bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of photoreceptor subtypes to spectral wavelength preference in
Drosophila
Authors: Yamaguchi Satoko , Desplan Claude , Heisenberg Martin
Published: March 2010

The visual systems of most species contain photoreceptors with distinct spectral sensitivities that allow animals to distinguish lights by their spectral composition. In
Drosophila,
photoreceptors R1–R6 have the same spectral sensitivity throughout the eye and are responsible for motion detection. In contrast, photoreceptors R7 and R8 exhibit heterogeneity and are important for color vision. We investigated how photoreceptor types contribute to the attractiveness of light by blocking the function of certain subsets and by measuring differential phototaxis between spectrally different lights. In a “UV vs. blue” choice, flies with only R1–R6, as well as flies with only R7/R8 photoreceptors, preferred blue, suggesting a nonadditive interaction between the two major subsystems. Flies defective for UV-sensitive R7 function preferred blue, whereas flies defective for either type of R8 (blue- or green-sensitive) preferred UV. In a “blue vs. green” choice, flies defective for R8 (blue) preferred green, whereas those defective for R8 (green) preferred blue. Involvement of all photoreceptors [R1–R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis from motion detection that is mediated exclusively by R1–R6.
Ancient DNA analyses exclude humans as the driving force behind late Pleistocene musk ox (
Ovibos moschatus
) population dynamics
Authors: Campos Paula F. , Willerslev Eske , Sher Andrei , Orlando Ludovic , Axelsson Erik , Tikhonov Alexei , Aaris-Sørensen Kim , Greenwood Alex D. , Kahlke Ralf-Dietrich , Kosintsev Pavel , Krakhmalnaya Tatiana , Kuznetsova Tatyana , Lemey Philippe , MacPhee Ross , Norris Christopher A. , Shepherd Kieran , Suchard Marc A. , Zazula Grant D. , Shapiro Beth , Gilbert M. Thomas P.
Published: March 2010

The causes of the late Pleistocene megafaunal extinctions are poorly understood. Different lines of evidence point to climate change, the arrival of humans, or a combination of these events as the trigger. Although many species went extinct, others, such as caribou and bison, survived to the present. The musk ox has an intermediate story: relatively abundant during the Pleistocene, it is now restricted to Greenland and the Arctic Archipelago. In this study, we use ancient DNA sequences, temporally unbiased summary statistics, and Bayesian analytical techniques to infer musk ox population dynamics throughout the late Pleistocene and Holocene. Our results reveal that musk ox genetic diversity was much higher during the Pleistocene than at present, and has undergone several expansions and contractions over the past 60,000 years. Northeast Siberia was of key importance, as it was the geographic origin of all samples studied and held a large diverse population until local extinction at ≈45,000 radiocarbon years before present (
14
C YBP). Subsequently, musk ox genetic diversity reincreased at ca. 30,000
14
C YBP, recontracted at ca. 18,000
14
C YBP, and finally recovered in the middle Holocene. The arrival of humans into relevant areas of the musk ox range did not affect their mitochondrial diversity, and both musk ox and humans expanded into Greenland concomitantly. Thus, their population dynamics are better explained by a nonanthropogenic cause (for example, environmental change), a hypothesis supported by historic observations on the sensitivity of the species to both climatic warming and fluctuations.
Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase
Authors: Chagpar Ryaz B. , Links Philip H. , Pastor M. Chris , Furber Levi A. , Hawrysh Andrea D. , Chamberlain M. Dean , Anderson Deborah H.
Published: March 2010

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is deregulated in many human diseases including cancer, diabetes, obesity, and autoimmunity. PI3K consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The p110-PI3K enzyme generates the key signaling lipid phosphatidylinositol 3,4,5-trisphosphate, which is dephosphorylated by the PI3-phosphatase PTEN. Here we show another function for the p85α regulatory protein: it binds directly to and enhances PTEN lipid phosphatase activity. We demonstrate that ectopically expressed FLAG-tagged p85 coimmunoprecipitates endogenous PTEN in an epidermal growth factor dependent manner. We also show epidermal growth factor dependent coimmunoprecipitation of endogenous p85 and PTEN proteins in HeLa cells. Thus p85 regulates both p110-PI3K and PTEN-phosphatase enzymes through direct interaction. This finding underscores the need for caution in analyzing PI3K activity because anti-p85 immunoprecipitations may contain both p85:p110-PI3K and p85:PTEN-phosphatase enzymes and thus measure net PI3K activity. We identify the N-terminal SH3-BH region of p85α, absent in the smaller p55α and p50α isoforms, as the region that mediates PTEN binding and regulation. Cellular expression of p85ΔSH3-BH results in substantially increased magnitude and duration of pAkt levels in response to growth factor stimulation. The ability of p85 to bind and directly regulate both p110-PI3K and PTEN-PI3-phosphatase allows us to explain the paradoxical insulin signaling phenotypes observed in mice with reduced PI3K or PTEN proteins. This discovery will impact ongoing studies using therapeutics targeting the PI3K/PTEN/Akt pathway.
Protein localization and dynamics within a bacterial organelle
Authors: Hughes H. Velocity , Huitema Edgar , Pritchard Sean , Keiler Kenneth C. , Brun Yves V. , Viollier Patrick H.
Published: March 2010

Protein localization mechanisms dictate the functional and structural specialization of cells. Of the four polar surface organelles featured by the dimorphic bacterium
Caulobacter crescentus
, the stalk, a cylindrical extension of all cell envelope layers, is the least well characterized at the molecular level. Here we apply a powerful experimental scheme that integrates genetics with high-throughput localization to discover StpX, an uncharacterized bitopic membrane protein that modulates stalk elongation and is sequestered to the stalk. In stalkless mutants StpX is dispersed. Two populations of StpX were discernible within the stalk with different mobilities: an immobile one near the stalk base and a mobile one near the stalk tip. Molecular anatomy provides evidence that (
i
) the StpX transmembrane domain enables access to the stalk organelle, (
ii
) the N-terminal periplasmic domain mediates retention in the stalk, and (
iii
) the C-terminal cytoplasmic domain enhances diffusion within the stalk. Moreover, the accumulation of StpX and an N-terminally truncated isoform is differentially coordinated with the cell cycle. Thus, at the submicron scale the localization and the mobility of a protein are precisely regulated in space and time and are important for the correct organization of a subcellular compartment or organelle such as the stalk.
Fusion of biomimetic stealth probes into lipid bilayer cores
Authors: Almquist Benjamin D. , Melosh Nicholas A.
Published: March 2010

Many biomaterials are designed to regulate the interactions between artificial and natural surfaces. However, when materials are inserted through the cell membrane itself the interface formed between the interior edge of the membrane and the material surface is not well understood and poorly controlled. Here we demonstrate that by replicating the nanometer-scale hydrophilic-hydrophobic-hydrophilic architecture of transmembrane proteins, artificial “stealth” probes spontaneously insert and anchor within the lipid bilayer core, forming a high-strength interface. These nanometer-scale hydrophobic bands are readily fabricated on metallic probes by functionalizing the exposed sidewall of an ultrathin evaporated Au metal layer rather than by lithography. Penetration and adhesion forces for butanethiol and dodecanethiol functionalized probes were directly measured using atomic force microscopy (AFM) on thick stacks of lipid bilayers to eliminate substrate effects. The penetration dynamics were starkly different for hydrophobic versus hydrophilic probes. Both 5- and 10 nm thick hydrophobically functionalized probes naturally resided within the lipid core, while hydrophilic probes remained in the aqueous region. Surprisingly, the barrier to probe penetration with short butanethiol chains (
E

o
,5 nm

 = 21.8
k

b

T
,
E

o
,10 nm

 = 15.3
k

b

T
) was dramatically higher than longer dodecanethiol chains (
E

o
,5 nm

 = 14.0
k

b

T
,
E

o
,10 nm

 = 10.9
k

b

T
), indicating that molecular mobility and orientation also play a role in addition to hydrophobicity in determining interface stability. These results highlight a new strategy for designing artificial cell interfaces that can nondestructively penetrate the lipid bilayer.
Deciphering the folding transition state structure and denatured state properties of Nucleophosmin C-terminal domain
Authors: Scaloni Flavio , Federici Luca , Brunori Maurizio , Gianni Stefano
Published: March 2010

Nucleophosmin (NPM1), one of the most abundant nucleolar proteins, is a frequent target of oncogenic mutations in acute myeloid leukaemia (AML). Mutation-induced changes at the C-terminal domain of NPM1 (Cter-NPM1) compromise its stability and cause the aberrant translocation of NPM1 to the cytosol. Hence, this protein represents a suitable candidate to investigate the relations between folding and disease. Since Cter-NPM1 folds via a compact denatured state, stabilization of the folded state of the mutated variants demands detailed structural information on both the native and denatured states. Here, we present the characterization of the complete folding pathway of Cter-NPM1 and provide molecular details for both the transition and the denatured states. The structure of the transition state was assessed by Φ-value analysis, whereas residual structure in the denatured state was mapped by evaluating the effect of mutations as modulated by conditions promoting denatured state compaction. Data reveal that folding of Cter-NPM1 proceeds via an extended nucleus and that the denatured state retains significant malleable structure at the interface between the second and third helices. Our observations constitute the essential prerequisite for structure-based drug-design studies, aimed at identifying molecules that may rescue pathological NPM1 mutants by stabilizing the native-like state.
Epigenetic alterations regulate estradiol-induced enhancement of memory consolidation
Authors: Zhao Zaorui , Fan Lu , Frick Karyn M.
Published: March 2010

The involvement of epigenetic alterations in mediating effects of estrogens on memory is unknown. The present study determined whether histone acetylation and DNA methylation are critical for the potent estrogen 17β-estradiol (E2) to enhance object recognition memory. We show that dorsal hippocampal E2infusion increases acetylation of dorsal hippocampal histone H3, but not H4—an effect blocked by dorsal hippocampal inhibition of ERK activation. Further, intrahippocampal inhibition of ERK activation or DNA methyltransferase (DNMT) activity blocked the memory-enhancing effects of E2. Consistent with these effects, E2decreased levels of HDAC2 protein and increased DNMT expression in the dorsal hippocampus. These findings provide evidence that the beneficial effects of E2on memory consolidation are associated with epigenetic alterations, and suggest these can be triggered by dorsal hippocampal ERK signaling.
Hose in Hose
, an
S
locus–linked mutant of
Primula vulgaris
, is caused by an unstable mutation at the
Globosa
locus
Authors: Li Jinhong , Dudas Brigitta , Webster Margaret A. , Cook Holly E. , Davies Brendan H. , Gilmartin Philip M.
Published: March 2010

Hose in Hose
mutants of primrose and cowslip have been cultivated since the early 17th century and show dominant homeotic conversion of sepals to petals. The phenotype shows variable penetrance and expressivity and is linked to the
S
locus, which controls floral heteromorphy in
Primula
species. Here we demonstrate that the homeotic conversion of sepals to petals in
Hose in Hose
is associated with up-regulation of both
Primula
B-function MADS box genes
PvDef
and
PvGlo
in the first floral whorl. We have defined a restriction fragment length polymorphism associated with
PvGlo
that cosegregates with the
Hose in Hose
phenotype and have also identified and characterized a retrotransposon insertion in the
PvGlo
promoter which is associated with the up-regulated expression of
PvGlo
. Excision of this retrotransposon, associated with epigenetic changes at the locus, causes reversion toward normal calyces and restores wild-type flower development. These data define the molecular basis of the
Hose in Hose
mutation and provide an explanation for its long-documented phenotypic instability.
Structure and biochemical analysis of the heparin-induced E1 dimer of the amyloid precursor protein
Authors: Dahms Sven O. , Hoefgen Sandra , Roeser Dirk , Schlott Bernhard , Gührs Karl-Heinz , Than Manuel E.
Published: March 2010

The amyloid precursor protein (APP) is the key player in Alzheimer’s disease pathology, yet APP and its analogues are also essential for neuronal development and cell homeostasis in mammals. We have determined the crystal structure of the entire N-terminal APP-E1 domain consisting of the growth factor like and the copper binding domains at 2.7-Å resolution and show that E1 functions as a rigid functional entity. The two subdomains interact tightly in a pH-dependent manner via an evolutionarily conserved interface area. Two E1 entities dimerize upon their interaction with heparin, requiring 8–12 sugar rings to form the heparin-bridged APP-E1 dimer in an endothermic and pH-dependent process that is characterized by a low micromolar dissociation constant. Limited proteolysis confirms that the heparin-bridged E1 dimers obtained in solution correspond to a dimer contact in our crystal, enabling us to model this heparin-[APP-E1]2complex. Correspondingly, the APP-based signal transduction, cell–cell- and/or cell–ECM interaction should depend on dimerization induced by heparin, as well as on pH, arguing that APP could fulfill different functions depending on its (sub)cellular localization.
Non–Ca
2+
-conducting Ca
2+
channels in fish skeletal muscle excitation-contraction coupling
Authors: Schredelseker Johann , Shrivastav Manisha , Dayal Anamika , Grabner Manfred
Published: March 2010

During skeletal muscle excitation-contraction (EC) coupling, membrane depolarizations activate the sarcolemmal voltage-gated L-type Ca
2+
channel (Ca
V
1.1). Ca
V
1.1 in turn triggers opening of the sarcoplasmic Ca
2+
release channel (RyR1) via interchannel protein–protein interaction to release Ca
2+
for myofibril contraction. Simultaneously to this EC coupling process, a small and slowly activating Ca
2+
inward current through Ca
V
1.1 is found in mammalian skeletal myotubes. The role of this Ca
2+
influx, which is not immediately required for EC coupling, is still enigmatic. Interestingly, whole-cell patch clamp experiments on freshly dissociated skeletal muscle myotubes from zebrafish larvae revealed the lack of such Ca
2+
currents. We identified two distinct isoforms of the pore-forming Ca
V
1.1α
1S
subunit in zebrafish that are differentially expressed in superficial slow and deep fast musculature. Both do not conduct Ca
2+
but merely act as voltage sensors to trigger opening of two likewise tissue-specific isoforms of RyR1. We further show that non-Ca
2+
conductivity of both Ca
V
1.1α
1S
isoforms is a common trait of all higher teleosts. This non-Ca
2+
conductivity of Ca
V
1.1 positions teleosts at the most-derived position of an evolutionary trajectory. Though EC coupling in early chordate muscles is activated by the influx of extracellular Ca
2+
, it evolved toward Ca
V
1.1-RyR1 protein–protein interaction with a relatively small and slow influx of external Ca
2+
in tetrapods. Finally, the Ca
V
1.1 Ca
2+
influx was completely eliminated in higher teleost fishes.
Role of interactions in an electronic Fabry–Perot interferometer operating in the quantum Hall effect regime
Authors: Ofek Nissim , Bid Aveek , Heiblum Moty , Stern Ady , Umansky Vladimir , Mahalu Diana
Published: March 2010

Interference of edge channels is expected to be a prominent tool for studying statistics of charged quasiparticles in the quantum Hall effect (QHE). We present here a detailed study of an electronic Fabry–Perot interferometer (FPI) operating in the QHE regime [C. Chamon, et al. (1997)
Phys Rev B
55:2331–2334], with the phase of the interfering quasiparticles controlled by the Aharonov–Bohm effect. Our main finding is that Coulomb interactions among the electrons dominate the interference, even in a relatively large area FPI, leading to a strong dependence of the area enclosed by the interference loop on the magnetic field. In particular, for a composite edge structure, with a few independent edge channels propagating along the edge, interference of the outmost edge channel (belonging to the lowest Landau level) was
insensitive
to magnetic field—suggesting a constant enclosed flux. However, when any of the inner edge channels interfered, the enclosed flux
decreased
when the magnetic field
increased
. By intentionally varying the enclosed area with a biased metallic gate and observing the periodicity of the interference pattern, charges
e
(for integer filling factors) and
e
/3 (for a fractional filling factor) were found to be expelled from the FPI. Moreover, these observations provided also a novel way of detecting the charge of the interfering quasiparticles.
Ablation of IL-17A abrogates progression of spontaneous intestinal tumorigenesis
Authors: Chae Wook-Jin , Gibson Thomas F. , Zelterman Daniel , Hao Liming , Henegariu Octavian , Bothwell Alfred L. M.
Published: March 2010

The intrinsic role of endogenous IL-17A in spontaneous intestinal tumorigenesis has not been addressed previously to our knowledge. Ablation of IL-17A significantly reduced tumor development in mice bearing a heterozygote mutation in the adenomatous polyposis coli (APC) gene (Apc
Min/+
mice). There was also a decrease in inflammatory cytokines and proinflammatory mediators, reduced infiltration of lymphocytes including T cells, and preservation of intestinal architecture and the presence of APC protein in intestinal epithelial cells. Interestingly, IL-17A ablation also corrected immunological abnormalities such as splenomegaly and thymic atrophy in Apc
Min/+
mice. CD4 T cells from Apc
Min/+
mice showed hyperproliferative potential in vitro and in vivo and increased levels of IL-17A and IL-10. The effector CD4 T cells from Apc
Min/+
mice were more resistant to regulatory T cell–mediated suppression. Finally, these CD4 T cells induced colitis in immunodeficient mice upon adoptive transfer, whereas the ablation of IL-17A in CD4 T cells in Apc
Min/+
mice completely abolished this pathogenic potential in vivo. Taken together, our results show that CD4 T cell–derived IL-17A promotes spontaneous intestinal tumorigenesis with altered functions of CD4 T cells in Apc
Min/+
mice.
Shh pathway activation is present and required within the vertebrate limb bud apical ectodermal ridge for normal autopod patterning
Authors: Bouldin Cortney M. , Gritli-Linde Amel , Ahn Sohyun , Harfe Brian D.
Published: March 2010

Expression of Sonic Hedgehog (
Shh
) in the posterior mesenchyme of the developing limb bud regulates patterning and growth of the developing limb by activation of the Hedgehog (Hh) signaling pathway. Through the analysis of
Shh
and Hh signaling target genes, it has been shown that activation in the limb bud mesoderm is required for normal limb development to occur. In contrast, it has been stated that Hh signaling in the limb bud ectoderm cannot occur because components of the Hh signaling pathway and Hh target genes have not been found in this tissue. However, recent array-based data identified both the components necessary to activate the Hh signaling pathway and targets of this pathway in the limb bud ectoderm. Using immunohistochemistry and various methods of detection for targets of Hh signaling, we found that SHH protein and targets of Hh signaling are present in the limb bud ectoderm including the apex of the bud. To directly test whether ectodermal Hh signaling was required for normal limb patterning, we removed
Smo
, an essential component of the Hh signaling pathway, from the apical ectodermal ridge (AER). Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional postaxial cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development.
Pain perception is altered by a nucleotide polymorphism in
SCN9A
Authors: Reimann Frank , Cox James J. , Belfer Inna , Diatchenko Luda , Zaykin Dmitri V. , McHale Duncan P. , Drenth Joost P. H. , Dai Feng , Wheeler Jerry , Sanders Frances , Wood Linda , Wu Tian-Xia , Karppinen Jaro , Nikolajsen Lone , Männikkö Minna , Max Mitchell B. , Kiselycznyk Carly , Poddar Minakshi , te Morsche Rene H.M. , Smith Shad , Gibson Dustin , Kelempisioti Anthi , Maixner William , Gribble Fiona M. , Woods C. Geoffrey
Published: March 2010

The gene
SCN9A
is responsible for three human pain disorders. Nonsense mutations cause a complete absence of pain, whereas activating mutations cause severe episodic pain in paroxysmal extreme pain disorder and primary erythermalgia. This led us to investigate whether single nucleotide polymorphisms (SNPs) in
SCN9A
were associated with differing pain perception in the general population. We first genotyped 27
SCN9A
SNPs in 578 individuals with a radiographic diagnosis of osteoarthritis and a pain score assessment. A significant association was found between pain score and SNP rs6746030; the rarer A allele was associated with increased pain scores compared to the commoner G allele (
P
= 0.016). This SNP was then further genotyped in 195 pain-assessed people with sciatica, 100 amputees with phantom pain, 179 individuals after lumbar discectomy, and 205 individuals with pancreatitis. The combined
P
value for increased A allele pain was 0.0001 in the five cohorts tested (1277 people in total). The two alleles of the SNP rs6746030 alter the coding sequence of the sodium channel Nav1.7. Each was separately transfected into HEK293 cells and electrophysiologically assessed by patch-clamping. The two alleles showed a difference in the voltage-dependent slow inactivation (
P
= 0.042) where the A allele would be predicted to increase Nav1.7 activity. Finally, we genotyped 186 healthy females characterized by their responses to a diverse set of noxious stimuli. The A allele of rs6746030 was associated with an altered pain threshold and the effect mediated through C-fiber activation. We conclude that individuals experience differing amounts of pain, per nociceptive stimulus, on the basis of their
SCN9A
rs6746030 genotype.
Molecular insight into lignocellulose digestion by a marine isopod in the absence of gut microbes
Authors: King Andrew J. , Cragg Simon M. , Li Yi , Dymond Jo , Guille Matthew J. , Bowles Dianna J. , Bruce Neil C. , Graham Ian A. , McQueen-Mason Simon J.
Published: March 2010

The digestion of lignocellulose is attracting attention both in terms of basic research into its metabolism by microorganisms and animals, and also as a means of converting plant biomass into biofuels. Limnoriid wood borers are unusual because, unlike other wood-feeding animals, they do not rely on symbiotic microbes to help digest lignocellulose. The absence of microbes in the digestive tract suggests that limnoriid wood borers produce all the enzymes necessary for lignocellulose digestion themselves. In this study we report that analysis of ESTs from the digestive system of
Limnoria quadripunctata
reveals a transcriptome dominated by glycosyl hydrolase genes. Indeed, > 20% of all ESTs represent genes encoding putative cellulases, including glycosyl hydrolase family 7 (GH7) cellobiohydrolases. These have not previously been reported in animal genomes, but are key digestive enzymes produced by wood-degrading fungi and symbiotic protists in termite guts. We propose that limnoriid GH7 genes are important for the efficient digestion of lignocellulose in the absence of gut microbes. Hemocyanin transcripts were highly abundant in the hepatopancreas transcriptome. Based on recent studies indicating that these proteins may function as phenoloxidases in isopods, we discuss a possible role for hemocyanins in lignin decomposition.
Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells
Authors: Rosenbaum Anton I. , Zhang Guangtao , Warren J. David , Maxfield Frederick R.
Published: March 2010

Niemann-Pick type C disease (NPC) is a lysosomal storage disorder causing accumulation of unesterified cholesterol in lysosomal storage organelles. Recent studies have shown that hydroxypropyl-β-cyclodextrin injections in

npc1
−/−

mice are partially effective in treating this disease. Using cultured fibroblasts, we have investigated the cellular mechanisms responsible for reduction of cholesterol accumulation. We show that decreased levels of cholesterol accumulation are maintained for several days after removal of cyclodextrin from the culture medium. This suggests that endocytosed cyclodextrin can reduce the cholesterol storage by acting from inside endocytic organelles rather than by removing cholesterol from the plasma membrane. To test this further, we incubated both NPC1 and NPC2 mutant cells with cholesterol-loaded cyclodextrin for 1 h, followed by chase in serum-containing medium. Although the cholesterol content of the treated cells increased after the 1-h incubation, the cholesterol levels in the storage organelles were later reduced significantly. We covalently coupled cyclodextrin to fluorescent dextran polymers. These cyclodextrin–dextran conjugates were delivered to cholesterol-enriched lysosomal storage organelles and were effective at reducing the cholesterol accumulation. We demonstrate that methyl-β-cyclodextrin is more potent than hydroxypropyl-β-cyclodextrin in reducing both cholesterol and bis(monoacylglycerol) phosphate accumulation in NPC mutant fibroblasts. Brief treatment of cells with cyclodextrins causes an increase in cholesterol esterification by acyl CoA:cholesterol acyl transferase, indicating increased cholesterol delivery to the endoplasmic reticulum. These findings suggest that cyclodextrin-mediated enhanced cholesterol transport from the endocytic system can reduce cholesterol accumulation in cells with defects in either NPC1 or NPC2.
Monomeric red fluorescent proteins with a large Stokes shift
Authors: Piatkevich Kiryl D. , Hulit James , Subach Oksana M. , Wu Bin , Abdulla Arian , Segall Jeffrey E. , Verkhusha Vladislav V.
Published: March 2010

Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 μm the breast cancer cells show significant polarization towards vessels in living mice.
FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry
Authors: Martin Douglas S. , Fathi Reza , Mitchison Timothy J. , Gelles Jeff
Published: March 2010

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and “limping.”
Optimal architectures of elongated viruses
Authors: Luque Antoni , Zandi Roya , Reguera David
Published: March 2010

Many viruses protect their genetic material by a closed elongated protein shell. Unlike spherical viruses, the structure of these prolates is not yet well understood, and only a few of them have been fully characterized. We present the results of a simple phenomenological model, which describes the remarkable structures of prolate or bacilliform viral shells. Surprisingly, we find that the special well-defined geometry of these elongated viruses arises just as a consequence of free-energy minimization of a generic interaction between the structural units of the capsid. Hemispherical
T
-number caps centered along the 5-, 3-, and 2-fold axes with hexagonally ordered cylindrical bodies are found to be local energy minima, thus justifying their occurrence as optimal viral structures. Moreover, closed elongated viruses show a sequence of magic numbers for the end-caps, leading to strict selection rules for the length and structure of the body as well as for the number of capsomers and proteins of the capsid. The model reproduces the architecture of spherical and bacilliform viruses, both in vivo and in vitro, and constitutes an important step towards understanding viral assembly and its potential control for biological and nanotechnological applications.
Cis
lethal genetic interactions attenuate and alter p53 tumorigenesis
Authors: Wang Yuxun , Zhang Weijia , Edelmann Lisa , Kolodner Richard D. , Kucherlapati Raju , Edelmann Winfried
Published: March 2010

Rpa1
, an essential gene involved in DNA replication and genome maintenance, is syntenic and linked to
Trp53
in mice and humans. To study the genetic interaction between
Rpa1
and
Trp53
in tumorigenesis, we generated compound

Rpa1
L230P/+
; Trp53
+/−

mutant mice with the mutant alleles in either
trans
or
cis
configuration. We demonstrate that the

Rpa1
L230P

missense mutation significantly alters the tumor phenotype and spectrum of
Trp53
mutant mice by modifying the genetic mechanisms underlying tumorigenesis. Importantly, when the

Rpa1
L230P

and
Trp53
mutant alleles are in
cis
, the tumor phenotype is attenuated and altered and loss of heterozygosity (LOH) at the
Trp53
wild-type locus is selected against, whereas in the trans configuration,

Rpa1
L230P

enhances the

Trp53
+/−

tumor phenotype even though

Rpa1
L230P

is ultimately lost by LOH. These studies indicate that polymorphic genetic variants in cell essential genes can genetically affect closely linked tumor suppressor loci via allelic phasing, which can result in profound phenotypic variations in tumorigenesis.
Genomic imprinting of experience-dependent cortical plasticity by the ubiquitin ligase gene
Ube3a
Authors: Sato Masaaki , Stryker Michael P.
Published: March 2010

A defect in the maternal copy of a ubiqutin ligase gene
Ube3a
can produce a neurodevelopmental defect in human children known as Angelman syndrome. We investigated the role of the maternally expressed
Ube3a
gene in experience-dependent development and plasticity of the mouse visual system. As demonstrated by optical imaging, rapid ocular dominance (OD) plasticity after brief monocular deprivation (MD) was severely impaired during the critical period (CP) in the visual cortex (VC) of
Ube3a
maternal-deficient (m−/p+) mice. Prolonged MD elicited significant plasticity in m−/p+ mice that never matched the level seen in control animals. In older animals after the CP, 7-day MD elicited mild OD shifts in both control and m−/p+ mice; however, the OD shifts in m−/p+ mice lacked the strengthening of visual responses to the two eyes characteristic of normal adult plasticity. Anatomic effects of the maternal deficiency include reduced spine density on basal, but not apical, dendrites of pyramidal neurons in the binocular region of the VC. Imprinting of
Ube3a
expression was not fully established in the early postnatal period, consistent with the normal development of cortical retinotopy and visual acuity that we observed in m−/p+ mice, but was fully established by the onset of the CP. These results demonstrate that paternal and maternal genomes are not functionally equivalent for cortical plasticity, and that maternally expressed
Ube3a
is required for normal experience-dependent modification of cortical circuits during and after the CP.
Probing bacterial transmembrane histidine kinase receptor–ligand interactions with natural and synthetic molecules
Authors: Ng Wai-Leung , Wei Yunzhou , Perez Lark J. , Cong Jianping , Long Tao , Koch Matthew , Semmelhack Martin F. , Wingreen Ned S. , Bassler Bonnie L.
Published: March 2010

Bacterial histidine kinases transduce extracellular signals into the cytoplasm. Most stimuli are chemically undefined; therefore, despite intensive study, signal recognition mechanisms remain mysterious. We exploit the fact that quorum-sensing signals are known molecules to identify mutants in the
Vibrio cholerae
quorum-sensing receptor CqsS that display altered responses to natural and synthetic ligands. Using this chemical-genetics approach, we assign particular amino acids of the CqsS sensor to particular roles in recognition of the native ligand, CAI-1 (
S
-3 hydroxytridecan-4-one) as well as ligand analogues. Amino acids W104 and S107 dictate receptor preference for the carbon-3 moiety. Residues F162 and C170 specify ligand head size and tail length, respectively. By combining mutations, we can build CqsS receptors responsive to ligand analogues altered at both the head and tail. We suggest that rationally designed ligands can be employed to study, and ultimately to control, histidine kinase activity.
ATP is required for the release of the anaphase-promoting complex/cyclosome from inhibition by the mitotic checkpoint
Authors: Miniowitz-Shemtov Shirly , Teichner Adar , Sitry-Shevah Danielle , Hershko Avram
Published: March 2010

The mitotic (or spindle assembly) checkpoint system ensures accurate segregation of chromosomes by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. This system acts by inhibiting the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase to target securin for degradation. APC/C is inhibited by a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Mad2, and Cdc20. The molecular mechanisms of the inactivation of the mitotic checkpoint, including the release of APC/C from inhibition, remain obscure. It has been reported that polyubiquitylation by the APC/C is required for the inactivation of the mitotic checkpoint [Reddy SK, Rape M, Margansky WA, Kirschner MW (2007)
Nature
, 446:921–924]. We confirmed the involvement of polyubiquitylation, but found that another process, which requires ATP cleavage at the
β
–
γ
position (as opposed to
α
–
β
bond scission involved in ubiquitylation), is essential for the release of APC/C from checkpoint inhibition. ATP (
β
–
γ
) cleavage is required both for the dissociation of MCC components from APC/C and for the disassembly of free MCC, whereas polyubiquitylation is involved only in the former process. We find that the requirement for ATP (
β
–
γ
) cleavage is not due to the involvement of the 26S proteasome and that the phenomena observed are not due to sustained activity of protein kinase Cdk1/cyclin B, caused by inhibition of the degradation of cyclin B. Thus, some other energy-consuming process is needed for the inactivation of the mitotic checkpoint.
microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors, biased myeloid development, and acute myeloid leukemia
Authors: Han Yoon-Chi , Park Christopher Y. , Bhagat Govind , Zhang Jinping , Wang Yulei , Fan Jian-Bing , Liu Mofang , Zou Yongrui , Weissman Irving L. , Gu Hua
Published: March 2010

The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.
Reducing the algorithmic variability in transcriptome-based inference
Authors: Tuna Salih , Niranjan Mahesan
Published: March 2010

Motivation: High-throughput measurements of mRNA abundances from microarrays involve several stages of preprocessing. At each stage, a user has access to a large number of algorithms with no universally agreed guidance on which of these to use. We show that binary representations of gene expressions, retaining only information on whether a gene is expressed or not, reduces the variability in results caused by algorithmic choice, while also improving the quality of inference drawn from microarray studies.

Results: Binary representation of transcriptome data has the desirable property of reducing the variability introduced at the preprocessing stages due to algorithmic choice. We compare the effect of the choice of algorithms on different problems and suggest that using binary representation of microarray data with Tanimoto kernel for support vector machine reduces the effect of the choice of algorithm and simultaneously improves the performance of classification of phenotypes.

Contact:  mn@ecs.soton.ac.uk

Supplementary information:  Supplementary data are available at Bioinformatics online.
The effect of translocation-induced nuclear reorganization on gene expression
Authors: Harewood Louise , Schütz Frédéric , Boyle Shelagh , Perry Paul , Delorenzi Mauro , Bickmore Wendy A. , Reymond Alexandre
Published: March 2010

Translocations are known to affect the expression of genes at the breakpoints and, in the case of unbalanced translocations, alter the gene copy number. However, a comprehensive understanding of the functional impact of this class of variation is lacking. Here, we have studied the effect of balanced chromosomal rearrangements on gene expression by comparing the transcriptomes of cell lines from controls and individuals with the t(11;22)(q23;q11) translocation. The number of differentially expressed transcripts between translocation-carrying and control cohorts is significantly higher than that observed between control samples alone, suggesting that balanced rearrangements have a greater effect on gene expression than normal variation. Many of the affected genes are located along the length of the derived chromosome 11. We show that this chromosome is concomitantly altered in its spatial organization, occupying a more central position in the nucleus than its nonrearranged counterpart. Derivative 22-mapping chromosome 22 genes, on the other hand, remain in their usual environment. Our results are consistent with recent studies that experimentally altered nuclear organization, and indicated that nuclear position plays a functional role in regulating the expression of some genes in mammalian cells. Our study suggests that chromosomal translocations can result in hitherto unforeseen, large-scale changes in gene expression that are the consequence of alterations in normal chromosome territory positioning. This has consequences for the patterns of gene expression change seen during tumorigenesis-associated genome instability and during the karyotype changes that lead to speciation.
Susceptibility loci reported in genome‐wide association studies are associated with Crohn’s disease in Canadian children
Authors: AMRE D. K. , MACK D. R. , MORGAN K. , ISRAEL D. , DESLANDRES C. , SEIDMAN E. G. , LAMBRETTE P. , COSTEA I. , KRUPOVES A. , FEGURY H. , DONG J. , GRIMARD G. , LEVY E.
Published: March 2010

Aliment Pharmacol Ther 31, 1186–1191

Background  Recent genome‐wide association studies based on adult and paediatric populations have implicated >30 genes/loci as susceptibility loci for Crohn’s disease (CD).

Aims  To investigate whether reported genes/loci were also associated with CD in Canadian children.

Design and Methods  A case‐control design was implemented at three paediatric gastroenterology clinics in Canada. Children ≤18 years of age with a confirmed diagnosis of CD were recruited along with controls. Single nucleotide polymorphisms (SNPs) in five genome‐wide association studies reported genes/loci were genotyped. Associations between individual SNPs and CD were examined.

Results  A total of 406 cases and 415 controls were studied. The mean (±s.d.) age of the cases was 12.3 (±3.2) years. Most cases were male (56.6%), had ileo‐colonic disease (L3 ± L4, 52.0%) and inflammatory behaviour (B1 ± p, 86.9%) at diagnosis. Allelic association analysis (two‐tailed) showed that three of the five targeted SNPs were significantly associated with overall susceptibility for CD (ZNF365, r10995271, P = 0.001; PTPN2, rs1893217, P = 0.005; STAT3, rs744166, P = 0.01). Associations with SNP rs4613763 in the PTGER4 locus were marginally nonsignificant (P = 0.07). The ZNF365 and STAT3 SNPs were predominantly associated with ileal disease with or without colonic involvement.

Conclusion  The identified susceptibility genes/loci for adult‐onset CD also confer risk for paediatric‐onset CD.
Mitochondrial D‐loop variations in paediatric acute myeloid leukaemia: a potential prognostic marker
Authors: Sharawat Surender K. , Bakhshi Radhika , Vishnubhatla Sreenivas , Bakhshi Sameer
Published: March 2010

The D‐Loop region of mitochondrial DNA (mtDNA) is the regulatory region for its replication and transcription. There are two hypervariable regions (HV‐I, HV‐II) and the rate of mutation in these regions is 100‐ to 200‐fold that of nuclear DNA. In the current study, the entire D‐loop region of mtDNA was amplified in two overlapping polymerase chain reaction fragments and variations were evaluated in 44 paediatric acute myeloid leukaemia (AML) patients by direct DNA sequencing methods. Median age of the patients was 8·5 years (1–18 years) and the male:female ratio was 3·8:1. A total of 222 variations were observed at 118 positions in the D‐Loop of 35/44 (79·5%) AML patients. The most common variations were T→C (24·6%) and C→T (21·4%) followed by A→G (15·8%). There was no significant difference in the event‐free survival (EFS) of patients with or without any variations (P = 0·40). Three variations in HV‐I, namely 16126T→C (P = 0·05), 16224T→C (P < 0·01) and 16311T→C (P < 0·001), were significantly associated with inferior EFS. In conclusion, this is the largest study to show a high frequency of mtDNA variations in paediatric AML and their potential relevance as a prognostic marker in this disease.
Enhancements of pest risk analysis techniques
Authors: Schrader G. , MacLeod A. , Mittinty M. , Brunel S. , Kaminski K. , Kehlenbeck H. , Petter F. , Baker R.
Published: March 2010

International, regional and national standards for plant health pest risk assessment and examples of their use from countries around the globe were examined together with similar documents from related fields such as animal health, nature conservation and genetically modified organism (GMO) assessment to determine how the consistency of assessing risk, or components of risk, within and between assessments is addressed. A range of approaches was identified that could be adopted and adapted for use in a revised decision support scheme for quarantine pests by the European and Mediterranean Plant Protection Organization (EPPO) to aid consistency. No single scheme contained a mechanism to ensure or guarantee consistency and no single scheme contained all of the approaches identified to maximize consistency. If the approach of using a five division scale to describe individual components that contribute to evaluating the overall pest risk is to be maintained in the EPPO scheme, then the primary needs required in the EPPO scheme to enhance consistency are (i) the provision of examples that describe divisions within the scales, or alternative but equivalent descriptors that allow assessors to distinguish between divisions, and (ii) a mechanism to combine risk elements in a consistent and transparent way. Features that would help inexperienced assessors include a clear structure, clear rating guidance, questions posed unambiguously, provision of standardized answers and an easily applicable method to interpret and summarize risk ratings. Beyond improvements to the EPPO scheme, assessors using the scheme will need training. Providing links to information and suggesting data sources that would help assessors answer questions would also be helpful (this is being addressed in Work Package 1 of PRATIQUE).
Survey on the use of tests mentioned in EPPO diagnostic protocols
Authors: Petter F. , Suffert M.
Published: March 2010

A survey on the use of EPPO diagnostic protocols was conducted in the summer of 2008. Approximately 90 laboratories that were registered in the EPPO database on Diagnostic Expertise were asked to indicate the number of samples that they tested during the year 2007 and which recommended test, or other test, they used. The survey was organized on a selection of 58 diagnostic protocols in all disciplines of plant health diagnosis (bacteriology, mycology, entomology, nematology, virology and phytoplasmology). Forty‐four laboratories from 20 EPPO countries responded. The results are presented for 12 protocols giving the number of tests used for diagnosis. From this survey it can be concluded that many of the tests for detection mentioned in EPPO diagnostic protocols are widely used in plant pest diagnostic laboratories in the EPPO Region.