bluuebunny/crossref_metadata_2025 huggingface dataset:
The Primed Ebolavirus Glycoprotein (19-Kilodalton GP
1,2
): Sequence and Residues Critical for Host Cell Binding
Authors: Dube Derek , Brecher Matthew B. , Delos Sue E. , Rose Sean C. , Park Edward W. , Schornberg Kathryn L. , Kuhn Jens H. , White Judith M.
Published: January 2009

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP
1,2
), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature
454:
177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP
1
and GP
2
that remain after GP
1,2
is proteolytically primed by endosomal cathepsins for fusion and residues in GP
1
that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP
1,2
contains residues 33 to 190 of GP
1
and all residues of GP
2
. The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP
1
, by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP
1
residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP
1,2
-mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP
1,2
is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP
1
) as a highly conserved region containing a two-stranded β-sheet, the two intra-GP
1
disulfide bonds, and four critical Lys residues.
exo-Substituent effects in halogenated icosahedral (B12H122–) and octahedral (B6H62–) closo-borane skeletons: chemical reactivity studied by experimental and quantum chemical methods
Authors: Lepšík Martin , Srnec Martin , Hnyk Drahomír , Grüner Bohumír , Plešek Jaromír , Havlas Zdeněk , Rulíšek Lubomír
Published: January 2009

The exo-substituent effects in halogenated icosahedral B12H122– (B12) and octahedral B6H62– (B6) closo-borane skeletons were studied both experimentally and theoretically. Firstly, the equilibrium geometries of exo-substituted B12 and B6 clusters were obtained using quantum chemical calculations at the MP2/def2-SVP level. A comparison with the available X-ray crystallographic data revealed a very good agreement between the theoretical and experimental values. Secondly, other descriptors of the molecular structure of these borane compounds – 11B NMR chemical shifts – were experimentally determined and compared with the calculated values obtained by the ab initio/GIAO approach at the MP2/def2-TZVP level. It was shown that the calculated data reproduced the experiment very closely. Thirdly, we investigated experimentally the halogenation reactions of B12 and attempted to explain the observed ratios between the two obtained disubstituted products (meta/ortho ~ 4:1) by calculating their thermodynamic stabilities using the DFT/B3LYP method. These calculations showed the enhanced stability of the meta disubstituted B12 but did not explain why the para product had not been observed in the experiment. We thus turned our attention to the kinetic aspects of exo-substitution reactions by exploring the possible reaction pathways and transition states. In spite of the complexity of the plausible reaction mechanisms, reasonable agreement was obtained between the calculated activation barriers and the experimental observations concerning the halogenation reactions of the B6 and B12 molecules. It also allowed to exclude from considerations certain reaction pathways leading to the mono- and dihalogenated B12 and B6 species.
SLOW INVARIANT MANIFOLDS AS CURVATURE OF THE FLOW OF DYNAMICAL SYSTEMS
Authors: GINOUX JEAN-MARC , ROSSETTO BRUNO , CHUA LEON O.
Published: January 2009

Considering trajectory curves, integral of n-dimensional dynamical systems, within the framework of Differential Geometry as curves in Euclidean n-space, it will be established in this article that the curvature of the flow, i.e. the curvature of the trajectory curves of any n-dimensional dynamical system directly provides its slow manifold analytical equation the invariance of which will be then proved according to Darboux theory. Thus, it will be stated that the flow curvature method, which uses neither eigenvectors nor asymptotic expansions but only involves time derivatives of the velocity vector field, constitutes a general method simplifying and improving the slow invariant manifold analytical equation determination of high-dimensional dynamical systems. Moreover, it will be shown that this method generalizes the Tangent Linear System Approximation and encompasses the so-called Geometric Singular Perturbation Theory. Then, slow invariant manifolds analytical equation of paradigmatic Chua's piecewise linear and cubic models of dimensions three, four and five will be provided as tutorial examples exemplifying this method as well as those of high-dimensional dynamical systems.
FRACTAL DIMENSION OF GLOBAL ATTRACTORS FOR SOME DISSIPATIVE LATTICE SYSTEMS
Authors: ZHOU SHENGFAN
Published: January 2009

In this letter, by using a criteria for estimating the fractal dimension of a compact subset of a Hilbert space which is directly obtained from [Chueshov & Lasiecka, 2004], the upper bounds of fractal dimension of the global attractors for first-order and partly dissipative lattice systems, and second-order dissipative lattice systems, are obtained.
PYRAMIDALITY AND AROMATICITY IN POLYPHOSPHAPHOSPHOLES
Authors: LI WEI-QI , LIU LI-LI , FENG JI-KANG , LIU ZI-ZHONG , REN AI-MIN , ZHANG GANG , SUN CHIA-CHUNG
Published: January 2009

Ab initio calculations show that, for the P n( CH )4-n PH with n = 0 - 4, the fully optimized structures have decreasing pyramidality at the tri-coordinated phosphorus atom with n. The interaction between the tri-coordinated phosphorus [Formula: see text] and the carbons–phosphorus π-system [Formula: see text] with different n, the differences of the structure, electron properties and energy among the phospholes indicate that the ring strain and the delocalization of the lone pair electron of tri-coordinated phosphorus should be responsible for this phenomena. Various aromatic criteria such as geometry, magnetism, and energy show that these molecules are aromatic, and their aromaticities increase with the number of phosphorus atoms in the nonplanar pentagons. The electron circulation in the aromatic molecules can promote the delocalization of the lone pair. In contrast to the B3LYP prediction, we draw some similar conclusions, but the planarity and aromaticity of molecules increase largely at the MP2 lever due to the consideration of electron correlation.
THEORETICAL STUDIES OF HYDROGEN BONDING INTERACTION BETWEEN TRIMETHYLAMINE AND SUBSTITUTED PHENOLS, INFLUENCE OF THE SUBSTITUENTS ON THE HYDROGEN BOND PROPERTIES AND THE VIBRATIONAL SPECTRUM
Authors: PARVEEN SALMA , DAS SUBOJIT , CHANDRA ASIT K. , ZEEGERS-HUYSKENS THERESE
Published: January 2009

Hydrogen bonding interactions between trimethylamine (TMA) and a series of para substituted phenols (X– C6H4OH , X = H , CH3, NH2, Cl , CN , and NO2) are studied by using density functional theory with the hybrid B3LYP functional and the 6-31++G(d,p) basis set. Both electron donor and acceptor substituents (X) are chosen to study systematically the relation between the proton donor ability of the phenols and the strength of the OH … N hydrogen bond. The effect of hydrogen bonding on spectral and structural parameters and their inter relation are discussed. The natural bond orbital (NBO) analysis (occupation of σ* orbitals, hyperconjugative energies and atomic charges) is also carried out to elucidate the reason behind the spectral and structural changes due to hydrogen bond formation. Several correlations between hydrogen bond strength and bond properties are discussed.
REACTION THERMODYNAMICS IN CHEMICAL VAPOR DEPOSITION OF BORON CARBIDES WITH BCl3–C3H6 (PROPENE)-H2 PRECURSORS
Authors: WANG TAO , SU KEHE , DENG JUANLI , ZENG YAN , ZENG QINGFENG , CHENG LAIFEI , ZHANG LITONG
Published: January 2009

The gas phase reaction thermodynamics in the chemical vapor deposition (CVD) process of preparing boron carbides via the precursors of BCl 3– C 3 H 6(propene)– H 2 is investigated with a set of 325 gaseous species, in which the data for 135 species are evaluated in this work. The thermochemistry data are calculated with accurate model chemistry at G3(MP2) and G3//B3LYP levels. The concentration distribution of all of the 325 species is obtained with the principle of chemical equilibrium. The thermochemistry data include the heat capacities, entropies, enthalpies of formation, and Gibbs free energies of formation. The heat capacities and entropies at temperatures in 298.15–2000 K are evaluated with the standard statistical thermodynamics. The Gibbs free energies of formation in 298.15–2000 K are calculated with the classical thermodynamics based on the developed heat capacities and entropies. By including the crystal B 4 C , C (graphite), and B , the results for an example of the 3:1:2 precursors of BCl 3: C 3 H 6: H 2 show that the crystal B 4 C can be produced at temperatures higher than 700 K while the graphite has a higher molar value and can be produced at lower temperatures. It is also examined that the production of graphite can be controlled by changing the ratio of the injected reactants or pressure. It is interesting that BHCl 2, BCH 3 Cl 2, BH 2 Cl , and B 2 Cl 4 are found to be the most effective species in the CVD process, which is similar to those in the BCl 3– CH 4– H 2 system. The results predicted in this work are consistent with the experiments.
Regulation of ENaC-mediated sodium transport by glucocorticoids in Reissner's membrane epithelium
Authors: Kim Sung Huhn , Kim Kyunghee X. , Raveendran Nithya N. , Wu Tao , Pondugula Satyanarayana R. , Marcus Daniel C.
Published: January 2009

Reissner's membrane epithelium forms much of the barrier that produces and sustains the large ionic differences between cochlear endolymph and perilymph. We have reported that Reissner's membrane contributes to normal cochlear function by absorbing Na+from endolymph via amiloride-sensitive channels in gerbil inner ear. We used mouse Reissner's membrane to 1) identify candidate genes involved in the Na+transport pathway, 2) determine whether their level of expression was regulated by the synthetic glucocorticoid dexamethasone, and 3) obtain functional evidence for the physiological importance of these genes. Transcripts were present for α-, β-, and γ-subunits of epithelial Na+channel (ENaC); corticosteroid receptors GR (glucocorticoid receptor) and MR (mineralocorticoid receptor); GR agonist regulator 11β-hydroxysteroid dehydrogenase (HSD) type 1 (11β-HSD1); Na+transport control components SGK1, Nedd4-2, and WNKs; and K+channels and Na+-K+-ATPase. Expression of the MR agonist regulator 11β-HSD2 was not detected. Dexamethasone upregulated transcripts for α- and β-subunits of ENaC (∼6- and ∼3-fold), KCNK1 (∼3-fold), 11β-HSD1 (∼2-fold), SGK1 (∼2-fold), and WNK4 (∼3-fold). Transepithelial currents from the apical to the basolateral side of Reissner's membrane were sensitive to amiloride (IC50∼0.7 μM) and benzamil (IC50∼0.1 μM), but not EIPA (IC50∼34 μM); amiloride-blocked transepithelial current was not immediately changed by forskolin/IBMX. Currents were reduced by ouabain, lowered bath Na+concentration (from 150 to 120 mM), and K+channel blockers (XE-991, Ba2+, and acidification from pH 7.4 to 6.5). Dexamethasone-stimulated current and gene expression were reduced by mifepristone, but not spironolactone. These molecular, pharmacological, and functional observations are consistent with Na+absorption by mouse Reissner's membrane, which is mediated by apical ENaC and/or other amiloride-sensitive channels, basolateral Na+-K+-ATPase, and K+-permeable channels and is under the control of glucocorticoids. These results provide an understanding and a molecular definition of an important transport function of Reissner's membrane epithelium in the homeostasis of cochlear endolymph.
General redox environment and carotid body chemoreceptor function
Authors: Agapito Maria Teresa , Sanz-Alfayate Gloria , Gomez-Niño Angela , Gonzalez Constancio , Obeso Ana
Published: January 2009

Carotid body (CB) chemoreceptor cells detect physiological levels of hypoxia and generate a hyperventilation, homeostatic in nature, aimed to minimize the deleterious effects of hypoxia. Intimate mechanisms involved in oxygen sensing in chemoreceptor cells remain largely unknown, but reactive oxygen species (ROS) had been proposed as mediators of this process. We have determined glutathione levels and calculated glutathione redox potential ( EGSH; indicator of the general redox environment of cells) in rat diaphragms incubated in the presence of oxidizing agents of two types: nonpermeating and permeating through cell membranes; in the latter group, unspecific oxidants and inhibitors of ROS-disposing enzymes were used. Selected concentrations of oxidizing agents were tested for their ability to modify the normoxic and hypoxic activity of chemoreceptor cells measured in vitro as their rate of release of neurotransmitters. Results evidence variable relationships between EGSH and the activity of chemoreceptor cells. The independence of chemoreceptor cell activity from the EGSH would imply that the ability of the CB to play its homeostatic role is largely preserved in any pathological or toxicological contingency causing oxidative stress. Consistent with this suggestion, it was also found that CB-mediated hypoxic hyperventilation was not altered by treatment of intact animals with agents that markedly decreased the EGSH in all tissues assayed.
Intrauterine growth restriction due to uteroplacental insufficiency decreased white matter and altered NMDAR subunit composition in juvenile rat hippocampi
Authors: Schober Michelle E. , McKnight Robert A. , Yu Xing , Callaway Christopher W. , Ke Xingrao , Lane Robert H.
Published: January 2009

Uteroplacental insufficiency (UPI), the major cause of intrauterine growth restriction (IUGR) in developed nations, predisposes to learning impairment. The underlying mechanism is unknown. Neuronal N-methyl-d-aspartate receptors (NMDARs) are critical for synaptogenesis and learning throughout life. We hypothesized that UPI-induced IUGR alters rat hippocampal NMDAR NR2A/NR2B subunit ratio and/or NR1 mRNA isoform expression and synaptic density at day 21 (P21). To test this hypothesis, IUGR was induced by bilateral uterine artery ligation of the late-gestation Sprague-Dawley dam. At P21, hippocampal NMDAR subunit mRNA and protein were measured, as were levels of synaptophysin. Neuronal, synaptic, and glial density in CA1, CA3, and dentate gyrus (DG) was assessed by immunofluorescence. IUGR increased NR1 mRNA isoform NR1-3a and 1-3b expression in both sexes. In P21 males, IUGR increased protein levels of NR1 C2′ and decreased NR1 C2, NR2A, and the NR2A-to-NR2B ratio, whereas in females, IUGR increased NR2B protein. In males, IUGR was associated with decreased myelin basic protein-to-neuronal nuclei ratio in CA1, CA3, and DG. We conclude that IUGR has sex-specific effects and that neither neuronal loss nor decreased synaptic density appears to account for the changes in NMDAR subunits. Rather, it is possible that synaptic NMDAR subunit composition is altered. Our results suggest that apparent recovery in the IUGR hippocampus may be associated with synaptic hyperexcitability. We speculate that the NMDAR plays an important role in IUGR-associated cognitive impairment.
Developmental changes in hypothalamic leptin receptor: relationship with the postnatal leptin surge and energy balance neuropeptides in the postnatal rat
Authors: Cottrell E. C. , Cripps R. L. , Duncan J. S. , Barrett P. , Mercer J. G. , Herwig A. , Ozanne S. E.
Published: January 2009

In the adult brain, leptin regulates energy homeostasis primarily via hypothalamic circuitry that affects food intake and energy expenditure. Evidence from rodent models has demonstrated that during early postnatal life, leptin is relatively ineffective in modulating these pathways, despite the high circulating levels and the presence of leptin receptors within the central nervous system. Furthermore, in recent years, a neurotrophic role for leptin in the establishment of energy balance circuits has emerged. The precise way in which leptin exerts these effects, and the site of leptin action, is unclear. To provide a detailed description of the development of energy balance systems in the postnatal rat in relation to leptin concentrations during this time, endogenous leptin levels were measured, along with gene expression of leptin receptors and energy balance neuropeptides in the medial basal hypothalamus, using in situ hybridization. Expression of leptin receptors and both orexigenic and anorexigenic neuropeptides increased in the arcuate nucleus during the early postnatal period. At postnatal day 4 (P4), we detected dense leptin receptor expression in ependymal cells of the third ventricle (3V), which showed a dramatic reduction over the first postnatal weeks, coinciding with marked morphological changes in this region. An acute leptin challenge robustly induced suppressor of cytokine signaling-3 expression in the 3V of P4 but not P14 animals, revealing a clear change in the location of leptin action over this period. These findings suggest that the neurotrophic actions of leptin may involve signaling at the 3V during a restricted period of postnatal development.
Renal tubulointerstitial fibrosis: common but never simple
Authors: Hewitson Tim D.
Published: January 2009

Regardless of etiology, all patients with chronic renal disease show a progressive decline in renal function with time. Fibrosis, so-called scarring, is a key cause of this pathophysiology. Fibrosis involves an excess accumulation of extracellular matrix (primarily composed of collagen) and usually results in loss of function when normal tissue is replaced with scar tissue. While recent major advances have led to a much better understanding of this process, many problems remain. We for instance know little about why some wounds heal and others scar and little about how many putative antifibrotic agents work. This review discusses recent advances in our understanding of the mechanisms of tubulointerstitial fibrosis, focusing on the regulation and role of the myofibroblast in this process, the role of recently recognized endogenous antifibrotic factors, controversy surrounding the effects of metalloproteinases, and the opportunities presented by new treatment strategies that abrogate and may even reverse fibrosis.
Linearly Additive Shape and Color Signals in Monkey Inferotemporal Cortex
Authors: McMahon David B. T. , Olson Carl R.
Published: January 2009

How does the brain represent a red circle? One possibility is that there is a specialized and possibly time-consuming process whereby the attributes of shape and color, carried by separate populations of neurons in low-order visual cortex, are bound together into a unitary neural representation. Another possibility is that neurons in high-order visual cortex are selective, by virtue of their bottom-up input from low-order visual areas, for particular conjunctions of shape and color. A third possibility is that they simply sum shape and color signals linearly. We tested these ideas by measuring the responses of inferotemporal cortex neurons to sets of stimuli in which two attributes—shape and color—varied independently. We find that a few neurons exhibit conjunction selectivity but that in most neurons the influences of shape and color sum linearly. Contrary to the idea of conjunction coding, few neurons respond selectively to a particular combination of shape and color. Contrary to the idea that binding requires time, conjunction signals, when present, occur as early as feature signals. We argue that neither conjunction selectivity nor a specialized feature binding process is necessary for the effective representation of shape–color combinations.
Long-Lasting Hyperexcitability Induced by Depolarization in the Absence of Detectable Ca2+Signals
Authors: Kunjilwar Kumud K. , Fishman Harvey M. , Englot Dario J. , O'Neil Roger G. , Walters Edgar T.
Published: January 2009

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca2+signals produced by depolarization. Indeed, transient Ca2+signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca2+-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca2+transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca2+levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve–ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis–dependent hyperexcitability under conditions in which Ca2+entry is prevented (by bathing in nominally Ca2+-free solutions containing EGTA) and detectable Ca2+transients are eliminated (by adding BAPTA-AM). Disruption of Ca2+release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca2+-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.
Limb Stiffness Is Modulated With Spatial Accuracy Requirements During Movement in the Absence of Destabilizing Forces
Authors: Wong Jeremy , Wilson Elizabeth T. , Malfait Nicole , Gribble Paul L.
Published: January 2009

The motor system can use a number of mechanisms to increase movement accuracy and compensate for perturbing external forces, interaction torques, and neuromuscular noise. Empirical studies have shown that stiffness modulation is one adaptive mechanism used to control arm movements in the presence of destabilizing external force loads. Other work has shown that arm muscle activity is increased at movement end for reaching movements to small visual targets and that changes in stiffness at movement end are oriented to match changes in visual accuracy requirements such as target shape. In this study, we assess whether limb stiffness is modulated to match spatial accuracy requirements during movement, conveyed using visual stimuli, in the absence of external force loads. Limb stiffness was estimated in the middle of reaching movements to visual targets located at the end of a narrow (8 mm) or wide (8 cm) visual track. When greater movement accuracy was required, we observed modest but reliable increases in limb stiffness in a direction perpendicular to the track. These findings support the notion that the motor system uses stiffness control to augment movement accuracy during movement and does so in the absence of external unstable force loads, in response to changing accuracy requirements conveyed using visual cues.
Cryptococcus gattii: Emergence in Western North America: Exploitation of a Novel Ecological Niche
Authors: Datta Kausik , Bartlett Karen H. , Marr Kieren A.
Published: January 2009

The relatively uncommon fungal pathogenCryptococcus gattiirecently emerged as a significant cause of cryptococcal disease in human and animals in the Pacific Northwest of North America. Although genetic studies indicated its possible presence in the Pacific Northwest for more than 30 years,C. gattiias an etiological agent was largely unknown in this region prior to 1999. The recent emergence may have been encouraged by changing conditions of climate or land use and/or host susceptibility, and predictive ecological niche modeling indicates a potentially wider spread.C. gattiican survive wide climatic variations and colonize the environment in tropical, subtropical, temperate, and dry climates. Long-term climate changes, such as the significantly elevated global temperature in the last 100 years, influence patterns of disease among plants and animals and create niche microclimates habitable by emerging pathogens.C. gattiimay have exploited such a hitherto unrecognized but clement environment in the Pacific Northwest to provide a wider exposure and risk of infection to human and animal populations.
A Susceptibility Locus for Papillary Thyroid Carcinoma on Chromosome 8q24
Authors: He Huiling , Nagy Rebecca , Liyanarachchi Sandya , Jiao Hong , Li Wei , Suster Saul , Kere Juha , de la Chapelle Albert
Published: January 2009

Papillary thyroid carcinoma (PTC) displays higher heritability than most other cancers. To search for genes predisposing to PTC, we performed a genome-wide linkage analysis in a large family with PTC and melanoma. Among several peaks the highest was at 8q24, with a maximum nonparametric linkage (NPL) score of 7.03. Linkage analysis was then broadened to comprise 25 additional PTC families that produced a maximum NPL score of 3.2, P = 0.007 at the 8q24 locus. Fine mapping with microsatellite markers was compatible with linkage to the 8q24 locus in 10 of the 26 families. In the large family, a ∼320 Kb haplotype was shared by individuals with PTC, melanoma, or benign thyroid disease, but not by unaffected individuals. A 12 Kb haplotype of 8 SNP markers within the larger haplotype was shared by 9 of the 10 families in which the 8q24 locus was compatible with linkage. The shared haplotype is located within 2 known overlapping protein-coding genes, thyroglobulin (TG) and Src-like adaptor (SLA). Resequencing of the coding and control regions of TG and SLA did not disclose putative mutations in PTC patients. Embedded in the TG-SLA region are three likely noncoding RNA genes, one of which (AK023948) harbors the 8-SNP haplotype. Resequencing of AK023948 and one of the other RNA genes did not reveal candidate mutations. Gene expression analysis indicated that AK023948 is significantly down-regulated in most PTC tumors. The putative noncoding RNA gene AK023948 is a candidate suseptibility gene for PTC. [Cancer Res 2009;69(2):625–31]
Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia
Authors: Seeley E. Scott , Carrière Catherine , Goetze Tobias , Longnecker Daniel S. , Korc Murray
Published: January 2009

Primary cilia have been proposed to participate in the modulation of growth factor signaling pathways. In this study, we determined that ciliogenesis is suppressed in both pancreatic cancer cells and pancreatic intraepithelial neoplasia (PanIN) lesions in human pancreatic ductal adenocarcinoma (PDAC). Primary cilia were absent in these cells even when not actively proliferating. Cilia were also absent from mouse PanIN cells in three different mouse models of PDAC driven by an endogenous oncogenic Kras allele. Inhibition of Kras effector pathways restored ciliogenesis in a mouse pancreatic cancer cell line, raising the possibility that ciliogenesis may be actively repressed by oncogenic Kras. By contrast, normal duct, islet, and centroacinar cells retained primary cilia in both human and mouse pancreata. Thus, arrested ciliogenesis is a cardinal feature of PDAC and its precursor PanIN lesions, does not require ongoing proliferation, and could potentially be targeted pharmacologically. [Cancer Res 2009;69(2):422–30]
De novo Induction of Genetically Engineered Brain Tumors in Mice Using Plasmid DNA
Authors: Wiesner Stephen M. , Decker Stacy A. , Larson Jon D. , Ericson Katya , Forster Colleen , Gallardo Jose L. , Long Chunmei , Demorest Zachary L. , Zamora Edward A. , Low Walter C. , SantaCruz Karen , Largaespada David A. , Ohlfest John R.
Published: January 2009

Spontaneous mouse models of cancer show promise to more accurately recapitulate human disease and predict clinical efficacy. Transgenic mice or viral vectors have been required to generate spontaneous models of glioma, a lethal brain tumor, because nonviral gene transfer is typically transient. To overcome this constraint, we used the Sleeping Beauty transposable element to achieve chromosomal integration of human oncogenes into endogenous brain cells of immunocompetent mice. Genetically engineered, spontaneous brain tumors were induced with plasmid DNA in a matter of weeks in three separate mouse strains. The phenotype of tumors was influenced by the combination of oncogenes delivered, resembling human astrocytoma or glioblastoma in the majority of cases. At least five different genes can be cotransfected simultaneously including reporters, allowing measurement of tumor viability by in vivo imaging. This model can accelerate brain tumor research in a variety of ways such as generation of “humanized” models for high throughput drug screening and candidate gene validation with exceptional speed and flexibility. [Cancer Res 2009;69(2):431–9]
p18Ink4c and p53 Act as Tumor Suppressors in Cyclin D1–Driven Primitive Neuroectodermal Tumor
Authors: Saab Raya , Rodriguez-Galindo Carlos , Matmati Kelly , Rehg Jerold E. , Baumer Shannon H. , Khoury Joseph D. , Billups Catherine , Neale Geoffrey , Helton Kathleen J. , Skapek Stephen X.
Published: January 2009

The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to ∼4 months. Interestingly, the Cdk inhibitor p18Ink4c was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18Ink4c is a tumor suppressor in cyclin D1–driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption. [Cancer Res 2009;69(2):440–8]
Quantitative DNA Methylation Analysis of FOXP3 as a New Method for Counting Regulatory T Cells in Peripheral Blood and Solid Tissue
Authors: Wieczorek Georg , Asemissen Anne , Model Fabian , Turbachova Ivana , Floess Stefan , Liebenberg Volker , Baron Udo , Stauch Diana , Kotsch Katja , Pratschke Johann , Hamann Alf , Loddenkemper Christoph , Stein Harald , Volk Hans Dieter , Hoffmüller Ulrich , Grützkau Andreas , Mustea Alexander , Huehn Jochen , Scheibenbogen Carmen , Olek Sven
Published: January 2009

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2–treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings. [Cancer Res 2009;69(2):599–608]
Chromosomal Signatures of a Subset of High-Grade Premalignant Cervical Lesions Closely Resemble Invasive Carcinomas
Authors: Wilting Saskia M. , Steenbergen Renske D.M. , Tijssen Marianne , van Wieringen Wessel N. , Helmerhorst Theo J.M. , van Kemenade Folkert J. , Bleeker Maaike C.G. , van de Wiel Mark A. , Carvalho Beatriz , Meijer Gerrit A. , Ylstra Bauke , Meijer Chris J.L.M. , Snijders Peter J.F.
Published: January 2009

Cervical cancer develops from precancerous high-grade cervical intraepithelial neoplasia (CIN) harboring a transforming infection with high-risk human papillomavirus, which is characterized by p16INK4a overexpression. Once such a lesion has developed, progression toward an invasive squamous cell carcinoma (SCC) may take one or more decades, underlining the heterogeneity of these lesions in terms of duration of existence and progression risk. We performed array-based comparative genomic hybridization (array CGH) on 46 p16INK4a immunopositive CIN2/3 lesions to determine whether this heterogeneity is reflected in their chromosomal profiles. Chromosomal profiles of CIN2/3 lesions were related to those of invasive cervical SCC and promoter methylation of CADM1, a tumor suppressor gene known to be functionally involved in the tumorigenic phenotype of cervical cancer cells. Frequent alterations found in CIN2/3 lesions included gains located at chromosome 1, 3, 7, and 20 and losses located at 4, 11, 16, 17, and 19. Unsupervised hierarchical clustering identified two subsets of CIN2/3 lesions, chromosomal profiles of one of which closely resembled invasive SCCs. Gains of 1, 3q, and 20 were characteristic for CIN2/3 lesions with chromosomal signatures resembling carcinomas. In addition, dense promoter methylation of the CADM1 gene was significantly more frequent in these CIN2/3 lesions (P = 0.004). No chromosomal alterations were detected in six CIN1 lesions, five of which were completely p16INK4a immunonegative. These findings suggest that biomarkers associated with gains at chromosomes 1, 3q, and 20 are potential hallmarks of advanced p16INK4a-positive CIN2/3 lesions with a high short-term risk of progression. [Cancer Res 2009;69(2):647–55]
Tenascin-C Is a Novel RBPJκ-Induced Target Gene for Notch Signaling in Gliomas
Authors: Sivasankaran Balasubramanian , Degen Martin , Ghaffari Anthony , Hegi Monika E. , Hamou Marie-France , Ionescu Mihai-Constantin S. , Zweifel Christian , Tolnay Markus , Wasner Morten , Mergenthaler Susanne , Miserez André R. , Kiss Robert , Lino Maddalena M. , Merlo Adrian , Chiquet-Ehrismann Ruth , Boulay Jean-Louis
Published: January 2009

Tenascin-C (TNC) expression is known to correlate with malignancy in glioblastoma (GBM), a highly invasive and aggressive brain tumor that shows limited response to conventional therapies. In these malignant gliomas as well as in GBM cell lines, we found Notch2 protein to be strongly expressed. In a GBM tumor tissue microarray, RBPJk protein, a Notch2 cofactor for transcription, was found to be significantly coexpressed with TNC. We show that the TNC gene is transactivated by Notch2 in an RBPJk-dependent manner mediated by an RBPJk binding element in the TNC promoter. The transactivation is abrogated by a Notch2 mutation, which we detected in the glioma cell line Hs683 that does not express TNC. This L1711M mutation resides in the RAM domain, the site of interaction between Notch2 and RBPJk. In addition, transfection of constructs encoding activated Notch2 or Notch1 increased endogenous TNC expression identifying TNC as a novel Notch target gene. Overexpression of a dominant negative form of the transcriptional coactivator MAML1 or knocking down RBPJk in LN319 cells led to a dramatic decrease in TNC protein levels accompanied by a significant reduction of cell migration. Because addition of purified TNC stimulated glioma cell migration, this represents a mechanism for the invasive properties of glioma cells controlled by Notch signaling and defines a novel oncogenic pathway in gliomagenesis that may be targeted for therapeutic intervention in GBM patients. [Cancer Res 2009;69(2):458–65]
C-1027, A Radiomimetic Enediyne Anticancer Drug, Preferentially Targets Hypoxic Cells
Authors: Beerman Terry A. , Gawron Loretta S. , Shin Seulkih , Shen Ben , McHugh Mary M.
Published: January 2009

The hypoxic nature of cells within solid tumors limits the efficacy of anticancer therapies such as ionizing radiation and conventional radiomimetics because their mechanisms require oxygen to induce lethal DNA breaks. For example, the conventional radiomimetic enediyne neocarzinostatin is 4-fold less cytotoxic to cells maintained in low oxygen (hypoxic) compared with normoxic conditions. By contrast, the enediyne C-1027 was nearly 3-fold more cytotoxic to hypoxic than to normoxic cells. Like other radiomimetics, C-1027 induced DNA breaks to a lesser extent in cell-free, or cellular hypoxic, compared with normoxic environments. However, the unique DNA interstrand cross-linking ability of C-1027 was markedly enhanced under the same hypoxic conditions that reduced its DNA break induction. Although the unique chemistry of C-1027 allows it to concurrently generate both DNA breaks and cross-links in normoxic cells, a low oxygen environment represses the former and promotes the latter. Thus, treatment with C-1027 offers a facile approach for overcoming the radioresistance associated with poorly oxygenated cells. [Cancer Res 2009;69(2):OF593–6]
Overexpression of RhoA Induces Preneoplastic Transformation of Primary Mammary Epithelial Cells
Authors: Zhao Xiangshan , Lu Lin , Pokhriyal Nidhi , Ma Hui , Duan Lei , Lin Simon , Jafari Nadereh , Band Hamid , Band Vimla
Published: January 2009

Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions, including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably used immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human mammary epithelial cells (hMEC). We show here that ectopic expression of wild-type (WT) RhoA as well as a constitutively active RhoA mutant (G14V) in two independent primary hMEC strains led to their immortalization and preneoplastic transformation. These cells have continued to grow over 300 population doublings (PD) with no signs of senescence, whereas cells expressing the vector or dominant-negative RhoA mutant (T19N) senesced after 20 PDs. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well-known Rho effectors including Rho kinase, PKN, mDia1, and mDia2, was also capable of immortalizing hMECs. Notably, similar to parental normal cells, Rho-immortalized cells have WT p53 and intact G1 cell cycle arrest on Adriamycin treatment. Rho-immortalized cells were anchorage dependent and were unable to form tumors when implanted in nude mice. Lastly, microarray expression profiling of Rho-immortalized versus parental cells showed altered expression of several genes previously implicated in immortalization and breast cancer progression. Taken together, these results show that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked to cellular transformation and breast cancer. [Cancer Res 2009;69(2):483–91]
Apoptosis Repressor with Caspase Recruitment Domain Contributes to Chemotherapy Resistance by Abolishing Mitochondrial Fission Mediated by Dynamin-Related Protein-1
Authors: Wang Jian-Xun , Li Qian , Li Pei-Feng
Published: January 2009

One obstacle of cancer therapy is the development of cancer resistance to chemotherapy. The molecular mechanisms by which the resistance is developed remain to be fully understood. Apoptosis repressor with caspase recruitment domain (ARC) is an endogenous antiapoptotic protein. Here, we report that ARC contributes to chemotherapy resistance by abolishing mitochondrial fission mediated by dynamin-related protein-1 (Drp1). Our results show that both HeLa and human gastric cancer (SGC-7901) cells have a high expression level of ARC. Doxorubicin at a low dose can slightly induce apoptosis in HeLa and SGC-7901 cells. In contrast, knockdown of ARC by its RNA interference enables the same low dose of doxorubicin to significantly induce apoptosis in HeLa and SGC-7901 cells. These data indicate that ARC is responsible for the cell resistance to doxorubicin treatment. Mitochondrial fission has recently been shown to be involved in triggering apoptosis. In exploring the molecular mechanism by which ARC participates in antagonizing doxorubicin-induced apoptosis, we observed that doxorubicin is able to induce mitochondrial fission that can be inhibited by ARC. Our results further show that Drp1 accumulates in mitochondria and mediates the signal of doxorubicin to induce mitochondrial fission. ARC is able to prevent Drp1 accumulations in mitochondria. Finally, we identified that PUMA is required for Drp1 accumulations in mitochondria. ARC inhibits Drp1 accumulations in mitochondria by directly binding to PUMA. Taken together, our results reveal a chemotherapy-resistant model in which ARC inhibits PUMA-mediated Drp1 accumulations in mitochondria and the consequent mitochondrial fission. [Cancer Res 2009;69(2):492–500]
Targeting Ornithine Decarboxylase Impairs Development of MYCN-Amplified Neuroblastoma
Authors: Rounbehler Robert J. , Li Weimin , Hall Mark A. , Yang Chunying , Fallahi Mohammad , Cleveland John L.
Published: January 2009

Neuroblastoma is a pediatric malignancy that arises from the neural crest, and patients with high-risk neuroblastoma, which typically harbor amplifications of MYCN, have an extremely poor prognosis. The tyrosine hydroxylase (TH) promoter-driven TH-MYCN transgenic mouse model faithfully recapitulates many hallmarks of human MYCN-amplified neuroblastoma. A key downstream target of Myc oncoproteins in tumorigenesis is ornithine decarboxylase (Odc), the rate-limiting enzyme of polyamine biosynthesis. Indeed, sustained treatment with the Odc suicide inhibitor α-difluoromethylornithine (DFMO) or Odc heterozygosity markedly impairs lymphoma development in Eμ-Myc transgenic mice, and these effects are linked to the induction of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1, which is normally repressed by Myc. Here, we report that DFMO treatment, but not Odc heterozygosity, impairs MYCN-induced neuroblastoma and that, in this malignancy, transient DFMO treatment is sufficient to confer protection. The selective anticancer effects of DFMO on mouse and human MYCN-amplified neuroblastoma also rely on its ability to disable the proliferative response of Myc, yet in this tumor context, DFMO targets the expression of the p21Cip1 Cdk inhibitor, which is also suppressed by Myc oncoproteins. These findings suggest that agents, such as DFMO, that target the polyamine pathway may show efficacy in high-risk, MYCN-amplified neuroblastoma. [Cancer Res 2009;15(4):547–53]
Basal Subtype and MAPK/ERK Kinase (MEK)-Phosphoinositide 3-Kinase Feedback Signaling Determine Susceptibility of Breast Cancer Cells to MEK Inhibition
Authors: Mirzoeva Olga K. , Das Debopriya , Heiser Laura M. , Bhattacharya Sanchita , Siwak Doris , Gendelman Rina , Bayani Nora , Wang Nicholas J. , Neve Richard M. , Guan Yinghui , Hu Zhi , Knight Zachary , Feiler Heidi S. , Gascard Philippe , Parvin Bahram , Spellman Paul T. , Shokat Kevan M. , Wyrobek Andrew J. , Bissell Mina J. , McCormick Frank , Kuo Wen-Lin , Mills Gordon B. , Gray Joe W. , Korn W. Michael
Published: January 2009

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies. [Cancer Res 2009;69(2):565–72]