bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary strategy of the swamp antechinus (Antechinus minimus maritimus) (Marsupialia:Dasyuridae) in coastal and inland heathland habitats
Authors: Allison Louise M. , Gibson Lesley A. , Aberton John G.
Published: March 2006

In the coastal heathlands of southern Victoria, populations of a rare dasyurid, the swamp antechinus (Antechinus minimus maritimus), are restricted to small and disjunct patches of suitable habitat. Although characteristics of their preferred habitat in terms of vegetation structure and composition have been described, little is known of their diet preferences. Diet and food availability of the species was examined at two coastal and two inland sites, during winter and spring by way of faecal analyses and pitfall trapping. Only minor differences in diet were observed between the coastal and inland habitats and this was consistent across season. There was, however, variation in food availability between the two habitat types, with generally higher frequencies of invertebrates occurring within the inland than in the coastal habitat during spring. Even so, when diet was directly compared with food availability, the differences observed within individual categories within each season were similar in magnitude and direction for both habitats, suggesting that inland and coastal populations of the swamp antechinus have similar dietary strategies. Insect larvae, Diplopoda and Coleoptera appeared to be favoured dietary items being almost consistently over-represented in the diet compared to their availability, whereas Collembola, Amphipoda, Dermaptera and Formicidae were avoided as they were never consumed in proportion to their availability. Although dietary preferences were evident, there was no clear selection of any one particular prey item, and so the swamp antechinus was considered a dietary generalist. Also, as the species sampled from most of the range of prey items available to them, it fits the qualitative criterion of opportunism. The generalist strategy of the swamp antechinus is likely to be advantageous in an environment subject to disturbance.
Running in pregnancy transiently increases postnatal hippocampal neurogenesis in the offspring
Authors: Bick-Sander Anika , Steiner Barbara , Wolf Susanne A. , Babu Harish , Kempermann Gerd
Published: March 2006

Voluntary wheel running of mice in pregnancy and lactation led to a twofold increase in hippocampal precursor-cell proliferation and in the number of Prox1-expressing lineage-determined cells at postnatal day 8 (P8). At P36, the number of newly generated granule cells approximately doubled, resulting in a 40% higher total number of granule cells in pups from running dams as compared with controls. Cell proliferation at embryonic day 15 (E15), in contrast, was decreased in the progeny of exercising mice, and the birth weight was reduced. At P49, body weight had normalized, and hippocampal neurogenesis was not different between the two groups. mRNA for FGF2 was expressed at higher levels at E15 and P8 in runner pups, whereas VEGF was increased only at E15. Insulin-like growth factor did not show differences at any time point. At P36, no differences for any of the factors were found. Our data indicate that maternal behavior and physical activity affects infantile growth-factor expression and can transiently stimulate postnatal hippocampal development in the offspring.
Interruption of tumor dormancy by a transient angiogenic burst within the tumor microenvironment
Authors: Indraccolo Stefano , Stievano Laura , Minuzzo Sonia , Tosello Valeria , Esposito Giovanni , Piovan Erich , Zamarchi Rita , Chieco-Bianchi Luigi , Amadori Alberto
Published: March 2006

Tumor growth is currently viewed as a phenomenon associated with neovascularization and sustained production of angiogenic factors, but whether a transient angiogenic switch may trigger tumor growth remains unclear. Here, we report that leukemia cells (MOLT-3) were poorly angiogenic and remained dormant when injected s.c. into immunodeficient mice. However, progressive growth of lymphoid tumors was invariably recorded when irradiated angiogenic cells from Kaposi’s sarcoma (KS-IMM) were locally coinjected with MOLT-3 cells or administered later. The persistence of KS-IMM cells
in vivo
was tracked by flow cytometry and real-time PCR analysis, and it was limited to a few days, during which angiogenesis was induced and preceded tumor growth. The engraftment of other types of poorly tumorigenic cancer cells was also greatly improved by irradiated KS-IMM cells. Moreover, short-term treatment with angiogenic factors, including basic FGF or VEGF, either given as recombinant factors or delivered by retroviral vectors, also accelerated tumor growth. These findings may emphasize that tumor angiogenesis is a process requiring a higher amount of angiogenic factors for its induction than maintenance.
Restriction of vaccinia virus replication by a
ced-3
and
ced-4
-dependent pathway in
Caenorhabditis
elegans
Authors: Liu Wan-Hsin , Lin Yi-Ling , Wang Jia-Pey , Liou Willisa , Hou Roger F. , Wu Yi-Chun , Liao Ching-Len
Published: March 2006

Genetic tractability and easy manipulation make
Caenorhabditis elegans
a good model to study host–pathogen interactions. Dozens of different bacterial species can pathogenically infect
C. elegans
under laboratory conditions, and all of these microbes are extracellular pathogens to nematodes. Viruses, on the other hand, are obligate intracellular parasites, and yet no viral infections have been reported for
C. elegans
. We established a procedure allowing vaccinia virus to enter and subsequently replicate in
C. elegans
. Virus replication was significantly enhanced in
ced-3
,
ced-4
,
ced-9(gf)
, and
egl-1(lf)
mutants, demonstrating that the core programmed cell death (PCD) genes
ced-3
,
ced-4
,
ced-9
, and
egl-1
control vaccinia virus replication in
C. elegans.
The ability of
ced-3
and
ced-4
alleles to restrict virus replication is correlated with their cell-killing activities. Moreover, the increase in vaccinia virus replication levels in the PCD-defective mutants was not likely to be caused by the extra live cells, as neither the inhibition of PCD by
icd-1
overexpression nor the presence of extra cells after extra cell divisions in
cul-1
or
lin-23
mutants had any significant effect on vaccinia virus replication. Therefore, the core PCD genes possess a unique function in controlling vaccinia virus replication in
C. elegans
.
Selective potentiation of Stat-dependent gene expression by collaborator of Stat6 (CoaSt6), a transcriptional cofactor
Authors: Goenka Shreevrat , Boothby Mark
Published: March 2006

The molecular mechanisms by which transcription is selectively activated and precisely controlled by signal transducer and activator of transcription (Stat) factors represent a central issue in cytokine-mediated cellular responses. Stat6 mediates responses to IL-4 and antagonizes Stat1 activated by IFN-γ. We have discovered that Stat6 binds to collaborator of Stat6 (CoaSt6), a protein that lacks conventional coactivator motifs but contains three iterations of a domain found in the variant histone macroH2A. Although macroH2A participates in transcriptional silencing, the macro domains of CoaSt6 increased IL-4-induced gene expression. Moreover, CoaSt6 amplified Stat6-mediated but not IFN-γ-induced gene expression, providing evidence of a selective coregulator of Stat-mediated gene transcription.
Global identification of noncoding RNAs in
Saccharomyces cerevisiae
by modulating an essential RNA processing pathway
Authors: Samanta Manoj Pratim , Tongprasit Waraporn , Sethi Himanshu , Chin Chen-Shan , Stolc Viktor
Published: March 2006

Noncoding RNAs (ncRNAs) perform essential cellular tasks and play key regulatory roles in all organisms. Although several new ncRNAs in yeast were recently discovered by individual studies, to our knowledge no comprehensive empirical search has been conducted. We demonstrate a powerful and versatile method for global identification of previously undescribed ncRNAs by modulating an essential RNA processing pathway through the depletion of a key ribonucleoprotein enzyme component, and monitoring differential transcriptional activities with genome tiling arrays during the time course of the ribonucleoprotein depletion. The entire
Saccharomyces cerevisiae
genome was scanned during cell growth decay regulated by promoter-mediated depletion of
Rpp1
, an essential and functionally conserved protein component of the RNase P enzyme. In addition to most verified genes and ncRNAs, expression was detected in 98 antisense and intergenic regions, 74 that were further confirmed to contain previously undescribed RNAs. A class of ncRNAs, located antisense to coding regions of verified protein-coding genes, is discussed in this article. One member,
HRA1
, is likely involved in 18S rRNA maturation.
The dependence receptor DCC requires lipid raft localization for cell death signaling
Authors: Furne Céline , Corset Véronique , Hérincs Zoltán , Cahuzac Nathalie , Hueber Anne-Odile , Mehlen Patrick
Published: March 2006

DCC
(deleted in colorectal cancer) is a putative tumor suppressor gene whose expression is lost in numerous cancers. DCC also encodes the main receptor for the neuronal navigation cue netrin-1. It has been shown that DCC belongs to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand is absent, thus conferring a state of cellular dependence on ligand availability. We recently proposed that DCC is a tumor suppressor because it induces the death of tumor cells that grow in settings of ligand unavailability. Moreover, it seems that the DCC/netrin-1 pair may also regulate neuron survival during nervous system development. However, the mechanisms by which DCC triggers cell death are still unknown. We show here that the localization of DCC to lipid rafts is a prerequisite for its proapoptotic activity, both in immortalized cells and in primary neurons. The presence of DCC in lipid rafts probably allows the formation of an adequate submembrane complex, because the interaction of caspase-9 with DCC is inhibited by the disorganization of lipid rafts. Thus, dependence receptors may require lipid raft localization for cell death signaling.
Engineered antibody Fc variants with enhanced effector function
Authors: Lazar Greg A. , Dang Wei , Karki Sher , Vafa Omid , Peng Judy S. , Hyun Linus , Chan Cheryl , Chung Helen S. , Eivazi Araz , Yoder Sean C. , Vielmetter Jost , Carmichael David F. , Hayes Robert J. , Dahiyat Bassil I.
Published: March 2006

Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcγ receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcγ receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement ofin vitroeffector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in anin vivopreclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.
BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates
Authors: Wu Leonard , Bachrati Csanad Z. , Ou Jiongwen , Xu Chang , Yin Jinhu , Chang Michael , Wang Weidong , Li Lei , Brown Grant W. , Hickson Ian D.
Published: March 2006

BLM
encodes a member of the highly conserved RecQ DNA helicase family, which is essential for the maintenance of genome stability. Homozygous inactivation of
BLM
gives rise to the cancer predisposition disorder Bloom’s syndrome. A common feature of many RecQ helicase mutants is a hyperrecombination phenotype. In Bloom’s syndrome, this phenotype manifests as an elevated frequency of sister chromatid exchanges and interhomologue recombination. We have shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase IIIα (hTOPO IIIα), can process recombination intermediates that contain double Holliday junctions into noncrossover products by a mechanism termed dissolution. Here we show that a recently identified third component of the human BLM/hTOPO IIIα complex, BLAP75/RMI1, promotes dissolution catalyzed by hTOPO IIIα. This activity of BLAP75/RMI1 is specific for dissolution catalyzed by hTOPO IIIα because it has no effect in reactions containing either
Escherichia coli
Top1 or Top3, both of which can also catalyze dissolution in a BLM-dependent manner. We present evidence that BLAP75/RMI1 acts by recruiting hTOPO IIIα to double Holliday junctions. Implications of the conserved ability of type IA topoisomerases to catalyze dissolution and how the evolution of factors such as BLAP75/RMI1 might confer specificity on the execution of this process are discussed.
Haptoglobin, a hemoglobin-binding plasma protein, is present in bony fish and mammals but not in frog and chicken
Authors: Wicher Krzysztof B. , Fries Erik
Published: March 2006

Hemoglobin (Hb) released from erythrocytes may cause oxidation of lipids and proteins. Haptoglobin (Hp), which occurs in the plasma of all mammals, binds free Hb and inhibits its oxidative activity. It is not known whether this protective protein also exists in lower vertebrates. By analyzing available genomic sequences, we have found that bony fish, but not more primitive animals, have a gene coding for a protein homologous to mammalian Hp. Furthermore, we show that this protein is present in the plasma of Japanese pufferfish (Takifugu rubripes) and that it binds Hb. These results, together with a phylogenetic analysis, suggest that Hp evolved from a complement-associated protein (mannose-binding lectin-associated serine proteinase, MASP), with the emergence of fish. Surprisingly, we found that both chicken (Gallus gallus) and the Western clawed frog (Xenopus tropicalis) lack the Hp gene. In chicken plasma, however, we identified a different type of Hb-binding protein, PIT54, which has been reported to be a potent antioxidant. PIT54 is a soluble member of the family of scavenger receptor cysteine-rich proteins, and we found that its gene exists only in birds. We also show that the plasma of ostrich (Strutio camelus), a primitive bird, contains both PIT54 and Hp. Collectively, our data suggest that PIT54 has successively taken over the function of Hp during the evolution of the avian lineage and has completely replaced the latter protein in chicken.
Gene evolution at the ends of wheat chromosomes
Authors: See Deven R. , Brooks Steven , Nelson James C. , Brown-Guedira Gina , Friebe Bernd , Gill Bikram S.
Published: March 2006

Wheat ESTs mapped to deletion bins in the distal 42% of the long arm of chromosome 4B (4BL) were ordered
in silico
based on
blastn
homology against rice pseudochromosome 3. The ESTs spanned 29 cM on the short arm of rice chromosome 3, which is known to be syntenic to long arms of group-4 chromosomes of wheat. Fine-scale deletion-bin and genetic mapping revealed that 83% of ESTs were syntenic between wheat and rice, a far higher level of synteny than previously reported, and 6% were nonsyntenic (not located on rice chromosome 3). One inversion spanning a 5-cM region in rice and three deletion bins in wheat was identified. The remaining 11% of wheat ESTs showed no sequence homology in rice and mapped to the terminal 5% of the wheat chromosome 4BL. In this region, 27% of ESTs were duplicated, and it accounted for 70% of the recombination in the 4BL arm. Globally in wheat, no sequence homology ESTs mapped to the terminal bins, and ESTs rarely mapped to interstitial chromosomal regions known to be recombination hot spots. The wheat–rice comparative genomics analysis indicated that gene evolution occurs preferentially at the ends of chromosomes, driven by duplication and divergence associated with high rates of recombination.
The folding energy landscape of apoflavodoxin is rugged: Hydrogen exchange reveals nonproductive misfolded intermediates
Authors: Bollen Yves J. M. , Kamphuis Monique B. , van Mierlo Carlo P. M.
Published: March 2006

Many native proteins occasionally form partially unfolded forms (PUFs), which can be detected by hydrogen/deuterium exchange and NMR spectroscopy. Knowledge about these metastable states is required to better understand the onset of folding-related diseases. So far, not much is known about where PUFs reside within the energy landscape for protein folding. Here, four PUFs of the relatively large apoflavodoxin (179 aa) are identified. Remarkably, at least three of them are partially misfolded conformations. The misfolding involves side-chain contacts as well as the protein backbone. The rates at which the PUFs interconvert with native protein have been determined. Comparison of these rates with stopped-flow data positions the PUFs in apoflavodoxin’s complex folding energy landscape. PUF1 and PUF2 are unfolding excursions that start from native apoflavodoxin but do not continue to the unfolded state. PUF3 and PUF4 could be similar excursions, but their rates of formation suggest that they are on a dead-end folding route that starts from unfolded apoflavodoxin and does not continue all of the way to native protein. All PUFs detected thus are off the protein’s productive folding route.
Orphaned ryanodine receptors in the failing heart
Authors: Song Long-Sheng , Sobie Eric A. , McCulle Stacey , Lederer W. J. , Balke C. William , Cheng Heping
Published: March 2006

Heart muscle is characterized by a regular array of proteins and structures that form a repeating functional unit identified as the sarcomere. This regular structure enables tight coupling between electrical activity and Ca
2+
signaling. In heart failure, multiple cellular defects develop, including reduced contractility, altered Ca
2+
signaling, and arrhythmias; however, the underlying causes of these defects are not well understood. Here, in ventricular myocytes from spontaneously hypertensive rats that develop heart failure, we identify fundamental changes in Ca
2+
signaling that are related to restructuring of the spatial organization of the cells. Myocytes display both a reduced ability to trigger sarcoplasmic reticulum Ca
2+
release and increased spatial dispersion of the transverse tubules (TTs). Remodeled TTs in cells from failing hearts no longer exist in the regularly organized structures found in normal heart cells, instead moving within the sarcomere away from the Z-line structures and leaving behind the sarcoplasmic reticulum Ca
2+
release channels, the ryanodine receptors (RyRs). These orphaned RyRs appear to be responsible for the dyssynchronous Ca
2+
sparks that have been linked to blunted contractility and, probably, Ca
2+
-dependent arrhythmias in diverse models of heart failure. We conclude that the increased spatial dispersion of the TTs and orphaned RyRs lead to the loss of local control and Ca
2+
instability in heart failure.
A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP
Authors: Ganesan Sundar , Ameer-beg Simon M. , Ng Tony T. C. , Vojnovic Borivoj , Wouters Fred S.
Published: March 2006

Förster resonance energy transfer (FRET) microscopy is a powerful technique that enables the visualization of signaling intermediates, protein interactions, and protein conformational and biochemical status. With the availability of an ever-increasing collection of fluorescent proteins, pairs of spectrally different variants have been used for the study of FRET in living cells. However, suitable spectral overlap, necessary for efficient FRET, is limited by the requirement for proper emission separation. Currently used FRET pairs represent compromises between these opposing spectral demands that reduce the maximally attainable FRET sensitivity. We present a previously undescribed FRET acceptor, a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). REACh allows the use of the photophysically superior FRET donor EGFP, with which it exhibits optimal spectral overlap, which obviates the need for narrow spectral filtering and allows additional fluorescent labels to be used within the same cell. The latter allows the generation of sophisticated bioassays for complex biological questions. We show that this dark acceptor is ideally suited for donor fluorescence lifetime imaging microscopy (FLIM) and confirm these measurements with an independent intensity-based donor fluorescence quenching resonance energy transfer (FqRET) assay. REACh also can be used in donor photobleaching kinetics-based FRET studies. By detecting FRET between a GFP-tagged ubiquitination substrate and REACh-labeled ubiquitin, we imaged the active ubiquitination machinery inside cells. This assay therefore can be used to study proteins whose function is regulated by ubiquitination.
Role of calcium in the regulation of mechanical power in insect flight
Authors: Gordon Shefa , Dickinson Michael H.
Published: March 2006

Most flying insect species use “asynchronous” indirect flight muscles (A-IFMs) that are specialized to generate high mechanical power at fast contraction frequencies. Unlike individual contractions of “synchronous” muscles, those of A-IFMs are not activated and deactivated in concert with neurogenically controlled cycling of myoplasmic [Ca
2+
] but rather are driven myogenically by oscillatory changes in length. The motor neurons of the A-IFMs, which fire at a rate much slower than contraction frequency, are thought to play the limited role of maintaining myoplasmic [Ca
2+
] above the critical threshold that maintains the muscle in a stretch-activatable state. Despite this asynchronous form of excitation–contraction coupling, animals can actively regulate power output as required for different flight behaviors, although the neurobiological and biophysical basis of this regulation is unknown. While presenting tethered flying fruit flies,
Drosophila melanogaster
, with visual stimuli, we recorded membrane potential spikes in identified A-IFM fibers. We show that mechanical power output rises and falls in concert with the firing frequency of all A-IFM fibers and cannot be explained by differential recruitment of separately innervated motor units. To explore the hypothesis that myoplasmic [Ca
2+
] might similarly rise and fall in concert with firing frequency, we genetically engineered
Drosophila
to express the FRET-based Ca
2+
indicator cameleon selectively within A-IFMs. The results show that Ca
2+
levels increase in proportion to muscle firing rate, both during spontaneous flight and when muscle spikes are elicited electrically. Collectively, these experiments on intact animals support an active role for [Ca
2+
] in regulating power output of stretch-activated A-IFM.
Activation of the cardiac ciliary neurotrophic factor receptor reverses left ventricular hypertrophy in leptin-deficient and leptin-resistant obesity
Authors: Raju Shubha V. Y. , Zheng Meizi , Schuleri Karl H. , Phan Alexander C. , Bedja Djahida , Saraiva Roberto M. , Yiginer Omer , Vandegaer Koenraad , Gabrielson Kathleen L. , O’Donnell Christopher P. , Berkowitz Dan E. , Barouch Lili A. , Hare Joshua M.
Published: March 2006

Disruption of the leptin signaling pathway within the heart causes left ventricular hypertrophy (LVH). Because human obesity is a syndrome of leptin resistance, which is not amenable to leptin treatment, the identification of parallel signal transduction pathways is of potential therapeutic value. Ciliary neurotrophic factor (CNTF), which acts parallel to leptin in the hypothalamus, is not previously recognized to have cardiac activity. We hypothesized that CNTF receptors are present on cardiomyocytes and their activation reverses LVH in both leptin-deficient
ob/ob
and leptin-resistant
db/db
mice. The localization of CNTF receptors (CNTFRα) to the sarcolemma in C57BL/6,
ob/ob
and
db/db
was confirmed
in situ
with immunohistochemistry, and immunoblotting (60 and 40 kDa) on isolated myocytes.
ob/ob
mice were randomly assigned to receive s.c. recombinant CNTF (CNTF
Ax15
; 0.1 mg·kg
−1
per day;
n
= 11) calorie-restriction (
n
= 9), or feeding ad libitum (
n
= 11).
db/db
mice were allocated to three similar groups (
n
= 8, 7, and 8, respectively) plus a leptin group (1 mg·kg
−1
per day;
n
= 7). Echocardiography showed that CNTF
Ax15
reduced cardiac hypertrophy [posterior wall thickness decreased by 29 ± 8% (
P
< 0.01) in
ob/ob
and by 21 ± 3% in
db/db
mice (
P
< 0.01)], which was consistent with the reduction of myocyte width. Western blotting showed that leptin and CNTF
Ax15
activated Stat3 and ERK1/2 pathway in cultured adult mice cardiomyocytes and cardiac tissue from in
ob/ob
and
db/db
mice. Together, these findings support the role of a previously undescribed signaling pathway in obesity-associated cardiac hypertrophy and have therapeutic implications for patients with obesity-related cardiovascular disease and other causes of LVH.
Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins
Authors: Aguilar Rubén C. , Longhi Silvia A. , Shaw Jonathan D. , Yeh Lan-Yu , Kim Sean , Schön Arne , Freire Ernesto , Hsu Ariel , McCormick William K. , Watson Hadiya A. , Wendland Beverly
Published: March 2006

Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins,
ENT1
and
ENT2
; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of
ent1
Δ
ent2
Δ cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity.
Latent extinction risk and the future battlegrounds of mammal conservation
Authors: Cardillo Marcel , Mace Georgina M. , Gittleman John L. , Purvis Andy
Published: March 2006

Global conservation prioritization usually emphasizes areas with highest species richness or where many species are thought to be at imminent risk of extinction. However, these strategies may overlook areas where many species have biological traits that make them particularly sensitive to future human impact but are not yet threatened because such impact is currently low. In this article, we identify such areas for the world’s mammals using latent extinction risk, the discrepancy between a species’ current extinction risk and that predicted from models on the basis of biological traits. Species with positive latent risk are currently less threatened than their biology would suggest, usually because they inhabit regions or habitats still comparatively unmodified by human activity. Using large new geographic, biological, and phylogenetic databases for nearly 4,000 mammal species, we map the global geographic distribution of latent risk to reveal areas where the mammal fauna is still relatively unthreatened but has high inherent sensitivity to disturbance. These hotspots include large areas such as the Nearctic boreal forests and tundra that are unrepresented in most current prioritization schemes, as well as high-biodiversity areas such as the island arc from Indonesia to the south Pacific. Incorporating latent extinction risk patterns into conservation planning could help guard against future biodiversity loss by anticipating and preventing species declines before they begin.
A sulfated metabolite produced bystf3negatively regulates the virulence ofMycobacteriumtuberculosis
Authors: Mougous Joseph D. , Senaratne Ryan H. , Petzold Christopher J. , Jain Madhulika , Lee Dong H. , Schelle Michael W. , Leavell Michael D. , Cox Jeffery S. , Leary Julie A. , Riley Lee W. , Bertozzi Carolyn R.
Published: March 2006

Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells.Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope ofM. tuberculosisand negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3′-phosphoadenosine-5′-phosphosulfate and a previously uncharacterized sulfotransferase,stf3. These findings extend the known functions of sulfated molecules as general modulators of cell–cell interactions to include those between a bacterium and a human host.
The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA
Authors: Schroeder Gottfried K. , Lad Chetan , Wyman Paul , Williams Nicholas H. , Wolfenden Richard
Published: March 2006

Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C–O cleavage, at a rate suggesting an upper limit of 10
−15
s
−1
for P–O cleavage of phosphodiester anions at 25°C. To evaluate the rate enhancement produced by P–O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P–O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C–O cleavage is sterically precluded so that hydrolysis occurs only by P–O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a
t
1/2
of 30,000,000 years at 25°C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (
k
cat
= 95 s
−1
) enhances the rate of phosphodiester hydrolysis by a factor of ≈10
17
. In alkaline solution, thymidylyl-3′-5′-thymidine (TpT) has been reported to decompose 10
5
-fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.
UV causation of melanoma in
Xiphophorus
is dominated by melanin photosensitized oxidant production
Authors: Wood Simon R. , Berwick Marianne , Ley Ronald D. , Walter Ronald B. , Setlow Richard B. , Timmins Graham S.
Published: March 2006

Controversy continues both as to which wavelengths of sunlight cause melanoma and the mechanisms by which these different wavelengths act. Direct absorption of UVB by DNA is central in albino animal models, but melanin-pigmented models have shown major contributions by wavelengths longer than UVB that are thought to be mediated by photosensitized oxidant production. The only model for which the action spectrum of melanoma causation is known is a genetically melanoma-susceptible specific cross of
Xiphophorus
fish. We used electron paramagnetic resonance to quantitatively detect the UV induction of reactive melanin radicals
in situ
in the melanin-containing cells in the skin of this model and derived the action spectrum for melanin-photosensitized oxidant production (Φ
ox
). This action spectrum was identical to that for melanoma induction (Φ
mel
). These results confirm the hypothesis that melanin-photosensitized radical production is the major causative step of melanoma in this model and demonstrate that the wavelengths and mechanisms of melanoma causation in different models are dependent on the presence of melanin. This approach should be applicable to humans, thus providing an accurate surrogate for Φ
mel
for prevention studies.
Pharmacological promotion of inclusion formation: A therapeutic approach for Huntington’s and Parkinson’s diseases
Authors: Bodner Ruth A. , Outeiro Tiago Fleming , Altmann Stephen , Maxwell Michele M. , Cho Stephanie H. , Hyman Bradley T. , McLean Pamela J. , Young Anne B. , Housman David E. , Kazantsev Aleksey G.
Published: March 2006

Misfolded proteins accumulate in many neurodegenerative diseases, including huntingtin in Huntington’s disease and α-synuclein in Parkinson’s disease. The disease-causing proteins can take various conformations and are prone to aggregate and form larger cytoplasmic or nuclear inclusions. One approach to the development of therapeutic intervention for these diseases has been to identify chemical compounds that reduce the size or number of inclusions. We have, however, identified a compound that promotes inclusion formation in cellular models of both Huntington’s disease and Parkinson’s disease. Of particular interest, this compound prevents huntingtin-mediated proteasome dysfunction and reduces α-synuclein-mediated toxicity. These results demonstrate that compounds that increase inclusion formation may actually lessen cellular pathology in both Huntington’s and Parkinson’s diseases, suggesting a therapeutic approach for neurodegenerative diseases caused by protein misfolding.
Identification of proangiogenic genes and pathways by high-throughput functional genomics: TBK1 and the IRF3 pathway
Authors: Korherr Christian , Gille Hendrik , Schäfer Rolf , Koenig-Hoffmann Kerstin , Dixelius Johan , Egland Kristi A. , Pastan Ira , Brinkmann Ulrich
Published: March 2006

A genome-wide phenotype screen was used to identify factors and pathways that induce proliferation of human umbilical vein endothelial cells (HUVEC). HUVEC proliferation is a recognized marker for factors that modulate vascularization. Screening “hits” included known proangiogenic factors, such as VEGF, FGF1, and FGF2 and additional factors for which a direct association with angiogenesis was not previously described. These include the kinase TBK1 as well as Toll-like receptor adaptor molecule and IFN regulatory factor 3. All three proteins belong to one signaling pathway that mediates induction of gene expression, including a mixture of secreted factors, which, in concert, mediate proliferative activity toward endothelial cells. TBK1 as the “trigger” of this pathway is induced under hypoxic conditions and expressed at significant levels in many solid tumors. This pattern of expression and the decreased expression of angiogenic factors in cultured cells upon RNA-interference-mediated ablation suggests that TBK1 is important for vascularization and subsequent tumor growth and a target for cancer therapy.
Global health benefits of mitigating ozone pollution with methane emission controls
Authors: West J. Jason , Fiore Arlene M. , Horowitz Larry W. , Mauzerall Denise L.
Published: March 2006

Methane (CH
4
) contributes to the growing global background concentration of tropospheric ozone (O
3
), an air pollutant associated with premature mortality. Methane and ozone are also important greenhouse gases. Reducing methane emissions therefore decreases surface ozone everywhere while slowing climate warming, but although methane mitigation has been considered to address climate change, it has not for air quality. Here we show that global decreases in surface ozone concentrations, due to methane mitigation, result in substantial and widespread decreases in premature human mortality. Reducing global anthropogenic methane emissions by 20% beginning in 2010 would decrease the average daily maximum 8-h surface ozone by ≈1 part per billion by volume globally. By using epidemiologic ozone-mortality relationships, this ozone reduction is estimated to prevent ≈30,000 premature all-cause mortalities globally in 2030, and ≈370,000 between 2010 and 2030. If only cardiovascular and respiratory mortalities are considered, ≈17,000 global mortalities can be avoided in 2030. The marginal cost-effectiveness of this 20% methane reduction is estimated to be ≈$420,000 per avoided mortality. If avoided mortalities are valued at $1 million each, the benefit is ≈$240 per tonne of CH
4
(≈$12 per tonne of CO
2
equivalent), which exceeds the marginal cost of the methane reduction. These estimated air pollution ancillary benefits of climate-motivated methane emission reductions are comparable with those estimated previously for CO
2
. Methane mitigation offers a unique opportunity to improve air quality globally and can be a cost-effective component of international ozone management, bringing multiple benefits for air quality, public health, agriculture, climate, and energy.
Design of natural killer T cell activators: Structure and function of a microbial glycosphingolipid bound to mouse CD1d
Authors: Wu Douglass , Zajonc Dirk M. , Fujio Masakazu , Sullivan Barbara A. , Kinjo Yuki , Kronenberg Mitchell , Wilson Ian A. , Wong Chi-Huey
Published: March 2006

Natural killer T (NKT) cells provide an innate-type immune response upon T cell receptor interaction with CD1d-presented antigens. We demonstrate through equilibrium tetramer binding and antigen presentation assays with Vα14i-positive NKT cell hybridomas that theSphingomonasglycolipid α-galacturonosyl ceramide (GalA-GSL) is a NKT cell agonist that is significantly weaker than α-galactosylceramide (α-GalCer), the most potent known NKT agonist. For GalA-GSL, a shorter fatty acyl chain, an absence of the 4-OH on the sphingosine tail and a 6′-COOH group on the galactose moiety account for its observed antigenic potency. We further determined the crystal structure of mCD1d in complex with GalA-GSL at 1.8-Å resolution. The overall binding mode of GalA-GSL to mCD1d is similar to that of the short-chain α-GalCer ligand PBS-25, but its sphinganine chain is more deeply inserted into the F′ pocket due to alternate hydrogen-bonding interactions between the sphinganine 3-OH with Asp-80. Subsequently, a slight lateral shift (>1 Å) of the galacturonosyl head group is noted at the CD1 surface compared with the galactose of α-GalCer. Because the relatively short C14fatty acid of GalA-GSL does not fully occupy the A′ pocket, a spacer lipid is found that stabilizes this pocket. The lipid spacer was identified by GC/MS as a mixture of saturated and monounsaturated palmitic acid (C16). Comparison of available crystal structures of α-anomeric glycosphingolipids now sheds light on the structural basis of their differential antigenic potency and has led to the design and synthesis of NKT cell agonists with enhanced cell-based stimulatory activities compared with α-GalCer.
Imidazenil and diazepam increase locomotor activity in mice exposed to protracted social isolation
Authors: Pinna Graziano , Agis-Balboa Roberto C. , Zhubi Adrian , Matsumoto Kinzo , Grayson Dennis R. , Costa Erminio , Guidotti Alessandro
Published: March 2006

In cortex and hippocampus, protracted (>4 weeks) social isolation of adult male mice alters the subunit expression of GABA type A receptors (GABAA-Rs) as follows: (i) the mRNAs encoding GABAA-R α1, α2, and γ2 subunits are decreased by ≈50%, whereas those encoding α4 and α5 subunits are increased by ≈100%; (ii) similarly, the synaptic membrane expression of the α1 subunit protein is down-regulated, and that of the α5 subunit protein is up-regulated; and (iii) the binding of [3H]flumazenil to hippocampal synaptic membranes is decreased. Behaviorally, socially isolated (SI) mice are resistant to the sedative effects of the positive allosteric GABAA-R modulators diazepam (DZP) and zolpidem. This resistance seems to be attributable to the decrease of α1-containing GABAA-Rs. Paradoxically, DZP, which, unlike zolpidem, acts at α5-containing GABAA-Rs, increases the locomotor activity of SI mice. Imidazenil, which fails to modulate α1-, α4-, and α6-containing GABAA-Rs but is a selective positive allosteric modulator of α5-containing GABAA-Rs, also increases locomotor activity in SI mice. Importantly, SI mice responded to muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one, and allopregnanolone similar to group-housed mice. These data suggest that a switch (a decrease in α1/α2 and γ2 and an increase in α4 and α5 subunits) in the composition of the heteropentameric GABAA-R subunit assembly without a change in total GABAA-R number occurs during social isolation. Thus, the repertoire of DZP and imidazenil actions in SI mice appears to be elicited by the allosteric modulation of GABAA-Rs overexpressing α5 subunits. Benzodiazepine response mediated by α1-containing GABAA-Rs is expected to be silent or reduced.
Structures of eukaryotic ribonucleotide reductase I define gemcitabine diphosphate binding and subunit assembly
Authors: Xu Hai , Faber Catherine , Uchiki Tomoaki , Racca Joseph , Dealwis Chris
Published: March 2006

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. Crucial for rapidly dividing cells, RNR is a target for cancer therapy. In eukaryotes, RNR comprises a heterooligomer of α
2
and β
2
subunits. Rnr1, the α subunit, contains regulatory and catalytic sites; Rnr2, the β subunit (in yeast, a heterodimer of Rnr2 and Rnr4), houses the diferric-tyrosyl radical crucial for catalysis. Here, we present three x-ray structures of eukaryotic Rnr1 from
Saccharomyces cerevisiae
: one bound to gemcitabine diphosphate (GemdP), the active metabolite of the mechanism-based chemotherapeutic agent gemcitabine; one with an Rnr2-derived peptide, and one with an Rnr4-derived peptide. Our structures reveal that GemdP binds differently from its analogue, cytidine diphosphate; because of unusual interactions of the geminal fluorines, the ribose and base of GemdP shift substantially, and loop 2, which mediates substrate specificity, adopts different conformations when binding to GemdP and cytidine diphosphate. The Rnr2 and Rnr4 peptides, which block RNR assembly, bind differently from each other but have unique modes of binding not seen in prokaryotic RNR. The Rnr2 peptide adopts a conformation similar to that previously reported from an NMR study for a mouse Rnr2-based peptide. In yeast, the Rnr2 peptide binds at subsites consisting of residues that are highly conserved among yeast, mouse, and human Rnr1s, suggesting that the mode of Rnr1–Rnr2 binding is conserved among eukaryotes. These structures provide new insights into subunit assembly and a framework for structure-based drug design targeting RNR.
Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides
Authors: Schirmer Andreas , Kennedy Jonathan , Murli Sumati , Reid Ralph , Santi Daniel V.
Published: March 2006

Resorcylic acid lactones containing a cis-enone are susceptible to Michael addition reactions and are potent inhibitors of several protein kinases. A structural-bioinformatics analysis identified a conserved Cys residue in the ATP-binding site of the kinases reported to be inhibited by cis-enone resorcylic acid lactones but absent in those that are not. Mining of the kinome database revealed that a subset of some 46 kinases contained this Cys residue. Screening a panel of 124 kinases with the resorcylic acid lactone hypothemycin showed that 18 of 19 targets containing the conserved Cys were inhibited. Kinetic analyses showed time-dependent inhibition, a hallmark of covalent inactivation, and biochemical studies of the interaction of extracellular signal-regulated kinase (ERK)2 with hypothemycin confirmed covalent adduct formation. Resorcylic acid lactones are unique among kinase inhibitors in that they target mitogen-activated protein (MAP) kinase pathways at four levels: mitogen receptors, MAP kinase kinase (MEK)1/2 and ERK1/2, and certain downstream ERK substrates. Cell lines dependent on the activation of Tyr kinase mitogen receptor targets of the resorcylic acid lactones were unusually sensitive toward hypothemycin and showed the expected inhibition of kinase phosphorylation due to inhibition of the mitogen receptors and/or MEK1/2 and ERK1/2. Among cells without mitogen receptor targets, those harboring an ERK pathway-activating B-RAF V600E mutation were selectively and potently inhibited by hypothemycin. Hypothemycin also prevented stimulated activation of the p38 cascade through inhibition of the Cys-containing targets MEK3/6 and TGF-β-activated kinase 1 and of the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) cascade through inhibition of MEK4/7.
Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified
Aedes aegypti
Authors: Franz Alexander W. E. , Sanchez-Vargas Irma , Adelman Zach N. , Blair Carol D. , Beaty Barry J. , James Anthony A. , Olson Ken E.
Published: March 2006

Mosquitoes (
Aedes aegypti
) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The
A. aegypti carboxypeptidase A
promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs’ white eye) strain by using the nonautonomous
mariner MosI
transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission
in vitro
of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic
A. aegypti
that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.