bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway
Authors: Orozco-Cardenas Martha , Ryan Clarence A.
Published: July 2002

Hydrogen peroxide (H
2
O
2
) generated in response to wounding can be detected at wound sites and in distal leaf veins within 1 hr after wounding. The response is systemic and maximizes at about 4–6 hr in both wounded and unwounded leaves, and then declines. The timing of the response corresponds with an increase in wound-inducible polygalacturonase (PG) mRNA and enzyme activity previously reported, suggesting that oligogalacturonic acid (OGA) fragments produced by PG are triggering the H
2
O
2
response. Systemin, OGA, chitosan, and methyl jasmonate (MJ) all induce the accumulation of H
2
O
2
in leaves. Tomato plants transformed with an antisense prosystemin gene produce neither PG activity or H
2
O
2
in leaves in response to wounding, implicating systemin as a primary wound signal. The antisense plants do produce both PG activity and H
2
O
2
when supplied with systemin, OGA, chitosan, or MJ. A mutant tomato line compromised in the octadecanoid pathway does not exhibit PG activity or H
2
O
2
in response to wounding, systemin, OGA, or chitosan, but does respond to MJ, indicating that the generation of H
2
O
2
requires a functional octadecanoid signaling pathway. Among 18 plant species from six families that were assayed for wound-inducible PG activity and H
2
O
2
generation, 14 species exhibited both wound-inducible PG activity and the generation of H
2
O
2
. Four species, all from the Fabaceae family, exhibited little or no wound-inducible PG activity and did not generate H
2
O
2
. The time course of wound-inducible PG activity and H
2
O
2
in
Arabidopsis thaliana
leaves was similar to that found in tomato. The cumulative data suggest that systemic wound signals that induce PG activity and H
2
O
2
are widespread in the plant kingdom and that the response may be associated with the defense of plants against both herbivores and pathogens.
Autoregulation of pituitary corticotroph SOCS-3 expression: Characterization of the murine SOCS-3 promoter
Authors: Auernhammer C. J. , Bousquet C. , Melmed S.
Published: July 2002

Pituitary corticotroph SOCS-3 is a novel intracellular regulator of leukemia inhibitory factor (LIF)-mediated proopiomelanocortin gene expression and adrenocorticotropic hormone (ACTH) secretion, inhibiting LIF-activated Janus kinase-signal transducers and activators of transcription (STAT) signaling in a negative autoregulatory loop. We now demonstrate in corticotroph AtT-20 cells that LIF-stimulated endogenous SOCS-3 mRNA expression is blocked in stable transfectants of SOCS-3 wild type or in dominant negative STAT-3 mutants, respectively. We characterized ≈3.8-kb genomic 5′ sequence of murine SOCS-3, including ≈2.9-kb sequence upstream of the transcription start site (+1), which was determined by 5′ rapid amplification of cDNA ends and RNase protection assay. Different 5′ constructs were cloned into the pGL3Basic vector, and luciferase activity was assayed in transiently transfected ACTH-secreting corticotroph AtT-20 cells. A STAT-1/STAT-3 binding element, located at nucleotides −72 to −64, was essential for LIF stimulation of SOCS-3 promoter activity. LIF induced 10-fold increased luciferase activity in a wild-type construct spanning −2757 to +929 bases. However, deletion or point mutation of the STAT-1/STAT-3 binding element abrogated LIF action (2- to 3-fold). Electrophoretic mobility-shift assay analysis confirmed specific binding of STAT-1 and STAT-3 to this region. These results characterize the genomic 5′ region of murine SOCS-3 and identify an important STAT-1/STAT-3 binding element therein. Thus, LIF-stimulated SOCS-3 gene expression is at least in part mediated by STAT-3 and STAT-1. The cytokine inhibitor SOCS-3 acts in a negative loop to autoregulate its own gene expression, thus limiting its accumulation in the corticotroph cell. These results demonstrate a mechanism for corticotroph plasticity with rapid “on” and “off” ACTH induction in response to neuro-immuno-endocrine stimuli, such as LIF.
Cancer dormancy and cell signaling: Induction of p21waf1initiated by membrane IgM engagement increases survival of B lymphoma cells
Authors: Marches Radu , Hsueh Robert , Uhr Jonathan W.
Published: July 2002

The p21WAF1(p21) cyclin-dependent kinase inhibitor plays a major role in regulating cell cycle arrest. It was recently reported that the p53-independent elevation of p21 protein levels is essential in mediating the G1arrest resulting from signal transduction events initiated by the crosslinking of membrane IgM on Daudi Burkitt lymphoma cells. Although the role of p21 in cell cycle regulation is well documented, there is little information concerning its role in antibody-mediated apoptosis. In the present study, we examined the involvement of p21 in the regulation of apoptosis by suppressing its induction in anti-IgM-treated Daudi cells through a p21 antisense expression construct approach. Reduction in induced p21 protein levels resulted in diminished G1arrest and increased apoptosis. The increased susceptibility to anti-IgM-mediated apoptosis was associated with increased caspase-3-like activity and poly-(ADP)ribose polymerase cleavage. These data suggest that p21 may directly interfere with the caspase cascade, thus playing a dual role in regulating both cell cycle progression and apoptosis.
Mechanism of light regulation of Rubisco: A specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f
Authors: Zhang Ning , Portis Archie R.
Published: July 2002

Rubisco activase is a nuclear-encoded chloroplast protein that is required for the light activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)
in vivo
. In most plants examined to date, there are two isoforms of Rubisco activase arising from alternative splicing that differ only at the carboxyl terminus. Here we demonstrate with recombinant proteins that in
Arabidopsis
the larger isoform has a unique role in the regulation of Rubisco activity. At physiological ratios of ADP/ATP, the 46-kDa isoform has minimal ATP hydrolysis and Rubisco activation activity in comparison with the 43-kDa isoform. Analysis of a series of carboxyl-terminal deletion and Ala substitution mutants of the 46-kDa isoform revealed that the presence of Cys residues at positions 411 and 392 were essential to preserve a low ATP hydrolysis and Rubisco activation activity in the presence of ADP. Consequently, incubation of the 46-kDa isoform with DTT and thioredoxin-f increased both activities, whereas incubations with DTT alone or with thioredoxin-m were ineffective. Thioredoxin-f and DTT had no effect on the 43-kDa isoform. However, premixing both isoforms before conducting a reduction and oxidation cycle demonstrated that the activity of both isoforms could be regulated. Reduction and oxidation also modulated the activity of native activase proteins isolated from either
Arabidopsis
or spinach, but not tobacco, which only has the smaller isoform. These findings suggest that in plants containing both isoforms, Rubisco activase regulates the activity of Rubisco in response to light-induced changes in both the ADP/ATP ratio and the redox potential via thioredoxin-f.
Elections, information aggregation, and strategic voting
Authors: Feddersen Timothy , Pesendorfer Wolfgang
Published: July 2002

A central role of elections is the
aggregation of information dispersed within a population. This article
surveys recent work on elections as mechanisms for aggregating
information and on the incentives for voters to vote strategically in
such elections.
Characterization of otoconin-95, the major protein of murine otoconia, provides insights into the formation of these inner ear biominerals
Authors: Verpy Elisabeth , Leibovici Michel , Petit Christine
Published: July 2002

During the course of a study aimed at identifying inner ear-specific transcripts, a 1,906-bp murine cDNA predicted to encode a secreted 469-aa protein with two domains of homology with the secreted phospholipases A
2
was isolated. This transcript is specifically expressed in the inner ear from embryonic day 9.5. The encoded 95-kDa glycoprotein is the major protein of the utricular and saccular otoconia and thus was named otoconin-95. By immunohistofluorescence, otoconin-95 also was detected in the cupulae of the semicircular canals and in previously undescribed transient granular structures of the cochlea. Otoconin-95 was found to be synthesized by various nonsensory cell types, but not by the supporting cells of the sensory epithelia, which produce the otoconial precursor vesicles. In addition, multiple isoforms generated by differential splicing were observed in different combinations during development. Based on the present results, we propose a model for the formation of the otoconia.
Targeting a SWI/SNF-related chromatin remodeling complex to the β-globin promoter in erythroid cells
Authors: Lee Chang-Hun , Murphy Michael R. , Lee Jong-Soo , Chung Jay H.
Published: July 2002

Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human β-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the β-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the β-globin promoter. Because E-RC1 cannot remodel chromatin over the β-globin promoter without EKLF
in vitro
, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the
P
rote
IN PO
sition
I
dentification with
N
uclease
T
ail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the β-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the β-globin promoter in mouse erythroleukemia cells. When the β-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers.
Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus
Authors: Carfí Andrea , Smith Craig A. , Smolak Pamela J. , McGrew Jeffrey , Wiley Don C.
Published: July 2002

Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-Å resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a β-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a β-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.
Evidence that Myb-related CDC5 proteins are required for pre-mRNA splicing
Authors: Burns C. Geoffrey , Ohi Ryoma , Krainer Adrian R. , Gould Kathleen L.
Published: July 2002

The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G
2
/M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction
in vitro
. Furthermore, genetic depletion of the homolog of CDC5 in
Saccharomyces cerevisiae
,
CEF1
, blocks the first step of pre-mRNA processing
in vivo
. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing.
Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels
Authors: Gil Ziv , Silberberg Shai D. , Magleby Karl L.
Published: July 2002

The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, ≈1 μm with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage.
pH gating of ROMK (K
ir
1.1) channels: Control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome
Authors: Schulte U. , Hahn H. , Konrad M. , Jeck N. , Derst C. , Wild K. , Weidemann S. , Ruppersberg J. P. , Fakler B. , Ludwig J.
Published: July 2002

Inward-rectifier K
+
channels of the ROMK (K
ir
1.1) subtype are responsible for K
+
secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other K
ir
channels. Such anomalous titration of this lysine residue (Lys-80 in K
ir
1.1) is accomplished by the tertiary structure of the K
ir
protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in K
ir
1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pK
a
into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pK
a
of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of K
ir
channels as well as for the channel defects found in patients with antenatal Bartter syndrome.
Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests
Authors: Royo Joaquín , León José , Vancanneyt Guy , Albar Juan Pablo , Rosahl Sabine , Ortego Félix , Castañera Pedro , Sánchez-Serrano José J.
Published: July 2002

De novo
jasmonic acid (JA) synthesis is required for wound-induced expression of proteinase inhibitors and other defense genes in potato and tomato. The first step in JA biosynthesis involves lipoxygenase (LOX) introducing molecular oxygen at the C-13 position of linolenic acid. We previously have shown that, in potato, at least two gene families code for 13-LOX proteins. We have now produced transgenic potato plants devoid of one specific 13-LOX isoform (LOX-H3) through antisense-mediated depletion of its mRNA. LOX-H3 depletion largely abolishes accumulation of proteinase inhibitors on wounding, indicating that this specific LOX plays an instrumental role in the regulation of wound-induced gene expression. As a consequence, weight gain of Colorado potato beetles fed on antisense plants is significantly larger than those fed on wild-type plants. The poorer performance of LOX-H3-deficient plants toward herbivory is more evident with a polyphagous insect; larvae of beet armyworm reared on the antisense lines have up to 57% higher weight than those fed on nontransformed plants. LOX-H3 thus appears to regulate gene activation in response to pest attack, and this inducible response is likely to be a major determinant for reducing performance of nonspecialized herbivores. However, the regulatory role of LOX-H3 is not caused by its involvement in the wound-induced increase of JA, as wild-type and LOX-H3 deficient plants have similar jasmonate levels after wounding. LOX-H3-deficient plants have higher tuber yields. The apparent effect of suppressing the inducible defensive response on plant vigor suggests that it may pose a penalty in plant fitness under nonstress situations.
A neomorphic syntaxin mutation blocks volatile-anesthetic action in
Caenorhabditis elegans
Authors: van Swinderen Bruno , Saifee Owais , Shebester Laynie , Roberson Russell , Nonet Michael L. , Crowder C. Michael
Published: July 2002

The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no
in vivo
data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode
Caenorhabditis elegans
, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets.
Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2
Authors: Saxton Tracy M. , Pawson Tony
Published: July 2002

The SH2 domain-containing tyrosine phosphatase Shp2 plays a pivotal role during the gastrulation of vertebrate embryos. However, because of the complex phenotype observed in mouse mutant embryos, the precise role of Shp2 during development is unclear. To define the specific functions of this phosphatase,
Shp2
homozygous mutant embryonic stem cells bearing the
Rosa-26
LacZ transgene were isolated and used to perform a chimeric analysis. Here, we show that
Shp2
mutant cells amass in the tail bud of embryonic day 10.5 chimeric mouse embryos and that this accumulation begins at the onset of gastrulation. At this early stage,
Shp2
mutant cells collect in the primitive streak of the epiblast and thus show deficiencies in their contribution to the mesoderm lineage. In high-contribution chimeras, we show that overaccumulation of
Shp2
mutant cells at the posterior end of the embryo results in two abnormal phenotypes: spina bifida and secondary neural tubes. Consistent with a failure to undergo morphogenic movements at gastrulation, Shp2 is required for embryo fibroblast cells to mount a positive chemotactic response to acidic fibroblast growth factor
in vitro
. Our results demonstrate that Shp2 is required at the initial steps of gastrulation, as nascent mesodermal cells form and migrate away from the primitive streak. The aberrant behavior of Shp2 mutant cells at gastrulation may result from their inability to properly respond to signals initiated by fibroblast growth factors.
Specific association of the gene product of
PKD2
with the TRPC1 channel
Authors: Tsiokas Leonidas , Arnould Thierry , Zhu Chenwen , Kim Emily , Walz Gerd , Sukhatme Vikas P.
Published: July 2002

The function(s) of the genes (
PKD1
and
PKD
2) responsible for the majority of cases of autosomal dominant polycystic kidney disease is unknown. While
PKD1
encodes a large integral membrane protein containing several structural motifs found in known proteins involved in cell–cell or cell–matrix interactions, PKD2 has homology to PKD1 and the major subunit of the voltage-activated Ca
2+
channels. We now describe sequence homology between PKD2 and various members of the mammalian transient receptor potential channel (TRPC) proteins, thought to be activated by G protein-coupled receptor activation and/or depletion of internal Ca
2+
stores. We show that PKD2 can directly associate with TRPC1 but not TRPC3 in transfected cells and
in vitro
. This association is mediated by two distinct domains in PKD2. One domain involves a minimal region of 73 amino acids in the C-terminal cytoplasmic tail of PKD2 shown previously to constitute an interacting domain with PKD1. However, distinct residues within this region mediate specific interactions with TRPC1 or PKD1. The C-terminal domain is sufficient but not necessary for the PKD2–TRPC1 association. A more N-terminal domain located within transmembrane segments S2 and S5, including a putative pore helical region between S5 and S6, is also responsible for the association. Given the ability of the TRPC to form functional homo- and heteromultimeric complexes, these data provide evidence that PKD2 may be functionally related to TRPC proteins and suggest a possible role of PKD2 in modulating Ca
2+
entry in response to G protein-coupled receptor activation and/or store depletion.
Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-Å resolution
Authors: Ikemizu Shinji , Sparks Lisa M. , van der Merwe P. Anton , Harlos Karl , Stuart David I. , Jones E. Yvonne , Davis Simon J.
Published: July 2002

The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell–cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-Å resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC′C′′ β-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell–cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein–protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies.
High levels of patched gene mutations in basal-cell carcinomas from patients with xeroderma pigmentosum
Authors: Bodak Nathalie , Queille Sophie , Avril Marie Françoise , Bouadjar Bakar , Drougard Christiane , Sarasin Alain , Daya-Grosjean Leela
Published: July 2002

Recently,
hptc
, a human gene homologous to the
Drosophila
segment polarity gene
patched
(
ptc
), has been implicated in the nevoid basal-cell carcinoma (BCC) syndrome, and somatic mutations of
hptc
also have been found in sporadic BCCs, the most frequent cancers found in the white population. We have analyzed the
hptc
gene, postulated to be a tumor suppressor gene, in 22 BCCs from patients with the hyperphotosensitive genodermatosis xeroderma pigmentosum (XP). Patients with XP are deficient in the repair of UV-induced DNA lesions and are characterized by their predisposition to cancers in sun-exposed skin. Analysis using PCR–single-strand conformation polymorphism of the
hptc
gene identified 19 alterations in 16 of 22 (73%) of the BCCs examined. Only two (11%) deletions of the
hptc
gene were found in XP BCCs compared with >30% rearrangement observed in non-XP sporadic BCCs, and 17 of 19 (89%) were base substitutions. Among the 17 base substitutions, 11 (65%) were CC → TT tandem mutations, and 4 (23%) were C → T substitutions, all targeted at bipyrimidine sites. Hence, a significantly higher number (15 of 19; 79%) of UV-specific alterations are seen in XP tumors, in contrast to non-XP sporadic BCCs. Interestingly, we have found that in 7 of 14 (50%) XP BCCs analyzed, both
hptc
and the tumor suppressor gene
p53
are mutated. Not only have our data indicated the key role played by
hptc
in the development of BCCs, they also have substantiated the link between unrepaired UV-induced DNA lesions and skin carcinogenesis, as exemplified by the UV-specific alterations of different genes in the same tumors.
Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing
Authors: Gaur Rajesh K. , Beigelman Leonid , Haeberli Peter , Maniatis Tom
Published: July 2002

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2and N7groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.
Contribution of S opponent cells to color appearance
Authors: De Valois Russell L. , De Valois Karen K. , Mahon Luke E.
Published: July 2002

We measured the regions in isoluminant color space over which observers perceive red, yellow, green, and blue and examined the extent to which the colors vary in perceived amount within these regions. We compared color scaling of various isoluminant stimuli by using large spots, which activate all cone types, to that with tiny spots in the central foveola, where S cones, and thus S opponent (S
o
) cell activity, are largely or entirely absent. The addition of S
o
input to that from the L and M opponent cells changes the chromatic appearance of all colors, affecting each primary color in different chromatic regions in the directions and by the amount predicted by our color model. Shifts from white to the various chromatic stimuli we used produced sinusoidal variations in cone activation as a function of color angle for each cone type and in the responses of lateral geniculate cells. However, psychophysical color-scaling functions for 2° spots were nonsinusoidal, being much more peaked. The color-scaling functions are well fit by sine waves raised to exponents between 1 and 3. The same is true for the color responses of a large subpopulation of striate cortex cells. The narrow color tuning, the discrepancies between the spectral loci of the peaks of the color-scaling curves and those of lateral geniculate cells, and the changes in color appearance produced by eliminating S
o
input provide evidence for a cortical processing stage at which the color axes are rotated by a combination of the outputs of S
o
cells with those of L and M opponent cells in the manner that we postulated earlier. There seems to be an expansive response nonlinearity at this stage.
Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity
Authors: Osuga Jun-ichi , Ishibashi Shun , Oka Teruaki , Yagyu Hiroaki , Tozawa Ryuichi , Fujimoto Akihisa , Shionoiri Futoshi , Yahagi Naoya , Kraemer Frederic B. , Tsutsumi Osamu , Yamada Nobuhiro
Published: July 2002

Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis
not only of triacylglycerol stored in adipose tissue but also of
cholesterol esters in the adrenals, ovaries, testes, and macrophages.
To elucidate its precise role in the development of obesity and
steroidogenesis, we generated HSL knockout mice by homologous
recombination in embryonic stem cells. Mice homozygous for the mutant
HSL allele (HSL−/−) were superficially normal except that the males
were sterile because of oligospermia. HSL−/− mice did not have
hypogonadism or adrenal insufficiency. Instead, the testes completely
lacked neutral cholesterol ester hydrolase (NCEH) activities and
contained increased amounts of cholesterol ester. Many epithelial cells
in the seminiferous tubules were vacuolated. NCEH activities were
completely absent from both brown adipose tissue (BAT) and white
adipose tissue (WAT) in HSL−/− mice. Consistently, adipocytes were
significantly enlarged in the BAT (5-fold) and, to a lesser extent in
the WAT (2-fold), supporting the concept that the hydrolysis of
triacylglycerol was, at least in part, impaired in HSL−/− mice. The
BAT mass was increased by 1.65-fold, but the WAT mass remained
unchanged. Discrepancy of the size differences between cell and tissue
suggests the heterogeneity of adipocytes. Despite these morphological
changes, HSL−/− mice were neither obese nor cold sensitive.
Furthermore, WAT from HSL−/− mice retained 40% of triacylglycerol
lipase activities compared with the wild-type WAT. In conclusion, HSL
is required for spermatogenesis but is not the only enzyme that
mediates the hydrolysis of triacylglycerol stored in adipocytes.
RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: Evidenced
in vivo
by an anti-farnesyl cysteine antibody
Authors: Baron Rudi , Fourcade Emmanuelle , Lajoie-Mazenc Isabelle , Allal Cuider , Couderc Bettina , Barbaras Ronald , Favre Gilles , Faye Jean-Charles , Pradines Anne
Published: July 2002

Protein isoprenylation is a lipid posttranslational modification
required for the function of many proteins that share a
carboxyl-terminal CAAX motif. The X residue determines which isoprenoid
will be added to the cysteine. When X is a methionine or serine, the
farnesyl-transferase transfers a farnesyl, and when X is a leucine
or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl
group. But despite its CKVL motif, RhoB was reported to be both
geranylgeranylated and farnesylated. Thus, the determinants of RhoB
prenylation appear more complex than initially thought. To determine
the role of RhoB CAAX motif, we designed RhoB mutants with modified
CAAX sequence expressed in baculovirus-infected insect cells. We
demonstrated that RhoB was prenylated as a function of the three
terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or
CLLL motif of Rap1A was farnesylated or geranylgeranylated,
respectively. Next, we produced a specific polyclonal antibody against
farnesyl cysteine methyl ester allowing prenylation analysis avoiding
the metabolic labeling restrictions. We confirmed that the unique
modification of the RhoB CAAX box was sufficient to direct the RhoB
distinct prenylation in mammalian cells and, inversely, that a
RhoA-CKVL chimera could be alternatively prenylated. Moreover, the
immunoprecipitation of endogenous RhoB from cells with the
anti-farnesyl cysteine antibody suggested that wild-type RhoB is
farnesylated
in vivo
. Taken together, our results
demonstrated that the three last carboxyl amino acids are the main
determinants for RhoB prenylation and described an anti-farnesyl
cysteine antibody as a useful tool for understanding the cellular
control of protein farnesylation.
Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags
Authors: de Souza Sandro J. , Camargo Anamaria A. , Briones Marcelo R. S. , Costa Fernando F. , Nagai Maria Aparecida , Verjovski-Almeida Sergio , Zago Marco A. , Andrade Luis Eduardo C. , Carrer Helaine , El-Dorry Hamza F. A. , Espreafico Enilza M. , Habr-Gama Angelita , Giannella-Neto Daniel , Goldman Gustavo H. , Gruber Arthur , Hackel Christine , Kimura Edna T. , Maciel Rui M. B. , Marie Suely K. N. , Martins Elizabeth A. L. , Nóbrega Marina P. , Paçó-Larson Maria Luisa , Pardini Maria Inês M. C. , Pereira Gonçalo G. , Pesquero João Bosco , Rodrigues Vanderlei , Rogatto Silvia R. , da Silva Ismael D. C. G. , Sogayar Mari C. , de Fátima Sonati Maria , Tajara Eloiza H. , Valentini Sandro R. , Acencio Marcio , Alberto Fernando L. , Amaral Maria Elisabete J. , Aneas Ivy , Bengtson Mário Henrique , Carraro Dirce M. , Carvalho Alex F. , Carvalho Lúcia Helena , Cerutti Janete M. , Corrêa Maria Lucia C. , Costa Maria Cristina R. , Curcio Cyntia , Gushiken Tsieko , Ho Paulo L. , Kimura Elza , Leite Luciana C. C. , Maia Gustavo , Majumder Paromita , Marins Mozart , Matsukuma Adriana , Melo Analy S. A. , Mestriner Carlos Alberto , Miracca Elisabete C. , Miranda Daniela C. , Nascimento Ana Lucia T. O. , Nóbrega Francisco G. , Ojopi Élida P. B. , Pandolfi José Rodrigo C. , Pessoa Luciana Gilbert , Rahal Paula , Rainho Claudia A. , da Ro's Nancy , de Sá Renata G. , Sales Magaly M. , da Silva Neusa P. , Silva Tereza C. , da Silva Wilson , Simão Daniel F. , Sousa Josane F. , Stecconi Daniella , Tsukumo Fernando , Valente Valéria , Zalcberg Heloisa , Brentani Ricardo R. , Reis Luis F. L. , Dias-Neto Emmanuel , Simpson Andrew J. G.
Published: July 2002

Transcribed sequences in the human genome can be identified with
confidence only by alignment with sequences derived from cDNAs
synthesized from naturally occurring mRNAs. We constructed a set of
250,000 cDNAs that represent partial expressed gene sequences and that
are biased toward the central coding regions of the resulting
transcripts. They are termed ORF expressed sequence tags (ORESTES). The
250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181
(1.45%) were found to match sequences in chromosome 22 with at least
one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67
(44.6%) of the 150 related genes, and for 45 of the 148 (30.4%)
EST-predicted genes on this chromosome. Using a set of stringent
criteria to validate our sequences, we identified a further 219
previously unannotated transcribed sequences on chromosome 22. Of
these, 171 were in fact also defined by EST or full length cDNA
sequences available in GenBank but not utilized in the initial
annotation of the first human chromosome sequence. Thus despite
representing less than 15% of all expressed human sequences in the
public databases at the time of the present analysis, ORESTES sequences
defined 48 transcribed sequences on chromosome 22 not defined by other
sequences. All of the transcribed sequences defined by ORESTES
coincided with DNA regions predicted as encoding exons by
genscan
. (
http://genes.mit.edu/GENSCAN.html
).
A whole-genome microarray reveals genetic diversity among
Helicobacter pylori
strains
Authors: Salama Nina , Guillemin Karen , McDaniel Timothy K. , Sherlock Gavin , Tompkins Lucy , Falkow Stanley
Published: July 2002

Helicobacter pylori
colonizes the stomach of half of
the world's population, causing a wide spectrum of disease ranging
from asymptomatic gastritis to ulcers to gastric cancer. Although the
basis for these diverse clinical outcomes is not understood, more
severe disease is associated with strains harboring a pathogenicity
island. To characterize the genetic diversity of more and less virulent
strains, we examined the genomic content of 15
H. pylori
clinical isolates by using a whole genome
H. pylori
DNA
microarray. We found that a full 22% of
H. pylori
genes
are dispensable in one or more strains, thus defining a minimal
functional core of 1281
H. pylori
genes. While the core
genes encode most metabolic and cellular processes, the strain-specific
genes include genes unique to
H. pylori
, restriction
modification genes, transposases, and genes encoding cell surface
proteins, which may aid the bacteria under specific circumstances
during their long-term infection of genetically diverse hosts. We
observed distinct patterns of the strain-specific gene distribution
along the chromosome, which may result from different mechanisms of
gene acquisition and loss. Among the strain-specific genes, we have
found a class of candidate virulence genes identified by their
coinheritance with the pathogenicity island.
Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Iα inhibit NO-induced cerebral dilation
Authors: Dostmann Wolfgang R. G. , Taylor Mark S. , Nickl Christian K. , Brayden Joseph E. , Frank Ronald , Tegge Werner J.
Published: July 2002

Arrays of octameric peptide libraries on cellulose paper were
screened by using
32
P-autophosphorylated cGMP-dependent
protein kinase Iα (cGPK) to identify peptide sequences with high
binding affinity for cGPK. Iterative deconvolution of every amino acid
position in the peptides identified the sequence LRK
5
H
(W45) as having the highest binding affinity. Binding of W45 to cGPK
resulted in selective inhibition of the kinase with
K
i
values of 0.8 μM and 560 μM for cGPK
and cAMP-dependent protein kinase (cAPK), respectively. Fusion of W45
to membrane translocation signals from
HIV-1
tat protein
(YGRKKRRQRRRPP-LRK
5
H, DT-2) or
Drosophila
Antennapedia homeo-domain (RQIKIWFQNRRMKWKK-LRK
5
H, DT-3)
proved to be an efficient method for intracellular delivery of these
highly charged peptides. Rapid translocation of the peptides into
intact cerebral arteries was demonstrated by using fluorescein-labeled
DT-2 and DT-3. The inhibitory potency of the fusion peptides was even
greater than that for W45, with
K
i
values of
12.5 nM and 25 nM for DT-2 and DT-3, respectively. Both peptides were
still poor inhibitors of cAPK. Selective inhibition of cGPK by DT-2 or
DT-3 in the presence of cAPK was demonstrated
in vitro
.
In pressurized cerebral arteries, DT-2 and DT-3 substantially decreased
NO-induced dilation. This study provides functional characterization of
a class of selective cGPK inhibitor peptides in vascular smooth muscle
and reveals a central role for cGPK in the modulation of vascular
contractility.
Poly(ADP-ribosyl)ation basally activated by DNA strand breaks reflects glutamate–nitric oxide neurotransmission
Authors: Pieper Andrew A. , Blackshaw Seth , Clements Emily E. , Brat Daniel J. , Krug David K. , White Alison J. , Pinto-Garcia Patricia , Favit Antonella , Conover Jill R. , Snyder Solomon H. , Verma Ajay
Published: July 2002

Poly(ADP-ribose) polymerase (PARP) transfers ADP ribose groups from NAD
+
to nuclear proteins after activation by DNA strand breaks. PARP overactivation by massive DNA damage causes cell death via NAD
+
and ATP depletion. Heretofore, PARP has been thought to be inactive under basal physiologic conditions. We now report high basal levels of PARP activity and DNA strand breaks in discrete neuronal populations of the brain, in ventricular ependymal and subependymal cells and in peripheral tissues. In some peripheral tissues, such as skeletal muscle, spleen, heart, and kidney, PARP activity is reduced only partially in mice with PARP-1 gene deletion (PARP-1
−/−
), implicating activity of alternative forms of PARP. Glutamate neurotransmission involving
N
-methyl-
d
-aspartate (NMDA) receptors and neuronal nitric oxide synthase (nNOS) activity in part mediates neuronal DNA strand breaks and PARP activity, which are diminished by NMDA antagonists and NOS inhibitors and also diminished in mice with targeted deletion of nNOS gene (nNOS
−/−
). An increase in NAD
+
levels after treatment with NMDA antagonists or NOS inhibitors, as well as in nNOS
−/−
mice, indicates that basal glutamate-PARP activity regulates neuronal energy dynamics.
Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis
Authors: Voehringer D. W. , Hirschberg D. L. , Xiao J. , Lu Q. , Roederer M. , Lock C. B. , Herzenberg L. A. , Steinman L. , Herzenberg L. A.
Published: July 2002

Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the “caspase cascade.” Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.
National Testing and the Improvement of Classroom Teaching: Can they coexist?
Authors: Williams Julian , Ryan Julie
Published: July 2002

ABSTRACT Can children's responses to tests be used to provide useful diagnostic information for classroom teachers? This article describes an analysis of children's performance in the 1997 UK mathematics tests by 7 and 14 year‐olds. The children's responses and errors were scaled against their ability using Rasch methodology. These were then interpreted in terms of the literature on the psychology of mathematics education, especially that related to misconceptions, and an attempt was made to describe children's progression in thinking as it relates to their test performance. This work has been reported to all primary and secondary schools in England and Wales, and is intended to provide a description of what the children believe and know. As such it provides a resource to help raise teachers' awareness of their children's thinking and present openings for diagnostic assessment and teaching. Opportunities for and obstacles to developing this approach in future are discussed.
Implementation of School Choice Policy: Interpretation and response by parents of students with special educational needs
Authors: Bagley Carl , Woods Philip A. , Woods Glenys
Published: July 2002

Abstract In England a restructured school system has been functioning throughout the 1990s. An integral aspect of this restructuring is the creation of a more competitive public‐market school system aimed at enhancing parental opportunities for choice amongst publicly‐funded schools. What has been the experience of the restructured system by one particular group of parents with specific needs and preferences, namely parents of students with special educational needs (SEN)? This article is intended to provide empirically‐based insights into the preferences, perceptions and responses of such parents. It draws on analyses of quantitative and qualitative data generated by a large‐scale, more general research study on school choice, funded by the UK Economic and Social Research Council (ref. no. R000234079). The findings reveal the depth and range of problems and difficulties encountered by parents of SEN students as they attempt to exercise choice in a more competitive public‐market environment.
M capacity as a lifespan construct: A study of its decrease in ageing subjects
Authors: Morra Sergio , Vigliocco Gabriella , Penello Bianca
Published: July 2002

The performance of 93 ageing subjects (age range, 59-96) was studied in tests that had been proved to measure M capacity in children (Morra, 1994), (i.e. the Backward Digit Span, the Counting Span, and the Mr Cucumber Test). For the sake of comparison, the Forward Digit Span was also administered. Preliminary analyses showed that Backward Digit Span, Counting Span, and Mr Cucumber have a similar rate of decline with age, and are significantly correlated with one another. The goodness of ” t of three different linear structural equation models suggests that, also in the elderly, the scores in these three tests reflect the same latent construct (i.e. M capacity), which in turn is negatively affected by age and positively affected by education. In addition, the variance within each subject of these three scores is usually small. It is concluded that these three tests can also be used as measures of M capacity in ageing subjects.
Nutritional deficits and behavioural development
Authors: Wachs Theodore D.
Published: July 2002

Despite a high incidence level the study of relations between nutritional deficits and children’s behavioural development is a topic that has been relatively neglected by developmental researchers. Such neglect has implications for the generalisability of developmental theories, especially to less developed countries where the majority of the world’s children live and where nutritional deficits are more likely to occur. This paper reviews evidence on the role played by nutritional deficits in children’s development and the mechanisms underlying nutrition → development links. Future directions for collaborative research between clinical, developmental, and nutritional scientists are proposed.