a novel class of proteins required for dispersion of mitochondria
Authors: Zhu Qianlong , Hulen Durvis , Liu Tongyao , Clarke Margaret
Published: July 2002

The
cluA
gene of
Dictyostelium discoideum
encodes a novel 150-kDa protein. Disruption of
cluA
results in clustering of mitochondria near the cell center. This is a striking difference from normal cells, whose mitochondria are dispersed uniformly throughout the cytoplasm. The mutant cell populations also exhibit an increased frequency of multinucleated cells, suggesting an impairment in cytokinesis. Both phenotypes are reversed by transformation of
cluA
−
cells with a plasmid carrying a constitutively expressed
cluA
gene. The predicted sequence of the
cluA
gene product is homologous to sequences encoded by open reading frames in the genomes of
Saccharomyces cerevisiae
and
Caenorhabditis elegans
, but not to any known protein. The only exception is a short region with some homology to the 42-residue imperfect repeats present in the kinesin light chain, which probably function in protein–protein interaction. These studies identify a new class of proteins that appear to be required for the proper distribution of mitochondria.
Detection of single-molecule interactions using correlated thermal diffusion
Authors: Mehta A. D. , Finer J. T. , Spudich J. A.
Published: July 2002

Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events of individual myosin molecules. We use correlated diffusion to measure the stiffness of a single myosin molecule and estimate its thermal fluctuation in a poststroke state as comparable in amplitude to the measured stroke distance. The use of correlated diffusion to measure kinetics of single-molecule interactions and the stiffness of the interacting moieties should be applicable to any pair of interacting molecules, and not limited to biological motors.
Global air-sea flux of CO
2
: An estimate based on measurements of sea–air pCO
2
difference
Authors: Takahashi Taro , Feely Richard A. , Weiss Ray F. , Wanninkhof Rik H. , Chipman David W. , Sutherland Stewart C. , Takahashi Timothy T.
Published: July 2002

Approximately 250,000 measurements made for the pCO
2
difference between surface water and the marine atmosphere,
ΔpCO
2
, have been assembled for the global oceans.
Observations made in the equatorial Pacific during El Nino events have
been excluded from the data set. These observations are mapped on the
global 4° × 5° grid for a single virtual calendar year (chosen
arbitrarily to be 1990) representing a non-El Nino year. Monthly global
distributions of ΔpCO
2
have been constructed using an
interpolation method based on a lateral advection–diffusion transport
equation. The net flux of CO
2
across the sea surface has
been computed using ΔpCO
2
distributions and
CO
2
gas transfer coefficients across sea surface. The
annual net uptake flux of CO
2
by the global oceans thus
estimated ranges from 0.60 to 1.34 Gt-C⋅yr
−1
depending on different formulations used for wind speed dependence on
the gas transfer coefficient. These estimates are subject to an error
of up to 75% resulting from the numerical interpolation method used to
estimate the distribution of ΔpCO
2
over the global
oceans. Temperate and polar oceans of the both hemispheres are the
major sinks for atmospheric CO
2
, whereas the equatorial
oceans are the major sources for CO
2
. The Atlantic Ocean is
the most important CO
2
sink, providing about 60% of the
global ocean uptake, while the Pacific Ocean is neutral because of its
equatorial source flux being balanced by the sink flux of the temperate
oceans. The Indian and Southern Oceans take up about 20% each.
Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice
Authors: Barrachina María Dolores , Martínez Vicente , Wang Lixin , Wei Jen Yu , Taché Yvette
Published: July 2002

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.
RNA-peptide fusions for the
in vitro
selection of peptides and proteins
Authors: Roberts Richard W. , Szostak Jack W.
Published: July 2002

Covalent fusions between an mRNA and the peptide or protein that it encodes can be generated by
in vitro
translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. The stable linkage between the informational (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. Fusions between a synthetic mRNA and its encoded
myc
epitope peptide have been enriched from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. Covalent RNA-peptide fusions should provide an additional route to the
in vitro
selection and directed evolution of proteins.
Dystrobrevin and dystrophin: An interaction through coiled-coil motifs
Authors: Sadoulet-Puccio Hélène M. , Rajala Michael , Kunkel Louis M.
Published: July 2002

Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction. Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex. Although the nature of dystrobrevin’s association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins. Using the yeast two-hybrid system to identify protein–protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin. The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin. We further refined this site of interaction to these proteins’ homologous coiled-coil motifs that flank their respective syntrophin-binding sites. We also show that the interaction between the dystrobrevin and dystrophin coiled-coil domains is specific and is not due to a nonspecific coiled-coil domain interaction. From the accumulated evidence of protein–protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex.
Chemical complementation identifies a proton acceptor for redox-active tyrosine D in photosystem II
Authors: Kim Sunyoung , Liang Jie , Barry Bridgette A.
Published: July 2002

Through the use of site-directed mutagenesis and chemical rescue, we have identified the proton acceptor for redox-active tyrosine D in photosystem II (PSII). Effects of chemical rescue on the tyrosyl radical were monitored by EPR spectroscopy. We also have acquired the Fourier–transform infrared (FT-IR) spectrum associated with the oxidation of tyrosine D and concomitant protonation of the acceptor. Mutant and isotopically labeled PSII samples are used to assign vibrational lines in the 3,600–3,100 cm
−1
region to N-H modes of His-189 in the D2 polypeptide. When His-189 in D2 is changed to a leucine (HL189D2) in PSII, dramatic alterations of both EPR and FT-IR spectra are observed. When imidazole is introduced into HL189D2 samples, results from both EPR and FT-IR spectroscopy argue that imidazole is functionally reconstituted into an accessible pocket and that imidazole acts as a chemical mimic for His-189. Small perturbations of EPR and FT-IR spectra are consistent with access to this pocket in wild-type PSII, as well. Structures of the analogous site in bacterial reaction centers suggest that an accessible pocket, large enough to contain imidazole, is bordered by tyrosine D and His-189 in the D2 polypeptide. These data provide evidence that His-189 in the D2 polypeptide of PSII acts as a proton acceptor for redox-active tyrosine D and that proton transfer to the imidazole ring facilitates the efficient oxidation/reduction of tyrosine D.
A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening
Authors: Chen Yao-Tseng , Scanlan Matthew J. , Sahin Ugur , Türeci Özlem , Gure Ali O. , Tsang Solam , Williamson Barbara , Stockert Elisabeth , Pfreundschuh Michael , Old Lloyd J.
Published: July 2002

Ser
ological analysis of
re
combinant cDNA
ex
pression libraries (SEREX) using tumor mRNA and autologous patient serum provides a powerful approach to identify immunogenic tumor antigens. We have applied this methodology to a case of esophageal squamous cell carcinoma and identified several candidate tumor targets. One of these, NY-ESO-1, showed restricted mRNA expression in normal tissues, with high-level mRNA expression found only in testis and ovary tissues. Reverse transcription–PCR analysis showed NY-ESO-1 mRNA expression in a variable proportion of a wide array of human cancers, including melanoma, breast cancer, bladder cancer, prostate cancer, and hepatocellular carcinoma. NY-ESO-1 encodes a putative protein of
M
r 17,995 having no homology with any known protein. The pattern of NY-ESO-1 expression indicates that it belongs to an expanding family of immunogenic testicular antigens that are aberrantly expressed in human cancers in a lineage-nonspecific fashion. These antigens, initially detected by either cytotoxic T cells (MAGE, BAGE, GAGE-1) or antibodies [HOM-MEL-40(SSX2), NY-ESO-1], represent a pool of antigenic targets for cancer vaccination.
Supercoiling affects the accessibility of glutathione to DNA-bound molecules: Positive supercoiling inhibits calicheamicin-induced DNA damage
Authors: LaMarr W. A. , Yu L. , Nicolaou K. C. , Dedon P. C.
Published: July 2002

DNA superhelical tension, an important feature of genomic organization, is known to affect the interactions of intercalating molecules with DNA. However, the effect of torsional tension on nonintercalative DNA-binding chemicals has received less attention. We demonstrate here that the enediyne calicheamicin γ
1
I
, a strand-breaking agent specific to the minor groove, causes ≈50% more damage in negatively supercoiled plasmid DNA than in DNA with positive superhelicity. Furthermore, we show that the decrease in damage in positively supercoiled DNA is controlled at the level of thiol activation of the drug. Our results suggest that supercoiling may affect both the activity of nonintercalating genotoxins
in vivo
and the accessibility of glutathione and other small physiologic molecules to DNA-bound chemicals or reactions occurring in the grooves of DNA.
Chromatin components as part of a putative transcriptional repressing complex
Authors: Lehming Norbert , Le Saux Agnès , Schüller Jutta , Ptashne Mark
Published: July 2002

TheDrosophilaHMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast. We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1. The latter is aDrosophilaprotein that is involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.
Different patterns of truncated prion protein fragments correlate with distinct phenotypes in P102L Gerstmann–Sträussler–Scheinker disease
Authors: Parchi Piero , Chen Shu G. , Brown Paul , Zou Wenquan , Capellari Sabina , Budka Herbert , Hainfellner Johannes , Reyes Patricio F. , Golden Gregory T. , Hauw Jean J. , Gajdusek D. Carleton , Gambetti Pierluigi
Published: July 2002

The clinicopathological phenotype of the Gerstmann–Sträussler–Scheinker disease (GSS) variant linked to the codon 102 mutation in the prion protein (PrP) gene (GSS P102L) shows a high heterogeneity. This variability also is observed in subjects with the same prion protein gene
PRNP
haplotype and is independent from the duration of the disease. Immunoblot analysis of brain homogenates from GSS P102L patients showed two major protease-resistant PrP fragments (PrP-res) with molecular masses of ≈21 and 8 kDa, respectively. The 21-kDa fragment, similar to the PrP-res type 1 described in Creutzfeldt–Jakob disease, was found in five of the seven subjects and correlated with the presence of spongiform degeneration and “synaptic” pattern of PrP deposition whereas the 8-kDa fragment, similar to those described in other variants of GSS, was found in all subjects in brain regions showing PrP-positive multicentric amyloid deposits. These data further indicate that the neuropathology of prion diseases largely depends on the type of PrP-res fragment that forms
in vivo
. Because the formation of PrP-res fragments of 7–8 kDa with ragged N and C termini is not a feature of Creutzfeldt–Jakob disease or fatal familial insomnia but appears to be shared by most GSS subtypes, it may represent a molecular marker for this disorder.
A C-terminal motif found in the β
2
-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na
+
/H
+
exchanger regulatory factor family of PDZ proteins
Authors: Hall Randy A. , Ostedgaard Lynda S. , Premont Richard T. , Blitzer Jeremy T. , Rahman Nadeem , Welsh Michael J. , Lefkowitz Robert J.
Published: July 2002

The Na
+
/H
+
exchanger regulatory factor (NHERF) binds to the tail of the β
2
-adrenergic receptor and plays a role in adrenergic regulation of Na
+
/H
+
exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the β
2
receptor. Mutagenesis studies of the β
2
receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the β
2
receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.