bluuebunny/crossref_metadata_2025 huggingface dataset:
A conserved domain is present in different families of vesicular fusion proteins: A new superfamily
Authors: Weimbs Thomas , Low Seng Hui , Chapin Steven J. , Mostov Keith E. , Bucher Philipp , Hofmann Kay
Published: July 2002

We have analyzed conserved domains in t-SNAREs [soluble
N
-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors in the target membrane], proteins that are believed to be involved in the fusion of transport vesicles with their target membrane. By using a sensitive computer method, the generalized profile method, we were able to identify a new homology domain that is common in the two protein families previously identified to act as t-SNAREs, the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) families, which therefore constitute a new superfamily. This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure. The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile. In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains. This domain was also identified in several proteins that have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families. Several new yeast, nematode, and mammalian proteins were identified that belong to the new superfamily. The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins.
Vaccine effect using a live attenuated nef-deficient simian immunodeficiency virus of African green monkeys in the absence of detectable vaccine virus replicationin vivo
Authors: Beer Brigitte , Baier Michael , zur Megede Jan , Norley Stephen , Kurth Reinhard
Published: July 2002

Immunization of adult macaques with live attenuated simian immunodeficiency viruses (SIVs) lacking the nef genes has been shown to protect against challenge with full-length pathogenic SIV. To test live attenuated virus vaccines for the first time in a natural host we have constructed a mutant SIV from African green monkeys (SIVagm) with a deletion of 125 bp in the nef gene (SIVagm3Δnef). This mutant showed moderately delayedin vitroreplication in the T cell line MOLT-4/8 and in primary peripheral blood mononuclear cells from African green monkeys (Cercopithecus aetiops) and pig-tailed macaques (Macaca nemestrina) compared with cloned wild-type SIVagm3. In contrast,in vivoreplication of SIVagm3Δnef in African green monkeys was severely impaired or undetectable and did not induce seroconversion. After challenge with wild-type SIVagm3 the SIVagm3Δnef preinoculated African green monkeys showed a memory antibody response that declined after week 2. In three of four African green monkeys the cell-associated virus load and in two of four African green monkeys the plasma virus load was dramatically decreased after the challenge compared with naive control animals. The remaining animal showed no evidence of productive challenge virus replication. This study demonstrates that a strong vaccine effect or protection in the SIVagm/African green monkey system is possible using a live attenuated vaccine in the absence of a productive infection and corresponding humoral immune response.
Aldol sensors for the rapid generation of tunable fluorescence by antibody catalysis
Authors: List Benjamin , Barbas Carlos F. , Lerner Richard A.
Published: July 2002

The synthesis of novel fluorogenic
retro
-aldol substrates for aldolase antibody 38C2 is described. These substrates are efficiently and specifically processed by antibody aldolases but not by natural cellular enzymes. Together, the fluorogenic substrates and antibody aldolases provide reporter gene systems that are compatible with living cells. The broad scope of the antibody aldolase allows for the processing of a range of substrates that can be designed to allow fluorescence monitoring at a variety of wavelengths. We also have developed the following concept in fluorescent protein tags. β-Diketones bearing a fluorescent tag are bound covalently by the aldolase antibody and not other proteins. We anticipate that proteins fused with the antibody can be tagged specifically and covalently within living cells with fluorophores of virtually any color, thereby providing an alternative to green fluorescent protein fusions.
DNA-binding domains of Fos and Jun do not induce DNA curvature: An investigation with solution and gel methods
Authors: Sitlani Ayesha , Crothers Donald M.
Published: July 2002

We demonstrate the use of a DNA minicircle competition binding assay, together with DNA cyclization kinetics and gel-phasing methods, to show that the DNA-binding domains (dbd) of the heterodimeric leucine zipper protein Fos–Jun do not bend the AP-1 target site. Our DNA constructs contain an AP-1 site phased by 1–4 helical turns against an A-tract-directed bend. Competition binding experiments reveal that (dbd)Fos–Jun has a slight preference for binding to linear over circular AP-1 DNAs, independent of whether the site faces in or out on the circle. This result suggests that (dbd)Fos–Jun slightly stiffens rather than bends its DNA target site. A single A-tract bend replacing the AP-1 site is readily detected by its effect on cyclization kinetics, in contrast to the observations for Fos–Jun bound at the AP-1 locus. In contrast, comparative electrophoresis reveals that Fos–Jun–DNA complexes, in which the A-tract bend is positioned close (1–2 helical turns) to the AP-1 site, show phase-dependent variations in gel mobilities that are comparable with those observed when a single A-tract bend replaces the AP-1 site. Whereas gel mobility variations of Fos–Jun–DNA complexes decrease linearly with increasing Mg
2+
contained in the gel, the solution binding preference of (dbd)Fos–Jun for linear over circular DNAs is independent of Mg
2+
concentration. Hence, gel mobility variations of Fos–Jun–DNA complexes are not indicative of (dbd)Fos–Jun-induced DNA bending (upper limit 5°) in the low salt conditions of gel electrophoresis. Instead, we propose that the gel anomalies depend on the steric relationship of the leucine zipper region with respect to a DNA bend.
Definition of amide protection factors for early kinetic intermediates in protein folding
Authors: Houry Walid A. , Sauder J. Michael , Roder Heinrich , Scheraga Harold A.
Published: July 2002

Hydrogen–deuterium exchange experiments have been used previously to investigate the structures of well defined states of a given protein. These include the native state, the unfolded state, and any intermediates that can be stably populated at equilibrium. More recently, the hydrogen–deuterium exchange technique has been applied in kinetic labeling experiments to probe the structures of transiently formed intermediates on the kinetic folding pathway of a given protein. From these equilibrium and nonequilibrium studies, protection factors are usually obtained. These protection factors are defined as the ratio of the rate of exchange of a given backbone amide when it is in a fully solvent-exposed state (usually obtained from model peptides) to the rate of exchange of that amide in some state of the protein or in some intermediate on the folding pathway of the protein. This definition is straightforward for the case of equilibrium studies; however, it is less clear-cut for the case of transient kinetic intermediates. To clarify the concept for the case of burst-phase intermediates, we have introduced and mathematically defined two different types of protection factors: one isPstruc, which is more related to thestructureof the intermediate, and the other isPapp, which is more related to thestabilityof the intermediate. Kinetic hydrogen–deuterium exchange data from disulfide-intact ribonuclease A and from cytochromecare discussed to explain the use and implications of these two definitions.
The
Rhizobium meliloti
ExoK and ExsH glycanases specifically depolymerize nascent succinoglycan chains
Authors: York Gregory M. , Walker Graham C.
Published: July 2002

The
Rhizobium meliloti
ExoK and ExsH glycanases have been proposed to contribute to production of low molecular weight (LMW) succinoglycan by depolymerizing high molecular weight succinoglycan chains in
R. meliloti
cultures. We expressed and purified ExoK and ExsH and determined that neither enzyme can extensively cleave succinoglycan prepared from
R. meliloti
cultures, although neutral/heat treatment and acid/heat treatment convert succinoglycan to forms that can be cleaved efficiently by both enzymes. These results were somewhat surprising, given that the
exoK
+
and
exsH
+
genes play a crucial role in production of LMW succinoglycan in
R. meliloti
cultures. We demonstrated by Western blot analyses that
R. meliloti
expresses ExoK and ExsH, that both proteins can be detected extracellularly, and that ExsH secretion depends on the
prsD
+
/
prsE
+
genes, consistent with previous predictions based on mutant analyses. Furthermore, we determined that the depolymerization activities associated with purified ExoK and ExsH are comparable with
exoK
+
and
exsH
+
-dependent depolymerization activities expressed in
R. meliloti
cultures. We resolved the apparent contradiction between the results of our previous genetic analyses and depolymerization assays by determining that ExoK and ExsH can cleave high molecular weight succinoglycan that is being produced actively by
R. meliloti
, but not succinoglycan that has accumulated in cultures, to yield LMW succinoglycan. We propose that ExoK and ExsH dynamically regulate the molecular weight distribution of succinoglycan by cleaving nascent succinoglycan only during a limited period after its synthesis, perhaps before it undergoes a time-dependent change in its conformation or aggregation state.
Proapoptotic activity of
Caenorhabditis elegans
CED-4 protein in
Drosophila
: Implicated mechanisms for caspase activation
Authors: Kanuka Hirotaka , Hisahara Shin , Sawamoto Kazunobu , Shoji Shin-ichi , Okano Hideyuki , Miura Masayuki
Published: July 2002

CED-4 protein plays an important role in the induction of programmed cell death in
Caenorhabditis elegans
through the activation of caspases. However, the precise mechanisms by which it activates caspases remain unknown. To investigate the conservation of CED-4 function in evolution, transgenic
Drosophila
lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induced massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate
Drosophila
caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in
Drosophila
S2 cells. Immunoprecipitation analysis showed that both CED-4 and CED-4 (K165R) bind directly to
Drosophila
caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation in S2 cells. Furthermore, CED-4 (K165R) partially prevented cell death induced by CED-4 in
Drosophila
compound eyes. Thus, CED-4 function is evolutionarily conserved in
Drosophila
, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases.
Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-γ-dependent mechanisms
Authors: Walker Patricia S. , Scharton-Kersten Tanya , Krieg Arthur M. , Love-Homan Laurie , Rowton Edgar D. , Udey Mark C. , Vogel Jonathan C.
Published: July 2002

Resistance to murine leishmaniasis correlates with development of a CD4+T helper 1 (Th1)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN), known to promote a Th1 immune response, could provide protection fromLeishmaniainfection, CpG-ODN and freeze-thawed (F/T)Leishmania majorwere coinjected intradermally into susceptible BALB/c mice. ALeishmania-specific Th1-predominant immune response was induced, and 40% of animals were protected from subsequent challenge with infectious organisms, with 0% protection of animals injected with F/TLeishmaniaorganisms and PBS, F/T organisms and control ODN, or F/T organisms alone. More striking protection (65–95%) was seen in mice first infected with intactLeishmaniaorganisms and then injected with CpG-ODN, either at the site of infection or at a remote site. To determine whether the therapeutic protection provided by CpG-ODN depended on IL-12 and IFN-γ production, both IFN-γ-deficient BALB/c mice and BALB/c mice treated with neutralizing anti-IL-12 mAb were first inoculated withLeishmaniaand then treated with either CpG-ODN, ODN, or PBS. None of these IFN-γ-deficient mice survived (0/20, 0/20, and 0/20 respectively). Furthermore, neutralization of IL-12 completely abolished the therapeutic protection provided by CpG-ODN (0/20 mice surviving). We conclude that immunostimulatory DNA sequences likely exert systemic effects via IL-12 and IFN-γ-dependent mechanisms and hold considerable promise as both vaccine adjuvants and potential therapeutic agents in the prevention and treatment of leishmaniasis.
Substrate-induced closure of the flap domain in the ternary complex structures provides insights into the mechanism of catalysis by 3-hydroxy-3-methylglutaryl–CoA reductase
Authors: Tabernero Lydia , Bochar Daniel A. , Rodwell Victor W. , Stauffacher Cynthia V.
Published: July 2002

3-hydroxy-3-methylglutaryl–CoA (HMG-CoA) reductase is the rate-limiting enzyme and the first committed step in the biosynthesis of cholesterol in mammals. We have determined the crystal structures of two nonproductive ternary complexes of HMG-CoA reductase, HMG-CoA/NAD
+
and mevalonate/NADH, at 2.8 Å resolution. In the structure of the
Pseudomonas mevalonii
apoenzyme, the last 50 residues of the C terminus (the flap domain), including the catalytic residue His381, were not visible. The structures of the ternary complexes reported here reveal a substrate-induced closing of the flap domain that completes the active site and aligns the catalytic histidine proximal to the thioester of HMG-CoA. The structures also present evidence that Lys267 is critically involved in catalysis and provide insights into the catalytic mechanism.
Intracellular σ
1
receptor modulates phospholipase C and protein kinase C activities in the brainstem
Authors: Morin-Surun M. P. , Collin T. , Denavit-Saubié M. , Baulieu E.-E. , Monnet F. P.
Published: July 2002

Most physiological effects of σ
1
receptor ligands are sensitive to pertussis toxin, suggesting a coupling with cell membrane-bound G proteins. However, the cloning of the σ
1
receptor has allowed the identification of an intracellular protein anchored on the endoplasmic reticulum. Here, we show, using the isolated adult guinea pig brainstem preparation, that activation of the σ
1
receptor results in its translocation from the cytosol to the vicinity of the cell membrane and induces a robust and rapid decrease in hypoglossal activity, which is mediated by phospholipase C. The subsequent activation of protein kinase C β
1
and β
2
isoforms and the phosphorylation of a protein of the same molecular weight as the cloned σ
1
receptor lead to a desensitization of the σ
1
motor response. Our results indicate that the intracellular σ
1
receptor regulates several components implicated in plasma membrane-bound signal transduction. This might be an example of a mechanism by which an intracellular receptor modulates metabotropic responses.
Natural experimental models: The global search for biomedical paradigms among traditional, modernizing, and modern populations
Authors: Garruto R. M. , Little M. A. , James G. D. , Brown D. E.
Published: July 2002

During the past four decades, biomedical scientists have slowly begun to recognize the unique opportunities for studying biomedical processes, disease etiology, and mechanisms of pathogenesis in populations with unusual genetic structures, physiological characteristics, focal endemic disease, or special circumstances. Such populations greatly extend our research capabilities and provide a natural laboratory for studying relationships among biobehavioral, genetic, and ecological processes that are involved in the development of disease. The models presented illustrate three different types of natural experiments: those occurring in traditionally living, modernizing, and modern populations. The examples are drawn from current research that involves population mechanisms of adaptation among East African Turkana pastoralists; a search for etiology and mechanisms of pathogenesis of an emerging disease among the Yakut people of Siberia; and psychosocial stress, hypertension, and cardiovascular disease in women working outside the home in New York City and among subpopulations in Hawaii. The models in general, and the examples in specific, represent natural laboratories in which relatively small intrapopulation differences and large interpopulation differences can be used to evaluate health and disease outcomes.
Correlation of deformability at a tRNA recognition site and aminoacylation specificity
Authors: Chang Kung-Yao , Varani Gabriele , Bhattacharya Subhra , Choi Hyunsic , McClain William H.
Published: July 2002

The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA. These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G⋅U wobble base pair located at the third position within the acceptor helix of alanine tRNA. We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition. Here we show that the mismatched pair C-C is an excellent substitute for G⋅U in alanine-tRNA-knockout cells. A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations. Structural heterogeneity also is present in the wild-type G⋅U RNA, whereas inactive Watson–Crick helices are structurally rigid. The correlation between functional and structural data suggests that the G⋅U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase. Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity.
Biosynthetic origin of conjugated double bonds: Production of fatty acid components of high-value drying oils in transgenic soybean embryos
Authors: Cahoon Edgar B. , Carlson Thomas J. , Ripp Kevin G. , Schweiger Bruce J. , Cook George A. , Hall Sarah E. , Kinney Anthony J.
Published: July 2002

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of α-eleostearic (18:3Δ
9

cis

,11

trans

,13

trans

) and α-parinaric (18:4Δ

9
cis
,11
trans
,13
trans
,15
cis

) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of
Momordica charantia
and
Impatiens balsamina
, tissues that accumulate large amounts of α-eleostearic and α-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Δ
12
-oleic acid desaturase was identified. Expression of full-length cDNAs for the
Momordica
(MomoFadX) and
Impatiens
(ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of α-eleostearic and α-parinaric acids, neither of which is present in untransformed soybean embryos. α-Eleostearic and α-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Δ
12
-oleic acid desaturase-type enzymes that we have termed “conjugase.”
HU-308: A specific agonist for CB
2
, a peripheral cannabinoid receptor
Authors: Hanuš L. , Breuer A. , Tchilibon S. , Shiloah S. , Goldenberg D. , Horowitz M. , Pertwee R. G. , Ross R. A. , Mechoulam R. , Fride E.
Published: July 2002

Two cannabinoid receptors have been identified: CB
1
, present in the central nervous system (CNS) and to a lesser extent in other tissues, and CB
2
, present outside the CNS, in peripheral organs. There is evidence for the presence of CB
2
-like receptors in peripheral nerve terminals. We report now that we have synthesized a CB
2
-specific agonist, code-named HU-308. This cannabinoid does not bind to CB
1
(
K
i
> 10 μM), but does so efficiently to CB
2
(
K
i
= 22.7 ± 3.9 nM); it inhibits forskolin-stimulated cyclic AMP production in CB
2
-transfected cells, but does so much less in CB
1
-transfected cells. HU-308 shows no activity in mice in a tetrad of behavioral tests, which together have been shown to be specific for tetrahydrocannabinol (THC)-type activity in the CNS mediated by CB
1
. However, HU-308 reduces blood pressure, blocks defecation, and elicits anti-inflammatory and peripheral analgesic activity. The hypotension, the inhibition of defecation, the anti-inflammatory and peripheral analgesic effects produced by HU-308 are blocked (or partially blocked) by the CB
2
antagonist SR-144528, but not by the CB
1
antagonist SR-141716A. These results demonstrate the feasibility of discovering novel nonpsychotropic cannabinoids that may lead to new therapies for hypertension, inflammation, and pain.
Antigen-driven CD4+ T cell and HIV-1 dynamics: Residual viral replication under highly active antiretroviral therapy
Authors: Ferguson Neil M. , deWolf Frank , Ghani Azra C. , Fraser Christophe , Donnelly Christl A. , Reiss Peter , Lange Joep M. A. , Danner Sven A. , Garnett Geoff P. , Goudsmit Jaap , Anderson Roy M.
Published: July 2002

Antigen-induced stimulation of the immune system can generate heterogeneity in CD4+ T cell division rates capable of explaining the temporal patterns seen in the decay of HIV-1 plasma RNA levels during highly active antiretroviral therapy. Posttreatment increases in peripheral CD4+ T cell counts are consistent with a mathematical model in which host cell redistribution between lymph nodes and peripheral blood is a function of viral burden. Model fits to patient data suggest that, although therapy reduces HIV replication below replacement levels, substantial residual replication continues. This residual replication has important consequences for long-term therapy and the evolution of drug resistance and represents a challenge for future treatment strategies.
Empirical laws of survival and evolution: Their universality and implications
Authors: Azbel' Mark Ya.
Published: July 2002

Presented analysis of human and fly life tables proves that with the specified accuracy their entire survival and mortality curves are uniquely determined by a single point (e.g., by the birth mortalityq0), according to the law, which is universal for species as remote as humans and flies. Mortality at any age decreases with the birth mortalityq0. According to life tables, in the narrow vicinity of a certainq0value (which is the same for all animals of a given species, independent of their living conditions), the curves change very rapidly and nearly simultaneously for an entire population of different ages. The change is the largest in old age. Because probability to survive to the mean reproductive age quantifies biological fitness and evolution, its universal rapid change withq0(which changes with living conditions) manifests a new kind of an evolutionary spurt of an entire population. Agreement between theoretical and life table data is explicitly seen in the figures. Analysis of the data on basic metabolism reduces it to the maximal mean lifespan (for animals from invertebrates to mammals), or to the maximal mean fission time (for bacteria), and universally scales them with the total number of body atoms only. Phenomenological origin of this unification and universality of metabolism, survival, and evolution is suggested. Their implications and challenges are discussed.
Probing cell-surface architecture through synthesis: An NMR-determined structural motif for tumor-associated mucins
Authors: Live David H. , Williams Lawrence J. , Kuduk Scott D. , Schwarz Jacob B. , Glunz Peter W. , Chen Xiao-Tao , Sames Dalibor , Kumar R. Ajay , Danishefsky Samuel J.
Published: July 2002

Cell-surface mucin glycoproteins are altered with the onset of oncogenesis. Knowledge of mucin structure could be used in vaccine strategies that target tumor-associated mucin motifs. Thus far, however, mucins have resisted detailed molecular analysis. Reported herein is the solution conformation of a highly complex segment of the mucin CD43. The elongated secondary structure of the isolated mucin strand approaches the stability of motifs found in folded proteins. The features required for the mucin motif to emerge are also described. Immunocharacterization of related constructs strongly suggests that the observed epitopes represent distinguishing features of tumor cell-surface architecture.
Msh2
status modulates both apoptosis and mutation frequency in the murine small intestine
Authors: Toft Neil J. , Winton Douglas J. , Kelly Jane , Howard Louise A. , Dekker Marleen , te Riele Hein , Arends Mark J. , Wyllie Andrew H. , Margison Geoffrey P. , Clarke Alan R.
Published: July 2002

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using
Msh2
null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation
in vivo
at the
Dlb-1
locus. Compared with wild-type mice,
Msh2
-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using
Msh2
-deficient cells
in vitro
suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that
Msh2
plays a role in the
in vivo
initiation of apoptosis after treatment with temozolomide,
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine, and cisplatin. This was not influenced by the
in vivo
depletion of
O
6
-alkylguanine-DNA-alkyltransferase after administration of
O
6
-benzylguanine . By analyzing mice mutant for both
Msh2
and
p53
, we found that the
Msh2
-dependent apoptotic response was primarily mediated through a
p53
-dependent pathway.
Msh2
also was required to signal delayed
p53
-independent death. Taken together, these studies characterize an
in vivo Msh2
-dependent apoptotic response to methylating agents and raise the possibility that
Msh2
deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.
Requirement for nitric oxide activation of p21
ras
/extracellular regulated kinase in neuronal ischemic preconditioning
Authors: Gonzalez-Zulueta Mirella , Feldman Alicia B. , Klesse Laura J. , Kalb Robert G. , Dillman James F. , Parada Luis F. , Dawson Ted M. , Dawson Valina L.
Published: July 2002

The mechanisms underlying neuronal ischemic preconditioning, a phenomenon in which brief episodes of ischemia protect against the lethal effects of subsequent periods of prolonged ischemia, are poorly understood. Ischemia can be modeled
in vitro
by oxygen-glucose deprivation (OGD). We report here that OGD preconditioning induces p21
ras
(Ras) activation in an
N
-methyl-
d
-aspartate receptor- and NO-dependent, but cGMP-independent, manner. We demonstrate that Ras activity is necessary and sufficient for OGD tolerance in neurons. Pharmacological inhibition of Ras, as well as a dominant negative mutant Ras, block OGD preconditioning whereas a constitutively active form of Ras promotes neuroprotection against lethal OGD insults. In contrast, the activity of phosphatidyl inositol 3-kinase is not required for OGD preconditioning because inhibition of phosphatidyl inositol 3-kinase with a chemical inhibitor or with a dominant negative mutant does not have any effect on the development of OGD tolerance. Furthermore, using recombinant adenoviruses and pharmacological inhibitors, we show that downstream of Ras the extracellular regulated kinase cascade is required for OGD preconditioning. Our observations indicate that activation of the Ras/extracellular regulated kinase cascade by NO is a critical mechanism for the development of OGD tolerance in cortical neurons, which may also play an important role in ischemic preconditioning
in vivo
.
Ribosomal subunit kinase-2 is required for growth factor-stimulated transcription of the c-
Fos
gene
Authors: Brüning Jens C. , Gillette Jennifer A. , Zhao Yi , Bjorbaeck Christian , Kotzka Jörg , Knebel Birgit , Avci Haluk , Hanstein Bettina , Lingohr Philipp , Moller David E. , Krone Wilhelm , Kahn C. Ronald , Müller-Wieland Dirk
Published: July 2002

Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the
Rsk-2
gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-
Fos
. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.
A Computational Framework for Unifying Vision and Language
Authors: Glasgow Janice
Published: July 2002

This paper presents a knowledge representation framework that provides an integrated approach to reasoning about vision and language in spatial domains. Images are stored using a representation that preserves information such as shape and distance; linguistic information is represented in a descriptive, semantic network. We incorporate a model‐based formalism as an intermediate representation, which can be used to transform visual to descriptive representations and vice versa.
Making Evidence‐based Practice Educational
Authors: Elliott John
Published: July 2002

Abstract The article examines David Hargreaves's ideas about the nature of ‘evidence‐based practice’ and the future direction for educational research. The first section explores Hargreaves's attempt to free an ‘engineering model’ of educational research, aimed at the production of actionable knowledge, from a naive and crude positivism. It draws on McIntyre's philosophical account of the character of generalisations in social science to question whether Hargreaves has succeeded. In doing so, the assumptions which underpin his conception of actionable knowledge, and preference for generalisations couched in the form of statistical probabilities, are discussed and critiqued. The article argues that they are embedded in the prevailing ideological climate of outcomesbased education. The rest of the article develops a different account of educational practice from the one that appears to underpin Hargreaves's ideas about ‘evidencebased’ practice and the role of educational research in supporting it. It does so by revisiting the work of Richard Peters, on the aims of education, and that of Lawrence Stenhouse, on curriculum design and ‘research‐based teaching’. Central to both Peters and Stenhouse's work is a view about the relationship between educational aims and processes, which is neglected in Hargreaves's account of the role of educational research in informing educational practice. The explanation appears to lie in Hargreaves's unquestioning commitment to an outcomes‐based view of education. The article shows how Stenhouse drew on Peters's educational theory to construct a more comprehensive view of educational research as ‘research‐based teaching’. Although the exposition of Peters and Stenhouse's work is set against Hargreaves's conception of ‘evidence‐based’ practice and the role of educational research, the article develops a position of ‘mutual accommodation’. In comparing Hargreaves's thinking with that of Stenhouse in particular, it indicates similarities as well as differences of perspective. One major theme in the article is that the current discourse about ‘evidence‐based’ teaching is uninformed by an articulate educational theory, and therefore excludes a thoughtful consideration of the implications of such a theory for educational research. The major aim of the article is to make a contribution towards rectifying this situation.
Adult-Child Interactions as Related to Adult’s Family History and Child’s Attachment
Authors: Shulman Shmuel , Becker Ada , Sroufe L. Alan
Published: July 2002

The objective of this study was to investigate separately the contribution of mother characteristics and child characteristics in dyadic interactions. This study was conducted in a unique setting, a co-operative nursery school in which each mother assumed the role of an assistant teacher once every three weeks. At the first stage, mother’s family history and child’s attachment were evaluated. Later on during school, mothers’ and children’s interactions were videotaped. The data allowed the analysis of interactions between mothers with different family histories and children from other families with different attachment types, as well as children’s interactions with different mothers. Results showed a complex interplay of mother and child characteristics within an adult-child interaction. Mothers were observed to be more involved with, and to express more anger toward, insecurely attached children, especially when their own child was classified as insecure. Inspection of children’s initiatives revealed that children preferred to turn to adults whose family history corresponded to the family history of their own mother. Results are discussed within the framework of attachment and family systems theories.
A Constraint-based Approach to Finnish CV Spoonerisms
Authors: Harrikari Heli
Published: July 2002

This paper provides a comprehensive Optimality Theoretic formalization of a Finnish language game. In opposition to earlier rule-based studies, the present analysis motivates the sometimes arbitrary-looking patterns of the game and brings together various phenomena under a unified set of universal constraints. The paper also demonstrates the close relationship between language games and natural languages by showing how the patterns of the game are analysable with the help of constraints of ordinary languages, or constraints that deviate only minimally from the original ones. The analysis of the game offers valuable external evidence for the phonology of Finnish with respect to the internal structure of long segments, vowel harmony, syllable markedness and the integrity of diphthongs.
Coordination in Optimality Theory
Authors: Gáspár Miklós
Published: July 2002

The paper offers an account of coordination within the framework of Optimality Theory, which makes use of violable and ranked constraints. Coordination is explained with the help of nine constraints, seven of which are needed in the theory independently of coordination, while the remaining two are coordination-specific constraints. Related phenomena of Unbalanced Coordination and Extraordinary Balanced Coordination are also discussed and differences among Norwegian, English and Hungarian are explained by the difference in the relative ranking of the four relevant constraints.
Annexin I and dexamethasone effects on phospholipase and cyclooxygenase activity in human synoviocytes
Authors: Sampey Annaleise V. , Hutchinson Paul , Morand Eric F.
Published: July 2002

Annexin I is a glucocorticoid‐induced mediator with anti‐inflammatory activity in animal models of arthritis. We studied the effects of a bioactive annexin I peptide, ac 2–26, dexamethasone (DEX), and interleukin‐1β (IL‐1β) on phospholipase A2 (PLA2) and cyclooxygenase (COX) activities and
prostaglandin E2 (PGE2) release in cultured human fibroblast‐like synoviocytes (FLS). Annexin I binding sites on human osteoarthritic (OA) FLS were detected by ligand binding flow cytometry. PLA2 activity was measured using 3H‐arachidonic acid release, PGE2 release and COX activity by ELISA, and COX2 content by flow cytometry. Annexin I binding sites were present on human OA FLS. Annexin I peptide ac 2–26 exerted a significant concentration‐dependent inhibition of FLS constitutive PLA2 activity, which was reversed by IL‐1β. In contrast, DEX inhibited IL‐1β‐induced PLA2 activity but not constitutive activity. DEX but not annexin I peptide inhibited IL‐1β‐induced PGE2 release. COX activity and COX2 expression were significantly increased by IL‐1β. Annexin I peptide demonstrated no inhibition of constitutive or IL‐1β‐induced COX activity. DEX exerted a concentration‐dependent inhibition of IL‐1β‐induced but not constitutive COX activity. Uncoupling of inhibition of PLA2 and COX by annexin I and DEX support the hypothesis that COX is rate‐limiting for PGE2 synthesis in FLS. The effect of annexin I but not DEX on constitutive PLA2 activity suggests a glucocorticoid‐independent role for annexin I in autoregulation of arachidonic acid production. The lack of effect of annexin I on cytokine‐induced PGE2 production suggests PGE2‐independent mechanisms for the anti‐inflammatory effects of annexin I in vivo.
Prevention of Alcohol Problems in the 21st Century: Challenges and Opportunities
Authors: Holder Harold D.
Published: July 2002

The community is the “new frontier” for alcohol and other drug prevention. New prevention initiatives at the community level suggest that effective strategies will often be quite different from national or state policies and will require a different perspective. Alcohol and other drug use is part of routine community life and must be considered in the context of the community, which is itself a dynamic and self‐adapting system. To develop effective community‐level interventions, prevention planners and policy makers must understand how various aspects of the community influence alcohol and other drug use and even contribute to alcohol and other drug problems. This paper outlines the basis for a systems approach to community prevention and the policy options that this approach suggests. It also examines the new science of complexity, differentiates between catchment and a systems approach to prevention, describes a public health model within a systems approach, and describes using local policy as a means to produce system changes as well as recent findings from community‐based prevention efforts that employed local alcohol policies.
A Study of the Acid Lability and Acute Toxicity of Dimethoxymethane in Rats
Authors: Poon Raymond , Moir David , Elwin James , Nadeau Brita , Singh Amreek , Yagminas Algis , Chu Ih
Published: July 2002

The pH stability and acute oral toxicity of dimethoxymethane (DMM) in rats were studied. In in vitro studies, DMM was found to be stable in artificial gastric juice at pH 2.5 and above but readily hydrolyzed at pH 2 and below, with 1 mole of DMM producing approximately 1 mole of formaldehyde and 2 moles of methanol. DMM incubated in sealed, excised stomachs appeared to equilibrate with the external incubation fluid over a 24-hour period without significant hydrolysis. In the toxicity study, three groups of female rats ( n = 5) were administered, by gavage, phosphate-buffered saline (control), DMM (31 mmoles/kg body weight [bw]/day), or methanol (MeOH) (30 mmoles/kg bw/day), respectively, for 3 consecutive days and then sacrificed on day 5. Transient ataxia lasting about 30 minutes was observed in animals following DMM administration. Increased urine volume was observed in the DMM group at the end of days 4 and 5. Markedly elevated level of formic acid was present in the urine of the MeOH group at days 1, 2, and 3, but not in the DMM group. Urinary ascorbic acid levels were increased in the DMM group on days 1, 2 and 3, and returned to baseline level at days 4 and 5. Microscopic examination detected no abnormal histological changes in the liver and kidneys of MeOH-or DMM-treated animals. No treatment effects were observed on the following endpoints: organ weights (liver, heart, thymus, kidneys), serum corticosterone, serum chemistry profile, hematology, hepatic phase 1 and phase 2 mixed-function oxidase activities, hepatic and serum thiobarbituric acid, hepatic glutathione, urinary protein, and urinary N-acetylglucosaminidase activity. These observations suggested that hydrolysis of DMM in the stomach required a gastric acidity below pH 2.5. At higher pH, intact DMM readily penetrated the stomach and therefore likely reached the circulation and other body tissues. There was no significant hydrolysis of orally administered DMM, and the acute effects were limited to transient ataxia and biochemical responses in the liver.
Italianità and internationalism: Production, design and mediation at Alessi, 1976–96
Authors: Lees-Maffei Grace
Published: July 2002

This article considers the Italian household product design company Alessi as quintessentially Italian and international. The interplay of mass-production and craft techniques at Alessi, an extended design process and the presentation of the family in company publicity exemplify the ambiguities identified within Italian design. The extent to which Alessi typifies Italian post-war design is questioned with reference to the company's international design team and the marketing of its products to foreign consumers as manifestations of italianità. The findings are based on primary research in Milan and the Alessi factory, including a series of interviews with designers, retailers, curators and media workers, combined with secondary material centred upon design history.
The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote
Authors: Hinchcliffe Edward H. , Cassels Grizzel O. , Rieder Conly L. , Sluder Greenfield
Published: July 2002

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.
Spindle Self-organization and Cytokinesis During Male Meiosis in asterless Mutants of Drosophila melanogaster
Authors: Bonaccorsi Silvia , Giansanti Maria Grazia , Gatti Maurizio
Published: July 2002

While Drosophila female meiosis is anastral, both meiotic divisions in Drosophila males exhibit prominent asters. We have identified a gene we call asterless (asl) that is required for aster formation during male meiosis. Ultrastructural analysis showed that asl mutants have morphologically normal centrioles. However, immunostaining with antibodies directed either to γ tubulin or centrosomin revealed that these proteins do not accumulate in the centrosomes, as occurs in wild-type. Thus, asl appears to specify a function required for the assembly of centrosomal material around the centrioles.

Despite the absence of asters, meiotic cells of asl mutants manage to develop an anastral spindle. Microtubules grow from multiple sites around the chromosomes, and then focus into a peculiar bipolar spindle that mediates chromosome segregation, although in a highly irregular way.

Surprisingly, asl spermatocytes eventually form a morphologically normal ana–telophase central spindle that has full ability to stimulate cytokinesis. These findings challenge the classical view on central spindle assembly, arguing for a self-organization of this structure from either preexisting or newly formed microtubules. In addition, these findings strongly suggest that the asters are not required for signaling cytokinesis.
Impairment of Inhibitory Synaptic Transmission in Mice Lacking Synapsin I
Authors: Terada Sumio , Tsujimoto Tetsuhiro , Takei Yosuke , Takahashi Tomoyuki , Hirokawa Nobutaka
Published: July 2002

Deletion of the synapsin I genes, encoding one of the major groups of proteins on synaptic vesicles, in mice causes late onset epileptic seizures and enhanced experimental temporal lobe epilepsy. However, mice lacking synapsin I maintain normal excitatory synaptic transmission and modulation but for an enhancement of paired-pulse facilitation. To elucidate the cellular basis for epilepsy in mutants, we examined whether the inhibitory synapses in the hippocampus from mutant mice are intact by electrophysiological and morphological means. In the cultured hippocampal synapses from mutant mice, repeated application of a hypertonic solution significantly suppressed the subsequent transmitter release, associated with an accelerated vesicle replenishing time at the inhibitory synapses, compared with the excitatory synapses. In the mutants, morphologically identifiable synaptic vesicles failed to accumulate after application of a hypertonic solution at the inhibitory preterminals but not at the excitatory preterminals. In the CA3 pyramidal cells in hippocampal slices from mutant mice, inhibitory postsynaptic currents evoked by direct electrical stimulation of the interneuron in the striatum oriens were characterized by reduced quantal content compared with those in wild type. We conclude that synapsin I contributes to the anchoring of synaptic vesicles, thereby minimizing transmitter depletion at the inhibitory synapses. This may explain, at least in part, the epileptic seizures occurring in the synapsin I mutant mice.