bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of single two‐pore domain TREK‐2 channels expressed in mammalian cells
Authors: Kang Dawon , Choe Changyong , Cavanaugh Eric , Kim Donghee
Published: June 2007

TREK‐2 (K2P10.1), a member of the two‐pore domain K+ (K2P) channel family, provides the background K+ conductance in many cell types, and is a target of neurotransmitters that act on receptors coupled to Gs and Gq. We report here that TREK‐2 exhibits small (TREK‐2S) and large (TREK‐2L) conductance phenotypes when expressed in mammalian cell lines (COS‐7, HEK293, HeLa) and in Xenopus oocytes. TREK‐2S phenotype shows a noisy open state with a mean conductance of 54 pS (+40 mV). TREK‐2L phenotype shows a full open state (202 pS) with several short‐lived sub‐conductance levels. Both phenotypes were strongly activated by arachidonic acid, membrane stretch (−40 mmHg) and intracellular acidification (pH 6.4). Phosphorylation of TREK‐2 produced by treatment of cells with activators of protein kinases A and C, and okadaic acid (a serine/threonine phosphatase inhibitor) decreased the current contributed by TREK‐2S and TREK‐2L, and caused partial switching of conductance levels from those of TREK‐2S and TREK‐2L to more intermediate values. Under this condition, TREK‐2 exhibited six conducting levels and one closed level. TREK‐2 mutants in which putative protein kinases A and C phosphorylation sites were mutated to alanines (S326A, S359A, S326A/S359A) displayed mostly TREK‐2S and TREK‐2L phenotypes. However, S326D and S359D mutants (as well as the double mutants) that mimic the phosphorylated state showed all six conducting levels and low channel activity. The S326A and S359A mutants did not significantly affect the intrinsic voltage dependence of TREK‐2 in Mg2+‐free solution. Phenotypes resembling TREK‐2S and TREK‐2L were also observed in cerebellar granule neurons that express TREK‐2 mRNA. These results show that TREK‐2 exhibits two primary modes of gating that give rise to two channel phenotypes under dephosphorylated conditions, and that its phosphorylation shifts the gating mode to include intermediate conducting levels. This represents a novel mechanism by which receptor agonists modulate the function of a K+ channel to alter cell excitability.
Exercise improves phosphatidylinositol‐3,4,5‐trisphosphate responsiveness of atypical protein kinase C and interacts with insulin signalling to peptide elongation in human skeletal muscle
Authors: Frøsig Christian , Sajan Mini P. , Maarbjerg Stine J. , Brandt Nina , Roepstorff Carsten , Wojtaszewski Jørgen F. P. , Kiens Bente , Farese Robert V. , Richter Erik A.
Published: June 2007

We investigated if acute endurance‐type exercise interacts with insulin‐stimulated activation of atypical protein kinase C (aPKC) and insulin signalling to peptide chain elongation in human skeletal muscle. Four hours after acute one‐legged exercise, insulin‐induced glucose uptake was ∼80% higher (N= 12, P < 0.05) in previously exercised muscle, measured during a euglycaemic–hyperinsulinaemic clamp (100 μU ml−1). Insulin increased (P < 0.05) both insulin receptor substrate (IRS)‐1 and IRS‐2 associated phosphatidylinositol (PI)‐3 kinase activity and led to increased (P < 0.001) phosphorylation of Akt on Ser473 and Thr308 in skeletal muscle. Interestingly, in response to prior exercise IRS‐2‐associated PI‐3 kinase activity was higher (P < 0.05) both at basal and during insulin stimulation. This coincided with correspondingly altered phosphorylation of the extracellular‐regulated protein kinase 1/2 (ERK 1/2), p70S6 kinase (P70S6K), eukaryotic elongation factor 2 (eEF2) kinase and eEF2. aPKC was similarly activated by insulin in rested and exercised muscle, without detectable changes in aPKC Thr410 phosphorylation. However, when adding phosphatidylinositol‐3,4,5‐triphosphate (PIP3), the signalling product of PI‐3 kinase, to basal muscle homogenates, aPKC was more potently activated (P= 0.01) in previously exercised muscle. Collectively, this study shows that endurance‐type exercise interacts with insulin signalling to peptide chain elongation. Although protein turnover was not evaluated, this suggests that capacity for protein synthesis after acute endurance‐type exercise may be improved. Furthermore, endurance exercise increased the responsiveness of aPKC to PIP3 providing a possible link to improved insulin‐stimulated glucose uptake after exercise.
Sponge Paleogenomics Reveals an Ancient Role for Carbonic Anhydrase in Skeletogenesis
Authors: Jackson Daniel J. , Macis Luciana , Reitner Joachim , Degnan Bernard M. , Wörheide Gert
Published: June 2007

Sponges (phylum Porifera) were prolific reef-building organisms during the Paleozoic and Mesozoic ∼542 to 65 million years ago. These ancient animals inherited components of the first multicellular skeletogenic toolkit from the last common ancestor of the Metazoa. Using a paleogenomics approach, including gene- and protein-expression techniques and phylogenetic reconstruction, we show that a molecular component of this toolkit was the precursor to the α-carbonic anhydrases (α-CAs), a gene family used by extant animals in a variety of fundamental physiological processes. We used the coralline demosponge
Astrosclera willeyana
, a “living fossil” that has survived from the Mesozoic, to provide insight into the evolution of the ability to biocalcify, and show that the α-CA family expanded from a single ancestral gene through several independent gene-duplication events in sponges and eumetazoans.
Synthesis of Pyridines Over Zeolites in Gas Phase
Authors: Slobodník Marek , Hronec Milan , Cvengrošová Zuzana , Bejblová Martina , Čejka Jiří
Published: June 2007

Pyridine and its methyl derivatives were synthesized in vapor phase from ethanol, formaldehyde and ammonia over zeolites H-ZSM-5, Beta, mordenite and over SiO2. Both the Si/Al molar ratio and modification of zeolite with zinc strongly influenced the selectivity in pyridine and alkylpyridines formation. The highest yield of pyridine was obtained over ZSM-5 modified with zinc and the highest pyridine-to-alkylpyridines ratio was achieved over H-ZSM-5 with high Si content (Si/Al ratio 140). The conversion of ethanol and the selectivity in pyridine formation were correlated with the acidity of used catalyst.
“The stroke is eighty nine”: understanding unprofessional behaviour through physician-authored prose
Authors: Rees C E , Knight L V
Published: June 2007

The unprofessional behaviour of medics is explored through their depiction in two physician-authored books—the novelBodiesand the autobiographyBedside stories: confessions of a junior doctor. Using the Integrative Model of Behavioural Prediction, not only the range and nature of professionalism lapses outlined in these books but also the reasons behind such unprofessional behaviours are examined. The books contained examples of lapses in professionalism outlined in research investigating the unprofessional behaviour of medical students, such as communication violations, objectification of patients and causing harm to patients. More interestingly, various reasons behind lapses in professionalism were found. Most examples of unprofessional behaviour were unintentional acts and therefore due to environmental constraints and skill deficits. Seemingly intentional acts were largely influenced by normative beliefs—that is, people feeling pressurised to act unprofessionally. Further research is needed to examine the depiction of lapses in professionalism in a wider range of physician-authored prose.
Light and electron microscopic observations of the infection of Camellia sasanqua by the fungus Exobasidum camelliae var. gracilis
Authors: Mims C.W. , Richardson E.A.
Published: June 2007

Only a very small percentage of the shoots of the numerous Camellia sasanqua Thunb. plants examined in this study were infected by Exobasidium camelliae var. gracilis Shirai, and prior to the onset of new leaf formation in the spring, it was impossible to distinguish these shoots from healthy shoots with the naked eye. However, beginning in early April, an infected shoot gave rise to a group of tightly clustered, pale-green leaves that were significantly larger and thicker than the dark-green healthy leaves. Light and transmission electron microscopic observations revealed the presence of intercellular hyphae of E. camelliae var. gracilis in the apical meristems of shoots that gave rise to enlarged infected leaves. Very slender intercellular hyphae were observed between the tightly packed cells of the apical meristem as well as between the cells of the tunica that covered the meristem. Hyphae also filled many of the intercellular spaces in the stem immediately below the apical meristem. Some hyphae formed distinctive haustoria that protruded for short distances into the invaded host cells. Hyphae also were present in the leaf primordia and proliferated in the intercellular spaces while the primordium differentiated into a leaf. Eventually a distinct pseudoparenchymatous layer of intercellular hyphae developed some four to six cell layers above the lower epidermis. This layer then gave rise to the basidia that made up the hymenium of E. camelliae var. gracilis. As basidia developed, the underlying cell layers of the leaf separated from the overlying hymenium and eventually sloughed away to expose the basidia. Localized infections of random leaves also were observed. In many cases only a small part of a leaf was infected. Localized infections also culminated in successful hymenium development.
The effect of growing season length and water level fluctuations on growth and survival of two rare and at risk Atlantic Coastal Plain flora species, Coreopsis rosea and Hydrocotyle umbellata
Authors: Lusk Jennifer M. , Reekie Edward G.
Published: June 2007

This study examined variables that may be limiting rare species of Nova Scotia’s Coastal Plain flora to determine why they have not expanded to other lakeshores, including dam reservoirs with appropriate slope and substrate, and to determine how reservoir management could improve habitat quality. A transplant experiment was conducted to examine whether variables related to growing season (establishment time, position on shoreline, and water-level fluctuation) were limiting two at-risk species, Coreopsis rosea Nutt. (Pink Tickseed) and Hydrocotyle umbellata L. (Water-pennywort). Establishment time as well as timing and duration of flooding were found to limit both species. Unseasonable flooding and later establishment had similar effects in reducing biomass. The contribution of time spent above water to the final biomass was 2–3.4 times greater than time spent below water. At reservoirs, earlier spring draw down would reduce flood stress, while opening the dam during periods of high precipitation would avoid plant damage due to unseasonable flooding. Exposure of the lower shoreline for 49–71 d or greater would benefit H. umbellata, and exposure of the upper shoreline for 100–112 d or greater would benefit C. rosea. To conserve current habitat, we must also minimize disturbances to watersheds affecting natural fluctuations of occupied lakes.
Correlated effects of selection for flower size in Raphanus raphanistrum
Authors: Lehtilä Kari , Holmén Bränn Kristina
Published: June 2007

The evolution of flower size may be constrained by trade-offs between flower size and other plant traits. The aim of this study was to determine how selection on flower size affects both reproductive and vegetative traits. Raphanus raphanistrum L. was used as the study species. Artificial selection for small and large petal size was carried out for two generations. We measured the realized heritability of flower size and recorded flower production, time to flowering, plant size, and seed production in the two selection lines. The realized heritability was h2 = 0.49. Our study, therefore, showed that R. raphanistrum has potential for rapid evolutionary change of floral size. The lines with large flowers produced smaller seeds and started to flower later than the lines with small flowers. There was no trade-off between flower size and flower number, but the lines selected for large flower size had more flowers and a larger plant size than lines selected for small flowers. Estimates of restricted maximum likelihood (REML) analysis of pedigrees also showed that flower size had a positive genetic correlation with start of flowering and plant height.
Compressibility behaviour of clays at large pressures
Authors: Tripathy Snehasis , Schanz Tom
Published: June 2007

In this study, a method is proposed on the basis of the diffuse double layer theory to determine the compressibility of clays for one-dimensional conditions for vertical pressures far greater than the testing range of conventional oeodometer tests. Experimental e–p curves of several reported bentonitic clays were considered to verify the proposed method. It is shown that the parameters required to use the diffuse double layer theory can be obtained from the experimental e–p data for a low range of pressure and those can in turn be used to compute void ratios at higher vertical pressures. The stepwise procedure to calculate the e–p relationships at high pressure is given. The results showed that the agreement between the calculated and experimental compression characteristics at large vertical pressures is very good for the clays considered in this study.Key words: clays, compressibility, consolidation, expansive soils.
Numerical modelling of soft soil stabilized by vertical drains, combining surcharge and vacuum preloading for a storage yard
Authors: Rujikiatkamjorn Cholachat , Indraratna Buddhima , Chu Jian
Published: June 2007

This paper presents a finite element analysis of a case study of a combined vacuum and surcharge load through prefabricated vertical drains (PVD) at a storage yard at Port of Tianjin, China. The top 15 m of soil at this site was very soft to soft and needed to be improved using preloading surcharges of more than 140 kPa. To avoid any stability problems associated with a high surcharge embankment, 80 kPa vacuum pressure combined with fill surcharge was applied (40 and 58 kPa for sections I and II, respectively). A plane strain analysis was performed using equivalent permeability and transformed unit-cell geometry. The converted (equivalent) parameters were incorporated in the finite element code ABAQUS, using the modified Cam-Clay theory. The performance of a trial embankment at the site of the storage yard is predicted on the basis of a constant vacuum pressure applied on the soil surface and distributed along the length of the drain. The predictions of settlement, pore-water pressure, and lateral displacement were compared with the available field data, and an acceptable agreement was found based on this numerical approach. The combination of vacuum and surcharge load can effectively shorten the preloading period, reduce the height of the embankment, and counterbalance excessive lateral displacements.Key words: consolidation, finite element analysis, plane strain method, soil improvement, vertical drains.
Desiccation-induced cracking and its effect on the hydraulic conductivity of clayey soils from Iran
Authors: Rayhani M HT , Yanful E K , Fakher A
Published: June 2007

Clay materials have many environmental applications, especially in situations where a hydraulic barrier is desired. However, as the plasticity of clay increases, cracks tend to develop during cycles of long dry spells. This is particularly a concern in the construction of covers or installation of landfill liners prior to waste filling. In the present study, specimens prepared from three natural clayey soils from Iran used for clay barrier construction, and one artificial clayey soil, were subjected to cycles of wetting and drying. Surface cracks of different dimensions formed as a result of drying. Specimens with the largest volumetric shrinkage strains typically contained the highest number of cracks. Specimens that developed cracks were subjected to hydraulic conductivity testing. The results showed that the dimension of cracks increased with increasing plasticity index and clay content and, so, the initial hydraulic conductivity increased with increasing plasticity index and cycles of drying and wetting. Cracking increased the hydraulic conductivity by 12–34 times, depending on the plasticity of the soil. After a long saturation time, the hydraulic conductivity of the soils decreased with an increase in saturation time, which could be associated with a self-healing process that affects the soils by different degrees.Key words: desiccation, cracking, plasticity, hydraulic conductivity, clay barriers, self-healing, volumetric shrinkage.
Involvement of Sp1 and Sp3 in differential regulation of human NHE3 promoter activity by sodium butyrate and IFN-γ/TNF-α
Authors: Amin Md. Ruhul , Dudeja Pradeep K. , Ramaswamy Krishnamurthy , Malakooti Jaleh
Published: June 2007

Previously, we reported that IFN-γ and TNF-α downregulate the expression of the human Na+/H+exchanger (NHE)3 gene by modulating Sp1/Sp3 transcription factors in C2BBe1 cells. It is reported that butyrate inhibits IFN-γ and TNF-α signaling pathways. In this study, we have investigated the effect of sodium butyrate (NaB) and IFN-γ/TNF-α on human NHE3 promoter activity. In transient transfection studies, NaB (5 mM) led to 10-fold stimulation of NHE3 promoter activity after incubation for 24 h. With 5′-deletion analysis, the NaB-responsive region was mapped to the NHE3 core promoter, bp −95 to + 5, which we had shown previously to confer responsiveness to IFN-γ/TNF-α. The stimulatory effect of NaB on the NHE3 promoter was reduced by 60% in the presence of IFN-γ/TNF-α. Mutually, the repressive effect of these cytokines was attenuated by NaB. Knockdown of Sp1 and Sp3 expression with small interfering RNA (siRNA) resulted in a significant resistance to NaB effects. NaB treatment showed no effect on Sp1 and Sp3 protein expression as assessed by Western blot analyses. Gel mobility shift assays with nuclear proteins from NaB-treated cells showed enhanced binding of Sp1 and Sp3 to the NHE3 promoter. The phosphatase inhibitor okadaic acid (200 nM) blocked the stimulatory effect of NaB on the NHE3 promoter. NaB effects on the NHE3 promoter were significantly attenuated by protein phosphatase (PP)1α- and PP2Aα-specific siRNA transfection. Our data suggest that the differential regulation of NHE3 gene expression by NaB and IFN-γ/TNF-α is mediated through alternative pathways that converge on Sp1/Sp3.
Uncoupling of the VEGF-endothelial nitric oxide axis in diabetic nephropathy: an explanation for the paradoxical effects of VEGF in renal disease
Authors: Nakagawa Takahiko
Published: June 2007

In many forms of experimental kidney diseases, renal VEGF is low, and administering VEGF can be shown to be protective. A paradox occurs in diabetes, in which renal VEGF levels are high and a deleterious effect of VEGF on kidney disease has been shown. We have hypothesized that endothelial dysfunction induced by hyperglycemia or other factors may underlie the pathogenic mechanisms of a high VEGF state. VEGF normally stimulates endothelial nitric oxide (NO) release and acts in concert with elevated NO levels as a trophic factor for vascular endothelium. The increased NO derived from the endothelial cell acts as an inhibitory factor that prevents excess endothelial cell proliferation, vascular smooth muscle cell proliferation, and macrophage infiltration. In the setting where NO bioavailability is reduced in diabetes, high levels of VEGF lead to excessive endothelial cell proliferation, stimulation of macrophage chemotaxis, and vascular smooth muscle cell activation. Consistent with this hypothesis is our recent observation that diabetes induced in endothelial NO-deficient mice results in clinical and histological features identical to human diabetic nephropathy. The discovery of the key role for impaired endothelial NO bioavailability in the stimulation of VEGF and VEGF-dependent disease may provide key insights into not only the pathogenesis of diabetic nephropathy but also the utility and hazard of administering VEGF as a treatment for kidney disease.
Properties and general use of the X-ray elastic factors
Authors: Ortner Balder
Published: June 2007

The equation ε(φ, ψ, hkl)=Fij(φ, ψ, hkl)σij can be directly deduced from Hooke’s law. It is shown that the matrix Fij(φ, ψ, hkl) which is usually called X-ray elastic factors, behaves as a second rank tensor. Since this behaviour is the only criterion for the question of whether or not it is a tensor, the F-matrix must be regarded as a second rank tensor. This allows us to make some statements about the structure of the F-matrix on the basis of Neumann’s principle, to find relationships among F-matrices in different measurement directions, and to apply the methods and strategies for the measurement of a second rank tensor. All this is shown in a few examples. It is further shown that a consistent use of the F-matrix can replace all methods for data evaluation which makes use of linear regressions and, in addition, avoids all difficulties and disadvantages of these methods. One of these disadvantages is that the sin2 ψ-method, as well as its derivatives, is generally not correct least square fits of the measured data. This is also shown in an example. The more complicated cases with stress or constitution gradients in the range of the probed volume or stress measurement after plastic deformation are not discussed.
High pressure deformation study of zirconium
Authors: Vogel Sven C. , Reiche Helmut , Brown Donald W.
Published: June 2007

In situ deformation studies of polycrystalline materials using diffraction are an established method to understand elastic and plastic deformation of materials. Studies of active deformation mechanisms, the interplay of deformation with texture, and ultimately the development of predictive capabilities for deformation modeling are an active field of research. Parameters studied by diffraction are typically lattice strains and texture evolution, which coupled with the macroscopic flow curve allow for improved understanding of the micro-mechanics of deformation. We performed a study of the uniaxial deformation of Zircaloy-2 at 2 GPa at the 13-BM-D beamline at the Advanced Photon Source. The deformation-DIA apparatus generates a confining hydrostatic pressure using a cubic anvil setup. Two differential rams allow an increase (compressive load) or decrease (tensile load) of the uniaxial straining in the vertical direction, allowing studies of plastic deformation at high pressures. In this paper, we describe how macroscopic strains, hydrostatic pressure, and uniaxial strains are derived and present some brief results.
An EXAFS study of photographic development in thermographic films
Authors: Blanton T. N. , Whitcomb D. R. , Misture S. T.
Published: June 2007

Silver K edge extended X-ray absorption fine structure (EXAFS) spectroscopy of films containing silver behenate (AgBeh) in the unprocessed, fully processed, and step-processed states has been performed. The results of the EXAFS analysis indicate that the intensity for the real-space peak for the Ag-O distance (∼2.3 Å) decreases while the real-space peak for the Ag-Ag distance (∼2.9 Å) grows with increasing thermal processing of the film. The changes observed in the real-space EXAFS signal indicate the growth of metallic silver at the expense of AgBeh. The X-ray absorption near-edge spectroscopy (XANES) portion of the signal shows that the absorption edge position varies stepwise, with unprocessed films and pure AgBeh having an edge location at 25 506 eV, films processed from steps 1 through 10 have an absorption edge at 25 508 eV, and the fully processed film has an edge location at 25 512 eV.
Unit cell expansion in ErT2 films
Authors: Rodriguez Mark A. , Browning James F. , Frazer Colleen S. , Snow Clark S. , Tissot Ralph G. , Boespflug Elaine P.
Published: June 2007

XRD analysis of plasma-vapor-deposited
ErT2 films during aging (T decay to
3He) reveals an hkl-dependent unit-cell expansion in which (200) grains expand out-of-plane as much as 0.01 Å more than (111) out-of-plane grains. Texture analysis of an aged
ErT2 film reveals a bimodal (111)/(200) out-of-plane preferred orientation. sin2 ψ analysis reveals significant in-plane macro-strain due to
3He
bubble formation/growth. The mechanistic origins regarding these observations are also discussed.
Validation of an alkali reaction, borate fusion, X-ray fluorescence method for silicon metal
Authors: Sieber John R. , Mackey Elizabeth A. , Marlow Anthony F. , Paul Rick , Martin Ryan
Published: June 2007

The value assignment of candidate Standard Reference Material (SRM®) 57b Silicon Metal provided an opportunity to develop an alkali reaction procedure as a precursor to borate fusion for the preparation of test specimens from the metal powder for X-ray fluorescence spectrometry (XRF). Suggested for this purpose by Blanchette in a 2002 Advances in X-ray Analysis article [45, 415–420 (2002)], the alkali reaction uses
LiOH∙H2O to convert Si to
Li2SiO3. Lithium silicate is fused with lithium borate flux without damage to platinum ware. Once specimens are fused and cast as beads, calibration standards are prepared to closely match the compositions of the specimens, allowing a linear calibration for each analyte. The XRF method yields results that are directly traceable to the mole through NIST SRM spectrometric solutions. The method was validated in two ways. First, the reaction was used on older SRMs for Si metal: SRM 57 and SRM 57a. Second, XRF results for candidate SRM 57b were compared to results obtained using prompt gamma-ray activation analysis (PGAA) and inductively coupled plasma optical emission spectrometry (ICPOES). Bias tests show the XRF results are accurate for the elements Al, S, Ca, Ti, Cr, Mn, Ni, Cu, and Zr. Levels of S, Ca, Cr, and Cu in candidate SRM 57b are near the limits of quantification of the borate fusion method. Iron results may be subject to a low bias. Phosphorus is not quantitatively retained during the alkali reaction and borate fusion. These elements, plus B, which cannot be determined after borate fusion, are listed in manufacturing specifications for Si metal.
Quantitative analysis with the transition edge sensor microcalorimeter X-ray detector
Authors: Jach Terrence , Ritchie Nicholas , Ullom Joel , Beall James A.
Published: June 2007

We report on the use of a microcalorimeter X-ray detector with a transition edge sensor in an electron probe to perform quantitative analysis. We analyzed two bulk samples of multielement glasses that have been previously characterized by chemical methods for use as standard reference materials. The spectra were analyzed against standards using three different correction schemes. In one of the standards, the reference line was easily resolved despite its proximity within 45 eV of another line. With the exception of direct measurements of oxygen (a particularly challenging element), the results are in agreement with the certified characterization to better than 1% absolute or 8% relative. This demonstrates the potential of microcalorimeter detectors as replacements for conventional energy dispersive detectors in applications requiring high energy resolution.
A Combination Hybrid-Based Vaccination/Adoptive Cellular Therapy to Prevent Tumor Growth by Involvement of T Cells
Authors: Savai Rajkumar , Schermuly Ralph Theo , Pullamsetti Soni Savai , Schneider Michael , Greschus Susanne , Ghofrani Hossein Ardeschir , Traupe Horst , Grimminger Friedrich , Banat Gamal-Andre
Published: June 2007

Cancer immunotherapy with dendritic cell–tumor cell fusion hybrids induces polyclonal stimulation against a variety of tumor antigens, including unknown antigens. Hybrid cells can prime CTLs, which subsequently develop antitumor responses. The aim of this study was to enhance the known antitumor effect of hybrid vaccination (HC-Vacc) and hybrid-primed adoptive T-cell therapy (HC-ACT) using the poorly immunogenic Lewis lung carcinoma (LLC1) model. The strategy used was a combination of a double HC-Vacc alternating with HC-ACT (HC-Vacc/ACT). Using flat-panel volumetric computer tomography and immunohistochemistry, we showed a significant retardation of tumor growth (85%). In addition, a significant delay in tumor development, a reduction in the number of pulmonary metastases, and increased survival times were observed. Furthermore, the tumors displayed significant morphologic changes and increased apoptosis, as shown by up-regulation of gene expression of the proapoptotic markers Fas, caspase-8, and caspase-3. The residual tumor masses seen in the HC-Vacc/ACT–treated mice were infiltrated with CD4+ and CD8+ lymphocytes and showed elevated IFNγ expression. Moreover, splenic enlargement observed in HC-Vacc/ACT–treated mice reflected the increased functionality of T cells, as also indicated by increased expression of markers for CTL activation, differentiation, and proliferation (Cd28, Icosl, Tnfrsf13, and Tnfsf14). Our findings indicate that the combination therapy of dendritic cell–tumor cell HC-Vacc/ACT is a very effective and a promising immunotherapeutic regimen against poorly immunogenic carcinomas. [Cancer Res 2007;67(11):5444–54]
Polychlorinated Biphenyl Levels in Peripheral Blood and Non-Hodgkin's Lymphoma: A Report from Three Cohorts
Authors: Engel Lawrence S. , Laden Francine , Andersen Aage , Strickland Paul T. , Blair Aaron , Needham Larry L. , Barr Dana B. , Wolff Mary S. , Helzlsouer Kathy , Hunter David J. , Lan Qing , Cantor Kenneth P. , Comstock George W. , Brock John W. , Bush David , Hoover Robert N. , Rothman Nathaniel
Published: June 2007

The incidence of non-Hodgkin's lymphoma (NHL) unrelated to HIV infection has steadily increased over the past several decades and remains substantially unexplained. Limited evidence suggests that increased concentrations of polychlorinated biphenyls (PCB) measured in blood or fat tissue are associated with increased risk of NHL. Although PCB congeners vary in their biological activity, the relation between individual congeners and NHL risk has not been examined previously using prospectively collected biospecimens. We examined congener-specific associations in three prospective cohorts. Prediagnostic serum or plasma concentrations of selected PCB congeners were measured among NHL cases and controls from these cohorts: Janus (190 cases and 190 controls) in Norway and CLUE I (74 cases and 147 controls) and the Nurses' Health Study (30 cases and 78 controls) in the United States. All blood samples were collected in the 1970s or 1980s. We used logistic regression to calculate odds ratios (OR) and 95% confidence intervals (95% CI) for the relations between risk of NHL and lipid-corrected plasma or serum concentrations. Several congeners (i.e., 118, 138, and 153) that were present at higher levels and were moderately to highly correlated with each other showed exposure-response trends with risk of NHL in all three cohorts. These associations were observed primarily among subjects diagnosed closer to the date of blood collection in the two cohorts with sufficient cases to permit stratification by time. Among cases diagnosed within the median years of follow-up (16 years in Janus and 12 years in CLUE I), ORs and 95% CIs for increasing fourths of concentration of congener 118 relative to the lowest fourth were as follows: 2.4 (0.9–6.5), 4.9 (1.6–15.3), and 5.3 (1.5–18.8; Ptrend < 0.005) in Janus and 8.1 (1.0–68.9), 6.6 (0.7–59.0), and 13.0 (1.6–106.8; Ptrend < 0.05) in CLUE I. Similar patterns were seen for congeners 138 and 153 and for total PCBs. Limited evidence of exposure-response trends was also observed for several other congeners. The primary 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane metabolite, p,p′-DDE, was not significantly associated with NHL in most analyses but slightly to moderately confounded the PCB associations. The results from these three cohorts suggest that concentrations of certain PCBs in blood are associated with increased risk of NHL. [Cancer Res 2007;67(11):5545–52]
A Role for the Fas/FasL System in Modulating Genetic Susceptibility to T-Cell Lymphoblastic Lymphomas
Authors: Villa-Morales María , Santos Javier , Pérez-Gómez Eduardo , Quintanilla Miguel , Fernández-Piqueras José
Published: June 2007

The Fas/FasL system mediates induced apoptosis of immature thymocytes and peripheral T lymphocytes, but little is known about its implication in genetic susceptibility to T-cell malignancies. In this article, we report that the expression of FasL increases early in all mice after γ-radiation treatments, maintaining such high levels for a long time in mice that resisted tumor induction. However, its expression is practically absent in T-cell lymphoblastic lymphomas. Interestingly, there exist significant differences in the level of expression between two mice strains exhibiting extremely distinct susceptibilities that can be attributed to promoter functional polymorphisms. In addition, several functional nucleotide changes in the coding sequences of both Fas and FasL genes significantly affect their biological activity. These results lead us to propose that germ-line functional polymorphisms affecting either the levels of expression or the biological activity of both Fas and FasL genes could be contributing to the genetic risk to develop T-cell lymphoblastic lymphomas and support the use of radiotherapy as an adequate procedure to choose in the treatment of T-cell malignancies. [Cancer Res 2007;67(11):5107–16]
Host Perforin Reduces Tumor Number but Does Not Increase Survival in Oncogene-Driven Mammary Adenocarcinoma
Authors: Street Shayna E.A. , Zerafa Nadeen , Iezzi Manuela , Westwood Jennifer A. , Stagg John , Musiani Piero , Smyth Mark J.
Published: June 2007

The concept of tumor immune surveillance has been supported by several recent studies in mice which show that immune effector mechanisms suppress hematologic malignancy. However, because the most common forms of human cancer are epithelial in origin, and comparatively very little data supports the immune surveillance of epithelial malignancies, we have chosen to evaluate the role of perforin-mediated cytotoxicity in the prevention of BALB/c Her2/neu-induced mammary cancer. Interestingly, perforin significantly delayed the onset of mammary tumorigenesis and reduced the number of mammary tumors without improving survival. Natural killer cell, but not CD8+ T cell, depletion resulted in a similar phenotype to perforin deficiency in this regard. Histologic analysis further indicated that the effect of perforin was most evident during the earliest stages of carcinogenesis rather than prior to or during the hyperplastic phase. This data suggests that perforin may mediate some suppression of epithelial carcinogenesis by intervening early in the tumor development process. [Cancer Res 2007;67(11):5454–60]
Apoptosis Suppression by Somatic Cell Transfer of Bcl-2 Promotes Sonic Hedgehog–Dependent Medulloblastoma Formation in Mice
Authors: McCall Todd D. , Pedone Carolyn A. , Fults Daniel W.
Published: June 2007

Medulloblastomas are malignant brain tumors that arise in the cerebellum in children. Aberrant activation of the Sonic hedgehog (Shh) signaling pathway, which normally stimulates proliferation of granule neuron precursors (GNP) during cerebellar development, induces tumors in mice that closely mimic human medulloblastomas. Shh-dependent medulloblastoma formation is enhanced by hyperactive insulin-like growth factor (IGF) signaling and ectopic expression of Myc oncogenes. This enhanced tumorigenesis stems from the sensitivity of GNPs to IGF and Myc levels in regulating proliferation. An emerging theme in cancer research is that oncogene-induced cell proliferation cannot initiate neoplastic transformation unless cellular programs that mediate apoptosis are disabled. Here, we report a high frequency of medulloblastoma formation in mice after postnatal overexpression of the antiapoptotic protein Bcl-2 in cooperation with Shh. Ectopic expression of Bcl-2 alone or in combination with N-Myc did not induce tumors, indicating that Shh has essential transforming functions in GNPs not supplied by the mitogenic stimulus of N-Myc combined with a strong antiapoptotic signal provided by Bcl-2. Expression of endogenous Bcl-2 was not up-regulated in Shh-induced tumors. Instead, elevated levels of phosphorylated Akt were found, suggesting that activated phosphatidylinositol 3-kinase signaling is one intrinsic mechanism for suppressing apoptosis in Shh-dependent medulloblastomas. Thus, blockade of apoptosis cooperates with Shh-stimulated proliferation to transform GNPs and induce aggressive medulloblastomas. These findings provide insights into the molecular signals that initiate medulloblastoma formation and they support the importance of blocking apoptosis in carcinogenesis. [Cancer Res 2007;67(11):5179–85]
Down-regulation of PU.1 by Methylation of Distal Regulatory Elements and the Promoter Is Required for Myeloma Cell Growth
Authors: Tatetsu Hiro , Ueno Shikiko , Hata Hiroyuki , Yamada Yasuhiro , Takeya Motohiro , Mitsuya Hiroaki , Tenen Daniel G. , Okuno Yutaka
Published: June 2007

The transcription factor PU.1 is essential for myeloid and B-cell development. Down-regulation of PU.1 by disruption of its 14-kb 5′ upstream regulatory element induced acute myeloid leukemia, T-cell lymphoma, and chronic lymphocytic leukemia–like disease in murine models. In the present study, we found that PU.1 was down-regulated in the majority of human myeloma cell lines and a subset of freshly isolated myeloma cells, in contrast to relatively high expression of PU.1 in normal plasma cells. Patients in this low PU.1 expression subset may have a poor prognosis. In human myeloma cell lines, the 17-kb 5′ upstream enhancer and the promoter region of the PU.1 gene were highly methylated, and this is consistent with disappearance of DNase I–hypersensitive sites in these regions. To elucidate the significance of down-regulation of PU.1, we generated stable myeloma cell lines with an inducible PU.1 expression system. Exogenous expression of PU.1 in PU.1 null myeloma cell lines, U266 and KMS12PE, induced complete growth arrest and cell death. Up-regulation of PU.1 by 5-aza-2′-deoxycytidine also induced growth arrest of KMS12PE and KHM11 myeloma cells. These data suggest that down-regulation of PU.1 is an essential step for the survival of a subset of myeloma cells and that up-regulation of PU.1 by demethylation agents or other types of agents may represent a new therapeutic strategy for treatment of multiple myeloma patients. [Cancer Res 2007;67(11):5328–36]
Mel-18 Acts as a Tumor Suppressor by Repressing Bmi-1 Expression and Down-regulating Akt Activity in Breast Cancer Cells
Authors: Guo Wei-Jian , Zeng Mu-Sheng , Yadav Ajay , Song Li-Bing , Guo Bao-Hong , Band Vimla , Dimri Goberdhan P.
Published: June 2007

The Bmi-1 oncogene is overexpressed in a number of malignancies including breast cancer. In addition to Bmi-1, mammalian cells also express four other polycomb group (PcG) proteins that are closely related to Bmi-1. Virtually nothing is known about the role of these PcG proteins in oncogenesis. We have recently reported that Mel-18, a Bmi-1–related PcG protein, negatively regulates Bmi-1 expression, and that its expression negatively correlates with Bmi-1 in proliferating and senescing human fibroblasts. Here, we report that the expression of Bmi-1 and Mel-18 inversely correlates in a number of breast cancer cell lines and in a significant number of breast tumor samples. Overexpression of Mel-18 results in repression of Bmi-1 and reduction of the transformed phenotype in malignant breast cancer cells. Furthermore, the repression of Bmi-1 by Mel-18 is accompanied by the reduction of Akt/protein kinase B (PKB) activity in breast cancer cells. Similarly, Bmi-1 knockdown using RNA interference approach results in down-regulation of Akt/PKB activity and reduction in transformed phenotype of MCF7 cells. Importantly, we show that overexpression of constitutively active Akt overrides tumor-suppressive effect of Mel-18 overexpression and the knockdown of Bmi-1 expression. Thus, our studies suggest that Mel-18 and Bmi-1 may regulate the Akt pathway in breast cancer cells, and that Mel-18 functions as a tumor suppressor by repressing the expression of Bmi-1 and consequently down-regulating Akt activity. [Cancer Res 2007;67(11):5083–9]
Plasma Folate, Vitamin B6, Vitamin B12, and Homocysteine and Pancreatic Cancer Risk in Four Large Cohorts
Authors: Schernhammer Eva , Wolpin Brian , Rifai Nader , Cochrane Barbara , Manson Jo Ann , Ma Jing , Giovannucci Ed , Thomson Cynthia , Stampfer Meir J. , Fuchs Charles
Published: June 2007

Folate deficiency induces DNA breaks and may alter cellular capacity for mutation and epigenetic methylation. Few studies have examined the influence of one-carbon nutrients on pancreatic cancer risk, although recent studies suggest a potential protective effect for one-carbon nutrients from food sources, but not from supplements. We conducted a prospective nested case-control study to examine plasma concentrations of folate, vitamin B6 [whose main circulating form is pyridoxal-5′-phosphate (PLP)], vitamin B12, and homocysteine in relationship to pancreatic cancer, using four large prospective cohorts. Multivariable adjusted odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using conditional logistic regression. All statistical tests were two sided. Among 208 cases and 623 controls, we observed no association between folate, PLP, vitamin B12, or homocysteine and pancreatic cancer risk. Comparing the highest to lowest quartiles of plasma concentration, the ORs were 1.20 (95% CI, 0.76–1.91) for folate, 0.80 (95% CI, 0.51–1.25) for B6, 0.91 (95% CI, 0.57–1.46) for B12, and 1.43 (95% CI, 0.90–2.28) for homocysteine. In analyses restricted to nonusers of multivitamins, we observe a modest inverse trend between folate, PLP, and B12 and pancreatic cancer risk. In contrast, no such inverse associations were observed among study subjects who reported multivitamin supplement use. Among all participants, plasma levels of folate, B6, B12, and homocysteine were not associated with a significant reduction in the risk of pancreatic cancer. Among participants who obtain these factors exclusively through dietary sources, there may be an inverse relation between circulating folate, B6, and B12 and risk. [Cancer Res 2007;67(11):5553–60]
Epigenetically Controlled Fibroblast Growth Factor Receptor 2 Signaling Imposes on the RAS/BRAF/Mitogen-Activated Protein Kinase Pathway to Modulate Thyroid Cancer Progression
Authors: Kondo Tetsuo , Zheng Lei , Liu Wei , Kurebayashi Junichi , Asa Sylvia L. , Ezzat Shereen
Published: June 2007

Fibroblast growth factor (FGF) signals play fundamental roles in development and tumorigenesis. Thyroid cancer is an example of a tumor with nonoverlapping genetic mutations that up-regulate mitogen-activated protein kinase (MAPK). Here, we show that FGF receptor 1 (FGFR1), which is expressed mainly in neoplastic thyroid cells, propagates MAPK activation and promotes tumor progression. In contrast, FGFR2 is down-regulated in neoplastic thyroid cells through DNA promoter methylation. Reexpression of FGFR2 competes with FGFR1 for the immediate substrate FGFR substrate 2 to impede signaling upstream of the BRAF/MAPK pathway. These data unmask an epigenetically controlled FGFR2 signal that imposes precisely on the intragenically modified BRAF/MAPK pathway to modulate thyroid cancer behavior. [Cancer Res 2007;67(11):5461–70]
Whole-Body Subcellular Multicolor Imaging of Tumor-Host Interaction and Drug Response in Real Time
Authors: Yang Meng , Jiang Ping , Hoffman Robert M.
Published: June 2007

To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)–expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. The Olympus IV100 Laser Scanning Microscope, with ultra-narrow microscope objectives (“stick objectives”), is used for three-color whole-body imaging of the two-color cancer cells interacting with the GFP-expressing stromal cells. In this model, drug response of both cancer and stromal cells in the intact live animal is also imaged in real time. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow. This new model system enables the first cellular and subcellular images of unperturbed tumors in the live intact animal. New visible real-time targets for novel anticancer agents are provided in this model, including the color-coded interacting cancer and stromal cells, tumor vasculature, and blood flow. This imageable model should lead to many new insights of in vivo cancer cell biology and to novel drug discovery. [Cancer Res 2007;67(11):5195–200]
Therapeutic Efficacy of Replication-Competent Retrovirus Vector–Mediated Suicide Gene Therapy in a Multifocal Colorectal Cancer Metastasis Model
Authors: Hiraoka Kei , Kimura Takahiro , Logg Christopher R. , Tai Chien-Kuo , Haga Kazunori , Lawson Gregory W. , Kasahara Noriyuki
Published: June 2007

Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition. [Cancer Res 2007;67(11):5345–53]