bluuebunny/crossref_metadata_2025 huggingface dataset:
Haplotype-based identification of a microsomal transfer protein marker associated with the human lifespan
Authors: Geesaman Bard J. , Benson Erica , Brewster Stephanie J. , Kunkel Louis M. , Blanché Hélène , Thomas Gilles , Perls Thomas T. , Daly Mark J. , Puca Annibale A.
Published: November 2003

We previously reported a genomewide linkage study for human longevity using 308 long-lived individuals (LLI) (centenarians or near-centenarians) in 137 sibships and identified statistically significant linkage within chromosome 4 near microsatellite D4S1564. This interval spans 12 million bp and contains ≈50 putative genes. To identify the specific gene and gene variants impacting lifespan, we performed a haplotype-based fine-mapping study of the interval. The resulting genetic association study identified a haplotype marker within microsomal transfer protein as a modifier of human lifespan. This same variant was tested in a second cohort of LLI from France, and although the association was not replicated, there was evidence for statistical distortion in the form of Hardy–Weinberg disequilibrium. Microsomal transfer protein has been identified as the rate-limiting step in lipoprotein synthesis and may affect longevity by subtly modulating this pathway. This study provides proof of concept for the feasibility of using the genomes of LLI to identify genes impacting longevity.
Comprehensive transposon mutant library of
Pseudomonas aeruginosa
Authors: Jacobs Michael A. , Alwood Ashley , Thaipisuttikul Iyarit , Spencer David , Haugen Eric , Ernst Stephen , Will Oliver , Kaul Rajinder , Raymond Christopher , Levy Ruth , Chun-Rong Liu , Guenthner Donald , Bovee Donald , Olson Maynard V. , Manoil Colin
Published: November 2003

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to
Pseudomonas aeruginosa
, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in
E. coli
, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.
ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA
Authors: Perriman Rhonda , Barta Imre , Voeltz Gia K. , Abelson John , Ares Manuel
Published: November 2003

Stable addition of U2 small nuclear ribonucleoprotein (snRNP) to form the prespliceosome is the first ATP-dependent step in splicing, and it requires the DEXD/H box ATPase Prp5p. However, prespliceosome formation occurs without ATP in extracts lacking the U2 snRNP protein Cus2p. Here we show that Prp5p is required for the ATP-independent prespliceosome assembly that occurs in the absence of Cus2p. Addition of recombinant Cus2p can restore the ATP dependence of prespliceosome assembly, but only if it is added before Prp5p. Prp5p with an altered ATP-binding domain (Prp5-GNTp) can support growth
in vivo
, but only in a
cus2
deletion strain, mirroring the
in vitro
results. Other Prp5 ATP-binding domain substitutions are lethal, even in the
cus2
deletion strain, but can be suppressed by U2 small nuclear RNA mutations that hyperstabilize U2 stem IIa. We infer that the presence of Cus2p and stem IIa-destabilized forms of U2 small nuclear RNA places high demands on the ATP-driven function of Prp5p. Because Prp5p is not dispensable
in vitro
even in the absence of ATP, we propose that the core Prp5p function in bringing U2 to the branchpoint is not directly ATP-dependent. The positive role of Cus2p in rescuing mutant U2 can be reconciled with its antagonistic effect on Prp5 function in a model whereby Cus2p first helps Prp5p to activate the U2 snRNP for prespliceosome formation but then is displaced by Prp5p before or during the stabilization of U2 at the branchpoint.
A genomic perspective on nutrient provisioning by bacterial symbionts of insects
Authors: Moran Nancy A. , Plague Gordon R. , Sandström Jonas P. , Wilcox Jennifer L.
Published: November 2003

Many animals show intimate interactions with bacterial symbionts that provision hosts with limiting nutrients. The best studied such association is that between aphids and
Buchnera aphidicola
, which produces essential amino acids that are rare in the phloem sap diet. Genomic studies of
Buchnera
have provided a new means for inferring metabolic capabilities of the symbionts and their likely contributions to hosts. Despite evolutionary reduction of genome size, involving loss of most ancestral genes,
Buchnera
retains capabilities for biosynthesis of all essential amino acids. In contrast, most genes duplicating amino acid biosynthetic capabilities of hosts have been eliminated. In
Buchnera
of many aphids, genes for biosynthesis of leucine and tryptophan have been transferred from the chromosome to distinctive plasmids, a feature interpreted as a mechanism for overproducing these amino acids through gene amplification. However, the extent of plasmid-associated amplification varies between and within species, and plasmid-borne genes are sometimes fewer in number than single copy genes on the (polyploid) main chromosome. This supports the broader interpretation of the plasmid location as a means of achieving regulatory control of gene copy number and/or transcription.
Buchnera
genomes have eliminated most regulatory sequences, raising the question of the extent to which gene expression is moderated in response to changing demands imposed by host nutrition or other factors. Microarray analyses of the
Buchnera
transcriptome reveal only slight changes in expression of nutritionrelated genes in response to shifts in host diet, with responses less dramatic than those observed for the related nonsymbiotic species,
Escherichia coli
.
The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development
Authors: Wang Aiming , Xia Qun , Xie Wenshuang , Datla Raju , Selvaraj Gopalan
Published: November 2003

Pollen fecundity is crucial to crop productivity and also to biodiversity in general. Pollen development is supported by the tapetum, a metabolically active sporophytic nurse layer that devotes itself to this process. The tapetum in cereals and a vast majority of other plants is of the nonamoeboid type. Unable to reach out to microspores, it secretes nutrients into the anther locule where the microspores reside and develop. Orbicules (Ubisch bodies), studied in various plants since their discovery ≈140 years ago, are a hallmark of the secretory tapetum. Their significance to tapetal or pollen development has not been established. We have identified in wheat and rice an anther-specific single-copy gene (per haploid genome equivalent) whose suppression in rice by RNA interference nearly eliminated the seed set. The flowers in the transgenics were normal for female functions, but the pollen collapsed and became less viable. Further characterization of the gene product, named RAFTIN, in wheat has shown that it is present in pro-orbicule bodies and it is accumulated in Ubisch bodies. Furthermore, it is targeted to microspore exine. Although the carboxyl portion of RAFTINs shares short, dispersed amino acid sequences (BURP domain) in common with a variety of proteins of disparate biological contexts, the occurrence RAFTIN
per se
is limited to cereals; neither the
Arabidopsis
genome nor the vast collection of ESTs suggests any obvious dicot homologs. Furthermore, our results show that RAFTIN is essential for the late phase of pollen development in cereals.
SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer
Authors: He Biao , You Liang , Uematsu Kazutsugu , Zang Keling , Xu Zhidong , Lee Amie Y. , Costello Joseph F. , McCormick Frank , Jablons David M.
Published: November 2003

Lung cancer is the leading cause of cancer death in the world, but the molecular mechanisms for its development have not been well characterized. The suppressors of cytokine signaling (SOCS) are inhibitors of cytokine signaling that function via the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Eight SOCS proteins with similar structures have been identified so far. SOCS family members, however, have distinct mechanisms of inhibition of JAK/STAT signaling. Abnormalities of the JAK/STAT pathway are associated with cancer. Inhibition of signaling results in growth suppression in various cell types. Recently, the involvement of SOCS-1 in carcinogenesis has been reported. Here, we report identification of frequent hypermethylation in CpG islands of the functional
SOCS-3
promoter that correlates with its transcription silencing in cell lines (lung cancer, breast cancer, and mesothelioma) and primary lung cancer tissue samples. Restoration of SOCS-3 in lung cancer cells where
SOCS-3
was methylation-silenced resulted in the down-regulation of active STAT3, induction of apoptosis, and growth suppression. Our results suggest that methylation silencing of
SOCS-3
is one of the important mechanisms of constitutive activation of the JAK/STAT pathway in cancer pathogenesis. The data also suggest that
SOCS-3
therapy may be useful in the treatment of cancer.
Exploring the functional complexity of cellular proteins by protein knockout
Authors: Zhang Jianxuan , Zheng Ning , Zhou Pengbo
Published: November 2003

Comprehensive dissection of protein functions entails more complicated manipulations than simply eliminating the protein of interest. Established knockdown technologies, such as RNA interference, antisense oligodeoxynucleotides, or ribozymes, are limited for specific applications such as modulating protein levels or specific targeting of a posttranslationally modified subpopulation. Here we show that the engineered Skp1, Cullin 1, and F-box-containing βTrCP substrate receptor ubiquitin-proteolytic system, designated protein knockout, could achieve not only total elimination but also rapid and systematic reduction of a given cellular protein. Stable expression of a single engineered βTrCP demonstrated simultaneous and sustained degradation of the entire retinoblastoma family proteins. Furthermore, the engineered βTrCP was capable of selecting hypo- but not hyperphosphorylated forms of retinoblastoma for degradation. The engineered βTrCP has been extensively modified to increase its specificity in substrate selection. This optimized protein-knockout system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in somatic cells.
Cancer risks attributable to low doses of ionizing radiation: Assessing what we really know
Authors: Brenner David J. , Doll Richard , Goodhead Dudley T. , Hall Eric J. , Land Charles E. , Little John B. , Lubin Jay H. , Preston Dale L. , Preston R. Julian , Puskin Jerome S. , Ron Elaine , Sachs Rainer K. , Samet Jonathan M. , Setlow Richard B. , Zaider Marco
Published: November 2003

High doses of ionizing radiation clearly produce deleterious consequences in humans, including, but not exclusively, cancer induction. At very low radiation doses the situation is much less clear, but the risks of low-dose radiation are of societal importance in relation to issues as varied as screening tests for cancer, the future of nuclear power, occupational radiation exposure, frequent-flyer risks, manned space exploration, and radiological terrorism. We review the difficulties involved in quantifying the risks of low-dose radiation and address two specific questions. First, what is the lowest dose of x- or γ-radiation for which good evidence exists of increased cancer risks in humans? The epidemiological data suggest that it is ≈10–50 mSv for an acute exposure and ≈50–100 mSv for a protracted exposure. Second, what is the most appropriate way to extrapolate such cancer risk estimates to still lower doses? Given that it is supported by experimentally grounded, quantifiable, biophysical arguments, a linear extrapolation of cancer risks from intermediate to very low doses currently appears to be the most appropriate methodology. This linearity assumption is not necessarily the most conservative approach, and it is likely that it will result in an underestimate of some radiation-induced cancer risks and an overestimate of others.
Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in
Caenorhabditis elegans
Authors: Liedtke Wolfgang , Tobin David M. , Bargmann Cornelia I. , Friedman Jeffrey M.
Published: November 2003

All animals detect osmotic and mechanical stimuli, but the molecular basis for these responses is incompletely understood. The vertebrate transient receptor potential channel vanilloid subfamily 4 (TRPV4) (VR-OAC) cation channel has been suggested to be an osmo/mechanosensory channel. To assess its function
in vivo
, we expressed TRPV4 in
Caenorhabditis elegans
sensory neurons and examined its ability to generate behavioral responses to sensory stimuli.
C. elegans
ASH neurons function as polymodal sensory neurons that generate a characteristic escape behavior in response to mechanical, osmotic, or olfactory stimuli. These behaviors require the TRPV channel OSM-9 because
osm-9
mutants do not avoid nose touch, high osmolarity, or noxious odors. Expression of mammalian TRPV4 in ASH neurons of
osm-9
worms restored avoidance responses to hypertonicity and nose touch, but not the response to odorant repellents. Mutations known to reduce TRPV4 channel activity also reduced its ability to direct nematode avoidance behavior. TRPV4 function in ASH required the endogenous
C. elegans
osmotic and nose touch avoidance genes
ocr-2, odr-3, osm-10
, and
glr-1
, indicating that TRPV4 is integrated into the normal ASH sensory apparatus. The osmotic and mechanical avoidance responses of TRPV4-expressing animals were different in their sensitivity and temperature dependence from the responses of wild-type animals, suggesting that the TRPV4 channel confers its characteristic properties on the transgenic animals' behavior. These results provide evidence that TRPV4 can function as a component of an osmotic/mechanical sensor
in vivo
.
Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray
Authors: Coser Kathryn R. , Chesnes Jessica , Hur Jingyung , Ray Sandip , Isselbacher Kurt J. , Shioda Toshi
Published: November 2003

To obtain comprehensive information on 17β-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0–100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed ≈3–5 times larger numbers of genes than it induced, whereas at higher concentrations (30–100 pM) it induced ≈1.5–2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, transforming growth factor α and stromal cell-derived factor 1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, depend on the E2 concentration, especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dosedependent aspects of E2 actions.
Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays
Authors: Nishizuka Satoshi , Charboneau Lu , Young Lynn , Major Sylvia , Reinhold William C. , Waltham Mark , Kouros-Mehr Hosein , Bussey Kimberly J. , Lee Jae K. , Espina Virginia , Munson Peter J. , Petricoin Emanuel , Liotta Lance A. , Weinstein John N.
Published: November 2003

Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling. To aid in such studies, we have developed a protocol for making reverse-phase protein lysate microarrays with larger numbers of spots than previously feasible. Our first application of these arrays was to profiling of the 60 human cancer cell lines (NCI-60) used by the National Cancer Institute to screen compounds for anticancer activity. Each glass slide microarray included 648 lysate spots representing the NCI-60 cell lines plus controls, each at 10 two-fold serial dilutions to provide a wide dynamic range. Mouse monoclonal antibodies and the catalyzed signal amplification system were used for immunoquantitation. The signal levels from the >30,000 data points for our first 52 antibodies were analyzed by usingp-scanand a quantitative dose interpolation method. Clustered image maps revealed biologically interpretable patterns of protein expression. Among the principal early findings from these arrays were two promising pathological markers for distinguishing colon from ovarian adenocarcinomas. When we compared the patterns of protein expression with those we had obtained for the same genes at the mRNA level by using both cDNA and oligonucleotide arrays, a striking regularity appeared: cell-structure-related proteins almost invariably showed a high correlation between mRNA and protein levels across the NCI-60 cell lines, whereas non-cell-structure-related proteins showed poor correlation.
Modeling current and future potential wintering distributions of eastern North American monarch butterflies
Authors: Oberhauser Karen , Peterson A. Townsend
Published: November 2003

Monarch butterflies overwinter in restricted areas in montane oyamel fir forests in central Mexico with specific microclimates that allow the butterflies to survive for up to 5 months. We use ecological niche modeling (ENM) to identify areas adequate for overwintering monarch colonies under both current and future climate scenarios. The ENM approach permits testing and validation of model predictivity, and yields quantitative, testable predictions regarding likely future climate change effects. Our models predicted monarch presence with a high degree of accuracy, and indicated that precipitation and diurnal temperature range were key environmental factors in making locations suitable for monarchs. When we projected monarch distribution onto future climate scenarios (Hadley Centre climate models), we found that conditions were likely to be inadequate across the entire current winter range, particularly owing to increased cool-weather precipitation that could cause increased mortality. This study applies ENM to understanding the seasonal dynamics of a migratory species under climate change, and uses ENM to identify key limiting environmental parameters in species' responses to climate change.
Site-1 protease is required for cartilage development in zebrafish
Authors: Schlombs Kornelia , Wagner Thomas , Scheel Jochen
Published: November 2003

gonzo
(
goz
) is a zebrafish mutant with defects in cartilage formation. The
goz
phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the
goz
phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (
s1p
). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in
goz
embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of
goz
are caused independently of the lipid defects.
Induction of frameshift and base pair substitution mutations by the major DNA adduct of the endogenous carcinogen malondialdehyde
Authors: VanderVeen Laurie A. , Hashim Muhammed F. , Shyr Yu , Marnett Lawrence J.
Published: November 2003

Instability of repetitive sequences is a hallmark of human cancer, and its enhancement has been linked to oxidative stress. Malondialdehyde is an endogenous product of oxidative stress that reacts with guanine to form the exocyclic adduct, pyrimido[1,2- α]purin-10(3
H
)-one (M
1
G). We used site-specifically modified single- and double-stranded vectors to investigate the mutagenic potential of M
1
G in bacteria and mammalian cells. M
1
G induced frameshift mutations (-1 and -2) when positioned in a reiterated (CpG)
4
sequence but not when positioned in a nonreiterated sequence in
Escherichia coli
and in COS-7 cells. The frequency of frameshift mutations was highest when M
1
G was placed at the third G in the sequence. M
1
G induced base pair substitutions at comparable frequencies in both sequence contexts in COS-7 cells. These studies indicate that M
1
G, an endogenously generated product of oxidative stress, induces sequence-dependent frameshift mutations and base pair substitutions in bacteria and in mammalian cells. This finding suggests a potential role for the M
1
G lesion in the induction of mutations commonly associated with human diseases.
Dietary Cu stabilizes brain superoxide dismutase 1 activity and reduces amyloid Aβ production in APP23 transgenic mice
Authors: Bayer Thomas A. , Schäfer Stephanie , Simons Andreas , Kemmling André , Kamer Thomas , Tepests Ralf , Eckert Anne , Schüssel Katrin , Eikenberg Oliver , Sturchler-Pierrat Christine , Abramowski Dorothee , Staufenbiel Matthias , Multhaup Gerd
Published: November 2003

The Cu-binding β-amyloid precursor protein (APP), and the amyloid Aβ peptide have been proposed to play a role in physiological metal regulation. There is accumulating evidence of an unbalanced Cu homeostasis with a causative or diagnostic link to Alzheimer's disease. Whereas elevated Cu levels are observed in APP knockout mice, APP overexpression results in reduced Cu in transgenic mouse brain. Moreover, Cu induces a decrease in Aβ levels in APP-transfected cellsin vitro. To investigate the influence of bioavailable Cu, transgenic APP23 mice received an oral treatment with Cu-supplemented sucrose-sweetened drinking water (1). Chronic APP overexpressionper sereduced superoxide dismutase 1 activity in transgenic mouse brain, which could be restored to normal levels after Cu treatment (2). A significant increase of brain Cu indicated its bioavailability on Cu treatment in APP23 mice, whereas Cu levels remained unaffected in littermate controls (3). Cu treatment lowered endogenous CNS Aβ before a detectable reduction of amyloid plaques. Thus, APP23 mice reveal APP-induced alterations linked to Cu homeostasis, which can be reversed by addition of dietary Cu.
Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats
Authors: Shklyaev Stanislav , Aslanidi George , Tennant Michael , Prima Victor , Kohlbrenner Eric , Kroutov Vadim , Campbell-Thompson Martha , Crawford James , Shek Eugene W. , Scarpace Philip J. , Zolotukhin Sergei
Published: November 2003

Adiponectin (Acrp30) is a physiologically active polypeptide hormone secreted by adipose tissue that shows insulin-sensitizing, antiinflammatory, and antiatherogenic properties. In humans, Acrp30 levels are inversely related to the degree of adiposity. In the current study, we tested the long-term weight-reducing and insulin-enhancing effects of Acrp30 cDNA delivered peripherally by a viral vector. To this end, we have generated a series of recombinant adeno-associated virus vectors of serotypes 1 and 5 encoding mouse Acrp30 cDNAs. The long-term expression of recombinant adeno-associated virus-Acrp30 vectors was tested after intramuscular or intraportal injection in female Sprague–Dawley rats with diet-induced obesity. We show that a single peripheral injection of 1012physical particles of Acrp30-encoding vectors resulted in sustained (up to 280 days) significant reduction in body weight, concomitant with the reduction in daily food intake. Acrp30 treatment resulted in higher peripheral insulin sensitivity measured by the i.p. glucose tolerance test in fasted animals. Ectopic expression of the Acrp30 transgene resulted in modulation of hepatic gluconeogenesis and lipogenesis, as demonstrated by the reduction of the expression of two key genes:PEPCK(phosphoenolpyruvate carboxykinase) andSREBP-1c(sterol regulatory element-binding protein 1c) in the liver. These data show successful peripheral therapy in a clinically relevant model for human obesity and insulin resistance.
Inhibition of interferon signaling by dengue virus
Authors: Muñoz-Jordán Jorge L. , Sánchez-Burgos Gilma G. , Laurent-Rolle Maudry , García-Sastre Adolfo
Published: November 2003

Dengue virus is a worldwide-distributed mosquito-borne flavivirus with a positive strand RNA genome. Its transcribed polyprotein is cleaved by host- and virus-encoded peptidases into 10 proteins, some of which are of unknown function. Although dengue virus-infected cells seem to be resistant to the antiviral action of IFN, the viral products that mediate this resistance are unknown. Therefore, we have analyzed the ability of the 10 dengue virus-encoded proteins to antagonize the IFN response. We found that expression in human A549 cells of the dengue virus nonstructural proteins NS2A, NS4A, or NS4B enhances replication of an IFN-sensitive virus. Moreover, expression of NS4B and, to a lesser extent, of NS2A and NS4A proteins results in down-regulation of IFN-β-stimulated gene expression. Cells expressing NS4B or infected with dengue virus do not exhibit nuclear signal transducer and activator of transcription (STAT) 1 on treatment with IFN-β or IFN-γ, indicating that NS4B might be involved in blocking IFN signaling during dengue virus infections. This protein, encoded by a positive strand RNA virus, is implicated as an IFN-signaling inhibitor.
Efficient oxidative folding of conotoxins and the radiation of venomous cone snails
Authors: Bulaj Grzegorz , Buczek Olga , Goodsell Ian , Jimenez Elsie C. , Kranski Jessica , Nielsen Jacob S. , Garrett James E. , Olivera Baldomero M.
Published: November 2003

The 500 different species of venomous cone snails (genus
Conus
) use small, highly structured peptides (conotoxins) for interacting with prey, predators, and competitors. These peptides are produced by translating mRNA from many genes belonging to only a few gene superfamilies. Each translation product is processed to yield a great diversity of different mature toxin peptides (≈50,000–100,000), most of which are 12–30 aa in length with two to three disulfide crosslinks.
In vitro
, forming the biologically relevant disulfide configuration is often problematic, suggesting that
in vivo
mechanisms for efficiently folding the diversity of conotoxins have been evolved by the cone snails. We demonstrate here that the correct folding of a
Conus
peptide is facilitated by a posttranslationally modified amino acid, γ-carboxyglutamate. In addition, we show that multiple isoforms of protein disulfide isomerase are major soluble proteins in
Conus
venom duct extracts. The results provide evidence for the type of adaptations required before cone snails could systematically explore the specialized biochemical world of “microproteins” that other organisms have not been able to systematically access. Almost certainly, additional specialized adaptations for efficient microprotein folding are required.
Addicting drugs utilize a synergistic molecular mechanism in common requiring adenosine and Gi-βγ dimers
Authors: Yao Lina , Fan Peidong , Jiang Zhan , Mailliard William S. , Gordon Adrienne S. , Diamond Ivan
Published: November 2003

The mesolimbic dopamine system and cAMP-dependent/protein kinase A (PKA) pathways are strongly implicated in addictive behaviors. Here we determine the role of dopamine D2 receptors (D2) in PKA signaling responses to δ-opioid (DOR) and cannabinoid (CB1) receptors. We find in NG108-15/D2 cells and in cultured primary neurons that a brief exposure to saturating concentrations of DOR and CB1 agonists increases cAMP, promotes PKA Cα translocation and increases cAMP-dependent gene expression. Activation of PKA signaling is mediated by Gi-βγ dimers. Importantly, subthreshold concentrations of DOR or CB1 agonists with D2 agonists, which are without effect when added separately, together activate cAMP/PKA signaling synergistically. There is also synergy between DOR or CB1 with ethanol, another addicting agent. In all instances, synergy requires adenosine activation of adenosine A2 receptors and is mediated by βγ dimers. Synergy by this molecular mechanism appears to confer hypersensitivity to opioids and cannabinoids while simultaneously increasing the sensitivity of D2 signaling when receptors are expressed on the same cells. This mechanism may account, in part, for drug-induced activation of medium spiny neurons in the nucleus accumbens.
Time-restricted role for dendritic activation of the mTOR-p70
S6K
pathway in the induction of late-phase long-term potentiation in the CA1
Authors: Cammalleri Maurizio , Lütjens Robert , Berton Fulvia , King Alvin R. , Simpson Cindy , Francesconi Walter , Sanna Pietro Paolo
Published: November 2003

Mammalian target of rapamycin (mTOR) is a key regulator of translational capacity. The mTOR inhibitor rapamycin can prevent forms of protein synthesis-dependent synaptic plasticity such as long-term facilitation in Aplysia and late-phase long-term potentiation (L-LTP) in the hippocampal CA1 region of rodents. In the latter model, two issues remain to be addressed: defining the L-LTP phase sensitive to rapamycin and identifying the site of rapamycin-sensitive protein synthesis. Here, we show that L-LTP is sensitive to application of rapamycin only during the induction paradigm, whereas rapamycin application after the establishment of L-LTP was ineffective. Second, we observed that Thr-389-phosphorylated p70 S6 kinase (p70
S6K
), the main active phosphoform of the mTOR effector p70
S6K
, was induced in an
N
-methyl-
d
-aspartateand phosphatidylinositol 3-kinase-dependent manner throughout the dendrites but not in the cell bodies of CA1 neurons in hippocampal slices after L-LTP induction. A similar dendrite-wide activation of p70
S6K
was induced in primary hippocampal neurons by depolarization with KCL or glutamate. In primary hippocampal neurons, the sites of dendritic activation of p70
S6K
appeared as discrete compartments along dendritic shafts like the hotspots for fast dendritic translation. Conversely, only a subset of dendritic spines also displayed activated p70
S6K
. Taken together, the present data suggest that the
N
-methyl-
d
-aspartate-, phosphatidylinositol 3-kinase-dependent dendritic activation of the mTOR-p70
S6K
pathway is necessary for the induction phase of protein synthesis-dependent synaptic plasticity. Newly synthesized proteins in dendritic shafts could be targeted selectively to activity-tagged synapses. Thus, coordinated activation of dendrite-wide translation and synaptic-specific activation is likely to be necessary for long-term synaptic plasticity.
Epstein-Barr virus provides a survival factor to Burkitt's lymphomas
Authors: Kennedy Gregory , Komano Jun , Sugden Bill
Published: November 2003

Epstein-Barr virus (EBV) has been causally associated with at least five human malignancies. The exact contributions made by EBV to these cancers remain unknown. We demonstrate that one viral protein found in all EBV-associated malignancies, Epstein-Barr nuclear antigen 1 (EBNA-1), is required for survival of one of these cancers, EBV-positive Burkitt's lymphoma. Inhibition of EBNA-1 decreases survival of these tumor cells by inducing apoptosis. Expression of EBNA-1 in uninfected cells also can inhibit apoptosis induced by expression of p53 in the absence of the EBV genome. Our findings demonstrate that EBNA-1 is critical for the continued survival of EBV-associated Burkitt's lymphoma, and, by extension, for the other B cell tumors with which EBV is associated. Efficient inhibitors of EBNA-1's functions would likely prove useful in the therapy of EBV-associated malignancies.
The ISWI ATPase Snf2h is required for early mouse development
Authors: Stopka Tomas , Skoultchi Arthur I.
Published: November 2003

Chromatin assembly and remodeling complexes alter histone–DNA interactions by using the energy of ATP hydrolysis catalyzed by nucleosome-dependent ATPase subunits. Several classes of ATP-dependent chromatin remodeling complexes exist, including the ISWI family. ISWI complexes disrupt histone–DNA interactions
in vitro
by facilitating nucleosome sliding. Snf2h is a widely expressed ISWI ATPase. We investigated the role of the
Snf2h
gene in mammalian development by generating a null mutation in mice.
Snf2h
heterozygous mutant mice are born at the expected frequency and appear normal.
Snf2h
-/-
embryos die during the periimplantation stage. Blastocyst outgrowth experiments indicate that loss of Snf2h results in growth arrest and cell death of both the trophectoderm and inner cell mass. To investigate the effect of decreased Snf2h levels in adult cells, we performed antisense inhibition of Snf2h in human hematopoietic progenitors. Reducing Snf2h levels inhibited CD34
+
progenitors from undergoing cytokine-induced erythropoiesis
in vitro
. Our results indicate that Snf2h is required for proliferation of early blastocyst-derived stem cells and adult human hematopoietic progenitors. Cells lacking Snf2h are thus prevented from further embryonic development and differentiation.
Differential responses to psychotherapy versus pharmacotherapy in patients with chronic forms of major depression and childhood trauma
Authors: Nemeroff Charles B. , Heim Christine M. , Thase Michael E. , Klein Daniel N. , Rush A. John , Schatzberg Alan F. , Ninan Philip T. , McCullough James P. , Weiss Paul M. , Dunner David L. , Rothbaum Barbara O. , Kornstein Susan , Keitner Gabor , Keller Martin B.
Published: November 2003

Major depressive disorder is associated with considerable morbidity, disability, and risk for suicide. Treatments for depression most commonly include antidepressants, psychotherapy, or the combination. Little is known about predictors of treatment response for depression. In this study, 681 patients with chronic forms of major depression were treated with an antidepressant (nefazodone), Cognitive Behavioral Analysis System of Psychotherapy (CBASP), or the combination. Overall, the effects of the antidepressant alone and psychotherapy alone were equal and significantly less effective than combination treatment. Among those with a history of early childhood trauma (loss of parents at an early age, physical or sexual abuse, or neglect), psychotherapy alone was superior to antidepressant monotherapy. Moreover, the combination of psychotherapy and pharmacotherapy was only marginally superior to psychotherapy alone among the childhood abuse cohort. Our results suggest that psychotherapy may be an essential element in the treatment of patients with chronic forms of major depression and a history of childhood trauma.
Modulation of RNA editing by functional nucleolar sequestration of ADAR2
Authors: Sansam Christopher L. , Wells K. Sam , Emeson Ronald B.
Published: November 2003

The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. Here we demonstrate that ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm.
Circadian modulation of long-term sensitization in
Aplysia
Authors: Fernandez Raymond I. , Lyons Lisa C. , Levenson Jonathan , Khabour Omar , Eskin Arnold
Published: November 2003

As the mechanisms for learning and memory are elucidated, modulation of learning and memory becomes a central issue. We studied the modulation of learning and memory by investigating the circadian regulation of short- and long-term sensitization of the siphon withdrawal reflex in
Aplysia
. We found that
Aplysia
exhibited diurnal and circadian rhythms of long-term sensitization (LTS) with significantly greater LTS occurring when animals were trained and tested during the day relative to those trained and tested at night. In contrast to the modulation of LTS, short-term sensitization was not regulated by the circadian clock. Time of training rather than time of testing determined the circadian rhythm of LTS. Animals trained during the subjective day demonstrated LTS when tested during either the day or the night. Conversely, when animals were trained during the night, LTS was not observed when animals were tested either at night or during the day. Thus, the circadian rhythm of LTS is a rhythm in learning rather than a rhythm in recall. The threshold required to elicit siphon withdrawal and the duration of siphon withdrawal were not regulated by the circadian clock. These results indicate that the circadian oscillator exerts strong modulatory influences on one form of long-term memory in
Aplysia
.
Regulation of survivin function by Hsp90
Authors: Fortugno Paola , Beltrami Elena , Plescia Janet , Fontana Jason , Pradhan Deepti , Marchisio Pier Carlo , Sessa William C. , Altieri Dario C.
Published: November 2003

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis. This interaction involves the ATPase domain of Hsp90 and the survivin baculovirus inhibitor of apoptosis repeat. Global suppression of the Hsp90 chaperone function or targeted Abmediated disruption of the survivin–Hsp90 complex results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis, and cell cycle arrest with mitotic defects. These data link the cellular stress response to an antiapoptotic and mitotic checkpoint maintained by survivin. Targeting the survivin–Hsp90 complex may provide a rational approach for cancer therapy.
Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells
Authors: Yang Meng , Li Lingna , Jiang Ping , Moossa A. R. , Penman Sheldon , Hoffman Robert M.
Published: November 2003

We have developed a simple yet powerful technique for delineating the morphological events of tumor-induced angiogenesis and other tumor-induced host processes with dual-color fluorescence. The method clearly images implanted tumors and adjacent stroma, distinguishing unambiguously the host and tumor-specific components of the malignancy. The dual-color fluorescence imaging is effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice. This model shows with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis and tumor-infiltrating lymphocytes. The GFP-expressing tumor vasculature, both nascent and mature, could be readily distinguished interacting with the RFP-expressing tumor cells. GFP-expressing dendritic cells were observed contacting RFP-expressing tumor cells with their dendrites. GFP-expressing macrophages were observed engulfing RFP-expressing cancer cells. GFP lymphocytes were seen surrounding cells of the RFP tumor, which eventually regressed. Dual-color fluorescence imaging visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations.
Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase
Authors: Mahmoud Soheil S. , Croteau Rodney B.
Published: November 2003

(+)-Pulegone is a central intermediate in the biosynthesis of (-)-menthol, the most significant component of peppermint essential oil. Depending on environmental conditions, this branch point metabolite may be reduced to (-)-menthone en route to menthol, by pulegone reductase (PR), or oxidized to (+)-menthofuran, by menthofuran synthase (MFS). To elucidate regulation of pulegone metabolism, we modified the expression of
mfs
under control of the CaMV 35S promoter in transformed peppermint plants. Overexpression and cosuppression of
mfs
resulted in the respective increase or decrease in the production of menthofuran, indicating that the control of MFS resides primarily at the level of transcription. Significantly, in both WT peppermint as well as in all transformed plants, the flux of (+)-pulegone through PR correlated negatively with the essential oil content of menthofuran, such that menthofuran, and pulegone increased, or decreased, in concert. These results suggested that menthofuran itself might influence the reduction of pulegone. Although (+)-menthofuran did not inhibit (+)-PR activity, stem feeding with menthofuran selectively decreased
pr
transcript levels in immature leaves, thereby accounting for decreased reductase activity and increased pulegone content. These data demonstrate that the metabolic fate of (+)-pulegone is controlled through transcriptional regulation of
mfs
and that menthofuran, either directly or indirectly, influences this process by down-regulating transcription from
pr
and/or decreasing
pr
message stability. The ability to reduce both menthofuran and pulegone levels is of commercial significance in improving essential oil quality; however, the physiological rationale for such complex regulation is presently unclear.
BDNF induces translocation of initiation factor 4E to mRNA granules: Evidence for a role of synaptic microfilaments and integrins
Authors: Smart Fiona M. , Edelman Gerald M. , Vanderklish Peter W.
Published: November 2003

In many cell types, translation can be regulated by a redistribution of translation initiation factors to actin-based cytoskeletal compartments that contain bound mRNAs. This process is evoked by extracellular signals and is regulated by determinants of cytoskeletal organization, such as integrins. In the present experiments, we provide evidence that similar mechanisms regulate local translation in dendrites during synaptic plasticity. Treatment of various neuronal preparations with the brain-derived neurotrophic factor (BDNF) resulted in redistribution of the critical eukaryotic initiation factor 4E (eIF4E) to an mRNA granule-rich cytoskeletal fraction isolated from detergent-solubilized samples. eIF4E linkage to cap structures mediates the recruitment of other translation factors in the initiation of translation events. Immunoprecipitation studies confirmed that eIF4E associates with mRNA granules and that BDNF increased this association. BDNF-induced redistribution of eIF4E was blocked by preincubation with either a peptide (GRGDSP) that inhibits integrin-matrix interactions or by a high concentration (20 μM) of the F-actin depolymerizing agent latrunculin A. Immunohistochemical studies in cultured neurons demonstrated that BDNF facilitated translocation of eIF4E into dendritic spines. Together, the findings suggest that BDNF regulates translation in dendrites by altering the localization of eIF4E relative to cytoskeletally bound mRNA granules. Integrins, which are known to be essential for stabilizing certain forms of synaptic plasticity, may be critical regulators of local translation events at synapses.
Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases
Authors: Finlin Brian S. , Crump Shawn M. , Satin Jonathan , Andres Douglas A.
Published: November 2003

Rem, Rem2, Rad, and Gem/Kir (RGK) represent a distinct GTPase family with largely unknown physiological functions. We report here that both Rem and Rad bind directly to Ca
2+
channel β-subunits (Ca
V
β)
in vivo
. No calcium currents are recorded from human embryonic kidney 293 cells coexpressing the L type Ca
2+
channel subunits Ca
V
1.2, Ca
V
β
2a
, and Rem or Rad, but Ca
V
1.2 and Ca
V
β
2a
transfected cells elicit Ca
2+
channel currents in the absence of these small G proteins. Importantly, Ca
V
3 (T type) Ca
2+
channels, which do not require accessory subunits for ionic current expression, are not inhibited by expression of Rem. Rem is expressed in primary skeletal myoblasts and, when overexpressed in C2C12 myoblasts, wild-type Rem inhibits L type Ca
2+
channel activity. Deletion analysis demonstrates a critical role for the Rem C terminus in both regulation of functional Ca
2+
channel expression and β-subunit association. These results suggest that all members of the RGK GTPase family, via direct interaction with auxiliary β-subunits, serve as regulators of L type Ca
2+
channel activity. Thus, the RGK GTPase family may provide a mechanism for achieving cross talk between Ras-related GTPases and electrical signaling pathways.