bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligands Differentially Modify the Nuclear Mobility of Estrogen Receptors α and β
Authors: Damdimopoulos Anastasios E. , Spyrou Giannis , Gustafsson Jan-Åke
Published: September 2007

Signaling of nuclear receptors depends on the structure of their ligands, with different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on estrogen receptor α (ERα) and ERβ mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERα, ligands can be classified into three distinct groups: 1) ligands that do not affect the mobility of the receptor, 2) ligands that cause a moderate effect, and 3) ligands that strongly impact mobility of ERα. Interestingly, we found that for ERβ such a classification was not possible because ERβ ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response toward the antiestrogens ICI and raloxifene, which was not attributable to differential subnuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERα, whereas ERβ retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteasome machinery. This novel finding that ERβ retains its mobility in the presence of antiestrogens could be important for its ability to regulate genes that do not contain classic estrogen response element sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.
Glucagon-Like Peptide-2 Activates β-Catenin Signaling in the Mouse Intestinal Crypt: Role of Insulin-Like Growth Factor-I
Authors: Dubé Philip E. , Rowland Katherine J. , Brubaker Patricia L.
Published: September 2007

Chronic administration of glucagon-like peptide-2 (GLP-2) induces intestinal growth and crypt cell proliferation through an indirect mechanism requiring IGF-I. However, the intracellular pathways through which IGF-I mediates GLP-2-induced epithelial tropic signaling remain undefined. Because β-catenin and Akt are important regulators of crypt cell proliferation, we hypothesized that GLP-2 activates these signaling pathways through an IGF-I-dependent mechanism. In this study, fasted mice were administered Gly2-GLP-2 or LR3-IGF-I (positive control) for 0.5–4 h. Nuclear translocation of β-catenin in non-Paneth crypt cells was assessed by immunohistochemistry and expression of its downstream proliferative markers, c-myc and Sox9, by quantitative RT-PCR. Akt phosphorylation and activation of its targets, glycogen synthase kinase-3β and caspase-3, were determined by Western blot. IGF-I receptor (IGF-IR) and IGF-I signaling were blocked by preadministration of NVP-AEW541 and through the use of IGF-I knockout mice, respectively. We found that GLP-2 increased β-catenin nuclear translocation in non-Paneth crypt cells by 72 ± 17% (P < 0.05) and increased mucosal c-myc and Sox9 mRNA expression by 90 ± 20 and 376 ± 170%, respectively (P < 0.05–0.01), with similar results observed with IGF-I. This effect of GLP-2 was prevented by blocking the IGF-IR as well as ablation of IGF-I signaling. GLP-2 also produced a time- and dose-dependent activation of Akt in the intestinal mucosa (P < 0.01), most notably in the epithelium. This action was reduced by IGF-IR inhibition but not IGF-I knockout. We concluded that acute administration of GLP-2 activates β-catenin and proliferative signaling in non-Paneth murine intestinal crypt cells as well as Akt signaling in the mucosa. However, IGF-I is required only for the GLP-2-induced alterations in β-catenin.
Estrogen and Progesterone are Critical Regulators of Stat5a Expression in the Mouse Mammary Gland
Authors: Santos Sarah J. , Haslam Sandra Z. , Conrad Susan E.
Published: September 2007

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.
Dehydroepiandrosterone Induces a Neuroendocrine Phenotype in Nerve Growth Factor-Stimulated Chromaffin Pheochromocytoma PC12 Cells
Authors: Ziegler Christian G. , Sicard Flavie , Lattke Peter , Bornstein Stefan R. , Ehrhart-Bornstein Monika , Krug Alexander W.
Published: September 2007

The adrenal androgen dehydroepiandrosterone (DHEA) is produced in the inner zone of the adrenal cortex, which is in direct contact to adrenal medullary cells. Due to their close anatomical proximity and tightly intermingled cell borders, a direct interaction of adrenal cortex and medulla has been postulated. In humans congenital adrenal hyperplasia due to 21-hydroxylase deficiency results in androgen excess accompanied by severe adrenomedullary dysplasia and chromaffin cell dysfunction. Therefore, to define the mechanisms of DHEA action on chromaffin cell function, we investigated its effect on cell survival and differentiation processes on a molecular level in the chromaffin cell line PC12. DHEA lessened the positive effect of NGF on cell survival and neuronal differentiation. Nerve growth factor (NGF)-mediated induction of a neuronal phenotype was inhibited by DHEA as indicated by reduced neurite outgrowth and decreased expression of neuronal marker proteins such as synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein-2. We examined whether DHEA may stimulate the cells toward a neuroendocrine phenotype. DHEA significantly elevated catecholamine release from unstimulated PC12 cells in the presence but not absence of NGF. Accordingly, DHEA enhanced the expression of the neuroendocrine marker protein chromogranin A. Next, we explored the possible molecular mechanisms of DHEA and NGF interaction. We demonstrate that NGF-induced ERK1/2 phosphorylation was reduced by DHEA. In summary, our data show that DHEA influences cell survival and differentiation processes in PC12 cells, possibly by interacting with the ERK1/2 MAPK pathway. DHEA drives NGF-stimulated cells toward a neuroendocrine phenotype, suggesting that the interaction of intraadrenal steroids and growth factors is required for the maintenance of an intact adrenal medulla.
Thrombospondin-1 Expression Is Increased during Follicular Atresia in the Primate Ovary
Authors: Thomas Fiona H. , Wilson Helen , Silvestri Audrey , Fraser Hamish M.
Published: September 2007

Thrombospondin (TSP)-1 is an antiangiogenic extracellular matrix glycoprotein that modulates several aspects of cellular function. The aim of this study was to determine the pattern of TSP-1 mRNA and protein expression as well as expression of its receptor CD36 in the marmoset ovary and to investigate the effects of inhibition of gonadotropins or VEGF activity on TSP-1 and CD36 expression in vivo. GnRH antagonist or VEGF Trap, a soluble decoy receptor, was administered on d 0 of the follicular phase of the cycle, and ovaries were collected at the end of the follicular phase (d 10). TSP-1 mRNA and protein were present in granulosa cells of preantral and antral follicles, with the highest staining at the late secondary and tertiary stages. Moreover, expression of TSP-1 mRNA and protein was significantly increased in tertiary follicles undergoing atresia. CD36 protein was detected in granulosa cells of preantral and antral follicles as well as in endothelial cells of large vessels. Inhibition of gonadotropin secretion or VEGF activity had no effect on TSP-1 expression; however, expression of CD36 protein was inhibited by the VEGF Trap. In conclusion, TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia; alternatively, TSP-1 may act on granulosa and/or endothelial cells to promote follicular atresia in the ovary. Angiogenesis is likely to involve a balance between pro- and antiangiogenic factors. Our results suggest that loss of VEGF activity does not regulate TSP-1 expression directly but may influence TSP-1 activity via down-regulation of the CD36 receptor.
CCAAT/Enhancer Binding Protein β, But Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis
Authors: Kuhl Adam J. , Ross Susan M. , Gaido Kevin W.
Published: September 2007

Prolonged in utero exposure of fetal male rats to dibutyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples, among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes and, therefore, reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semiquantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including steroidogenic acute regulatory, scavenger receptor B-1, cytochrome P450 side chain cleavage, and cytochrome P450 17A1, that are down-regulated after an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by EMSA, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein β (c/ebp β) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp β in DBP treated animals, whereas SF-1 was reduced in both diethyl phthalate (nontoxic) and DBP (toxic) treatments. These results suggest that inhibition of c/ebp β, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. In addition, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.
Oxidative Stress in the Thyroid Gland: From Harmlessness to Hazard Depending on the Iodine Content
Authors: Poncin Sylvie , Gérard Anne-Catherine , Boucquey Marie , Senou Maximin , Calderon Pedro Buc , Knoops Bernard , Lengelé Benoît , Many Marie-Christine , Colin Ides M.
Published: September 2007

In basal conditions, thyroid epithelial cells produce moderate amounts of reactive oxygen species (ROS) that are physiologically required for thyroid hormone synthesis. They are not necessarily toxic because they are continuously detoxified either in the process of hormone synthesis or by endogenous antioxidant systems. Using a rat model of goiter formation and iodine-induced involution, we found that compared with control thyroids, the oxidative stress, assessed by the detection of 4-hydroxynonenal, was strongly enhanced both in hyperplastic and involuting glands. The level of antioxidant defenses (glutathione peroxidases and peroxiredoxins) was also up-regulated in both groups, although somewhat less in the latter. Of note, increased oxidative stress came along with an inflammatory reaction, but only in involuting glands, suggesting that although antioxidant systems can adequately buffer a heavy load of ROS in goiter, it is not necessarily the case in involuting glands. The effects of 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), an endogenous ligand of peroxisome proliferated-activated receptor γ (PPARγ) with antiinflammatory properties, were then investigated in involuting glands. This drug strongly reduced both 4-hydroxynonenal staining and the inflammatory reaction, indicating that it can block iodine-induced cytotoxicity. When experiments were carried out with the PPARγ antagonist, bisphenol A diglycidyl ether, 15dPGJ2-induced effects remained unchanged, suggesting that these effects were not mediated by PPARγ. In conclusion, thyroid epithelial cells are well adapted to endogenously produced ROS in basal and goitrous conditions. In iodine-induced goiter involution, the increased oxidative stress is accompanied by inflammation that can be blocked by 15dPGJ2 through PPARγ-independent protective effects.
NFATc1 Induces Osteoclast Fusion Via Up-Regulation of Atp6v0d2 and the Dendritic Cell-Specific Transmembrane Protein (DC-STAMP)
Authors: Kim Kabsun , Lee Seoung-Hoon , Ha Kim Jung , Choi Yongwon , Kim Nacksung
Published: September 2007

NFATc1 has been characterized as a master regulator of nuclear factor κB ligand-induced osteoclast differentiation. Herein, we demonstrate a novel role for NFATc1 as a positive regulator of nuclear factor κB ligand-mediated osteoclast fusion as well as other fusion-inducing factors such as TNF-α. Exogenous overexpression of a constitutively active form of NFATc1 in bone marrow-derived monocyte/macrophage cells (BMMs) induces formation of multinucleated osteoclasts as well as the expression of fusion-mediating molecules such as the d2 isoform of vacuolar ATPase Vo domain (Atp6v0d2) and the dendritic cell-specific transmembrane protein (DC-STAMP). Moreover, inactivation of NFATc1 by cyclosporin A treatment attenuates expression of Atp6v0d2 and DC-STAMP and subsequent fusion process of osteoclasts. We show that NFATc1 binds to the promoter regions of Atp6v0d2 and DC-STAMP in osteoclasts and directly induces their expression. Furthermore, overexpression of Atp6v0d2 and DC-STAMP rescues cell-cell fusion of preosteoclasts despite reduced NFATc1 activity. Our data indicate for the first time that the NFATc1/Atp6v0d2 and DC-STAMP signaling axis plays a key role in the osteoclast multinucleation process, which is essential for efficient bone resorption.
Descriptions of reversed yielding in bending
Authors: Rees D W A
Published: September 2007

Existence of Bauschinger effect in bending-unbending of copper beams has been shown from experiment. In modelling of the Bauschinger effect, it is shown that a significant second plastic penetration can occur with the release of the moment required for an elasticplastic bending of a beam. The theory is given for both linear and parabolic hardening material models. The elastic and plastic strains are developed from each hardening model to express the beam curvature of the unstressed neutral axis. Conditions are expressed, using the normalized stress—strain response of a rectangular beam section, for which the release is purely elastic and elastic—plastic. Under the latter the depth to which a second zone of plasticity penetrates is given. Two stress distributions: one for applying the moment and the other for its release, are sufficient to derive the residual stress. Residuals found for parabolic hardening are believed to be more realistic than those from simpler linear or perfectly plastic models, particularly, where a second penetration is evident.
Dynamic analysis of a cabinet of a reactor protection system
Authors: Lee B Y , Kim W J , Shon J Y , Shin H M , Kwon S , Kim S H , Lee S B
Published: September 2007

The responses of a cabinet for a reactor protection system under seismic loadings are analysed by using the finite-element method, and its dynamic characteristics are evaluated. Analysed modes are compared with the measured data from a resonance search test. The structural safety of the cabinet is evaluated considering the required response spectrums of the operation-base and safe-shutdown earthquakes. The transient response of the cabinet is analysed by utilizing the measured acceleration of the vibration table representing the seismic motion of the ground. Time histories of accelerations at the locations of major internal parts are extracted. The transient responses are compared with those from the seismic test and the results were found to closely represent the system.
Static analysis of cross-ply laminated plate with integrated surface piezoelectric layers
Authors: Alibeigloo A , Shakeri M
Published: September 2007

Analytical study of the piezoelastic behaviour of a laminated rectangular plate with piezoelectric layers and finite length is presented. The solution is obtained applying Fourier series expansion to the state-space equation. The plate is assumed to be simply supported at four edges. The stress, displacement, and electric potential distributions are obtained by solving these state equations. Both the direct and the inverse piezoelectric effects are investigated and the influence of pizieoelectric layers on the mechanical behaviour of plate is studied. The method is validated by comparing numerical results of the plate without piezoelectric layers with that available in the literature.
Sliding mode synchronous control for fixture clamps system driven by hydraulic servo systems
Authors: Dachang Z H U
Published: September 2007

Although the two sides clips of a fixture clamps system have the same driving mechanisms, the synchronous error between the dual drivers of the two clips is generated by non-balanced forces. With position variation of clips and various uncertainty disturbances during the working process, the synchronous movement of the two clips is difficult. In the current paper, sliding mode synchronous controller is designed for fixture clamps system which is driven by hydraulic servo system. Setting the load dynamic error of one driver as external disturbance to the other driver, the departure from synchronization caused by the parameter variation and external forces between two clips is limited. The stability of the control system and the convergence of synchronous tracking errors are guaranteed by Lyapunov stability theory. Simulations illustrate the applicability of the proposed approach.
Identification of material damage in metal sheets
Authors: Omerspahic E
Published: September 2007

Two material models are examined to identify damage propagation in two selected sheet materials: a high-strength steel alloy, ZSte340, and an aluminium alloy, AA5182. The method of continuous uniaxial tension loading and unloading of sheet specimens was adopted for identification of the isotropic material damage parameters. Two damage evolution trends were identified, one linear and the other bilinear. Inverse modelling technique was applied for identification of material parameters. A representative cost function, based on a correlation between the experimental and simulation results obtained, was minimized. An optimization code, relying on response surface methodology, was used to identify the material parameters. Good agreement between the experimental and numerical results from structural simulations of the experiment was obtained up to the onset of diffuse necking. For materials that fracture before a localized necking zone has developed, the proposed methodology enables identification of the critical damage at failure.
Performance of a self-lifting linear air contact
Authors: Stolarski T A , Woolliscroft S P
Published: September 2007

An investigation into the performance of a self-levitating linear air bearing that functions on the squeeze film principle was conducted and a detailed set of results describing its floating characteristics at various operating parameters is presented. Experimentally assessed performance of the bearing was compared with performance predicted by a computer model of the bearing.
Effect of compressive force on the performance of a proton exchange membrane fuel cell
Authors: Ous T , Arcoumanis C
Published: September 2007

The effect of the compressive force on the performance of a proton exchange membrane fuel cell has been examined experimentally. The performance has been evaluated on two polarization regions of the cell: ohmic and mass transport. Cell voltage and current density as a function of pressure were measured under constant load and various inlet air humidity conditions. The pressure distribution on the surface of the gas diffusion layer was measured using a pressure detection film and the results show that increasing the pressure improves the performance of the cell. The improvement of the cell voltage in the ohmic region was found to be greater than that in the mass transport region, whereas for the cell current density, the mass transport region exhibited higher change. The increase in the cell specific power in the ohmic and mass transport regions, as pressure increases from 0 to 2MNm-2, is estimated to be 9 and 18mWcm−2, respectively. However, the fuel cell performance in these two regions declined dramatically when excessive pressure (≥5 MNm−2) was applied. The mass transport region proved to be more susceptible to this sharp decline under excessive pressure than the ohmic region.
Design and implementation of integrated non-uniform rational B-spline and digital differential analyser interpolators for computerized numerical control servocontrollers
Authors: Chen C-Y , Liao P-S , Cheng C-C , Jong G-F
Published: September 2007

The purpose of this article is to propose the design and implement an efficient and open real-time non-uniform rational B-spline (NURBS) interpolator for computerized numerical control (CNC) motion control. The proposed architecture of real-time NURBS interpolator integrates the advantages of digital differential analyser (DDA) and the native NURBS interpolator with the help of the data conversion module. First, the desired compensation positions are generated by NURBS interpolator concerning feedrate and curvature for manufacturing accuracy. A data conversion module is then applied to transform the position commands from float to integer. Using this integral-type position command, the pulse trains can be generated by DDA for the AC servomechanism in position mode. The simulation and experimental results validate that this novel real-time NURBS controller is effective and efficient enough to drive the general servomechanism without changing the control scheme of the traditional AC servomotor.
Study on overlapping of two sequential scans in laser forming
Authors: Shen H , Zhou J , Yao Z Q
Published: September 2007

Laser forming can utilize localized heating for the forming of high strength alloys and use combinations of straightandcurved lines to produce three-dimensional formedcomponents, which are of significant value to industries of aerospace, shipbuilding, and microelectronics. A three-dimensional non-linear, indirectly coupled thermal—structural model for the overlapping ratio of two sequential laser beams scanning is established, accounting for temperature dependency of material properties. Numerical results are verified by experimental data. Numerical investigations are carried out to examine the difference in plastic strain distributions under different overlapping ratios. The results show that the bending angle is larger when the size of plastic zone on the top surface is wider, although the plastic strain is smaller.
Ultrasensitive Flow-based Immunoassays Using Single-Molecule Counting
Authors: Todd John , Freese Bob , Lu Ann , Held Douglas , Morey Jennifer , Livingston Richard , Goix Philippe
Published: September 2007

Background: Immunoassay (IA) technology has expanded the clinical utility of protein biomarkers, but demands for increased sensitivity, dynamic reporting ranges, and small sample volumes have limited the potential clinical usefulness of many biomarkers. We assessed the performance, including limits of detection (LODs) and the dynamic reporting range, of an IA-based technology, Erenna Immunoassay System, for a series of biomarkers, including cardiac troponin I (cTnI).

Methods: Erenna IAs were used with 10 different and clinically important biomarkers to ascertain the LOD with various sample sizes (10 μL to 200 μL).

Results: The Erenna Immunoassay System generated LODs of 10–100 pg/L using 100 μL of sample. For cTnI, the LOD was 0.2 ng/L and a 10% CV was seen between 0.78 and 1.6 ng/L.

Conclusions: The Erenna IA-based technology reproducibly measures protein biomarkers with detection limits of 10–100 pg/L, with a dynamic range of >4.5 logs in sample volumes of 50–150 μL.
Assessment of Liquid Microbead Arrays for the Screening of Newborns for Spinal Muscular Atrophy
Authors: Pyatt Robert E , Mihal David C , Prior Thomas W
Published: September 2007

Background: Spinal muscular atrophy is a common neurodegenerative disorder that has recently been considered for inclusion in the next generation of newborn screening regimens. We sought to validate liquid microbead arrays for the identification of affected individuals by direct DNA analysis.

Methods: Assays were created to detect the homozygous deletions in exon 7 of the SMN1 gene found in approximately 95% of affected individuals by use of 2 different microbead chemistries on the Luminex 200: MultiCode-PLx and Tag-It. A series of 367 blood spots including 164 from affected individuals, 46 from known carriers, and 157 from unaffected individuals were then analyzed with each assay.

Results: The MultiCode-PLx assay required 4.2 h to perform and provided correct identification of all 164 samples from affected individuals. Correct exclusion was also made for all 46 carrier and 157 unaffected individual samples. The Tag-It assay required 6.8 h, detected all samples from affected individuals, and excluded all but 1 (99.5%) of the samples from carriers and unaffected individuals. Neither method was sensitive to increasing copy numbers of the SMN2 gene.

Conclusions: Both methods showed high sensitivity and specificity for the detection of patients with spinal muscular atrophy. For both methods, ample DNA was extracted from all blood spots for analysis, and SMN2 copy numbers did not interfere. Liquid bead arrays represent a robust method for DNA analysis in newborn screening laboratories.
Scanning the Cystic Fibrosis Transmembrane Conductance Regulator Gene Using High-Resolution DNA Melting Analysis
Authors: Montgomery Jesse , Wittwer Carl T , Kent Jana O , Zhou Luming
Published: September 2007

Background: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping.

Methods: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions. Common variants in 96 blood donors were identified in each exon by high-resolution melting on a LightScanner®. We then performed a subsequent blinded study on 30 samples enriched for disease-causing variants, including all 23 variants recommended by the American College of Medical Genetics and 8 additional, well-characterized variants.

Results: We identified 22 different sequence variants in 96 blood donors, including 4 novel variants and the disease-causing p.F508del. In the blinded study, all 40 disease-causing heterozygotes (29 unique) were detected, including 1 new probable disease-causing variant (c.3500-2A>T). The number of false-positive amplicons was decreased 96% by considering the 6 most common heterozygotes. The melting patterns of most heterozygotes were unique (37 of 40 pairs within the same amplicon), the exceptions being p.F508del vs p.I507del, p.G551D vs p.R553X, and p.W1282X vs c.4002A>G. The homozygotes p.G542X, c.2789 + 5G>A, and c.3849 + 10kbC>T were directly identified, but homozygous p.F508del was not. Specific genotyping of these exceptions, as well as genotyping of the 5T allele of intron 8, was achieved by unlabeled-probe and small-amplicon melting assays.

Conclusions: High-resolution DNA melting methods provide a rapid and accurate alternative for complete CFTR analysis. False positives can be decreased by considering the melting profiles of common variants.
The Feasibility of Using a Risk-informed Approach for Calculating Reactor Pressure Vessel Heatup and Cooldown Operating Curves
Authors: Gamble Ron , Server William , Carter Robert
Published: September 2007

The current procedures for calculating pressure-temperature (P/T) limits for normal reactor startup and shutdown are defined by deterministic fracture mechanics methodology in the ASME Code, Appendix G (in both Section XI and Section III). The recent pressurized thermal shock (PTS) re-evaluation effort used a very thorough probabilistic fracture mechanics (PFM) evaluation to develop a technical basis to increase the PTS screening criteria. The feasibility of applying this same PFM methodology to evaluate normal startup and shutdown operation for both pressurized water reactor (PWR) and boiling water reactor (BWR) pressure vessels is described in this paper. The approach taken in this study was to define a new risk-informed margin term to be applied to the stress intensity factor for membrane tension (KIm) in Appendix G. The margin on KIm was determined by finding the value that results in a vessel failure frequency equal to 10−6 failures/reactor-year when the reactor operates up to the pressure-temperature limits calculated using the risk-informed margin. This simple approach was selected because it results in a minimum change to the current ASME Code procedure, is easy to apply when revising plant operating P/T limits, and allows for an increase in the allowable P/T limits by as much as would be provided by any alternate procedure. The results from this initial study indicate the margin on KIm in Appendix G can be reduced from 2 to 1.5 for PWR vessels for shutdown and potentially reduced from 2 to as low as 1 for startup. The results indicate the margin on KIm in Appendix G can be reduced from 2 to approximately 1 for BWR vessels for shutdown and potentially startup. Additional analyses for PWR and BWR vessels will be needed to develop a comprehensive risk-informed basis for any revisions to the ASME Code, Appendix G.
Qualitative Sampling of the Building Envelope for Water Leakage
Authors: Haughton Lonnie L. , Murphy Colin R.
Published: September 2007

Industry standards for intrusive water leakage evaluations of the building envelope are promulgated by ASTM E 2128, “Standard Guide for Evaluating Water Leakage of Building Walls,” which lays out a purposeful step-by-step methodology by which information is accumulated and succeeding sampling locations are identified by a skilled professional; however, some laypersons have criticized building envelope survey findings that are not derived statistically from random sampling. This paper notes that there are relatively few building envelope investigations for which statistical random sampling, in and of itself, is a legitimate or practical methodology for achieving a comprehensive understanding of the sources and mechanisms of water leakage and, therefore, the use of quantitative (i.e., statistical) survey protocols to evaluate the validity of purposeful qualitative sampling of the building envelope is not appropriate. Further, this paper demonstrates that a building envelope evaluation that has been carried out in conformance with ASTM E 2128 satisfies current rules of evidence that require an expert’s sampling methodology and analysis to be based upon “scientifically valid” principles. In addition, this paper identifies fundamental assumptions that guide the initial steps of most water leakage evaluations, proposes basic categories for prioritizing the sampling, and discusses the potentially complementary roles of limited surveys by plaintiff and defense experts.
Façade Renovation of the Wexner Center for the Arts
Authors: Li Shawn S. , McClelland Neil
Published: September 2007

The Wexner Center for the Arts went through a major façade renovation to fix the water leakage and condensation problems. The work included replacing glazed skylights, curtain walls, and roofs. This paper describes how renovation work has been done with modern technologies to study thermal and lighting performance on the new façade. In addition to the computational analysis, a full scale mock-up was constructed in a laboratory for air, water, and structural test, and to evaluate the condensation resistance performance. Currently there are no industry standards for condensation evaluation for a mock-up of this size. The mock-up test method refers to ASTM C 1199, “Standard Test Method for Measuring the Steady-State Thermal Transmittance of Fenestration Systems Using Hot Box Methods,” and AAMA 1053 test procedures but with modifications to suit the project. The new façade of the Wexner Center is a perfect example of how façade design can be done with modern analyses tools and a coordinated design approach to deliver an environmentally efficient cladding system. The paper also describes the issues for the current test standards to the projects that have specific design conditions.
The role of nitric oxide on matrix metalloproteinase 2 (MMP2) and MMP9 in placenta and fetus from diabetic rats
Authors: Pustovrh M C , Jawerbaum A , White V , Capobianco E , Higa R , Martínez N , López-Costa J J , González E
Published: September 2007

Matrix metalloproteinases (MMPs) play an important role in tissue remodeling that accompanies the rapid growth, differentiation, and structural changes of the placenta and several fetal organs. In the present study, we investigated whether the diabetic maternal environment may alter the regulatory homeostasis exerted by nitric oxide (NO) on MMPs activity in the feto-placental unit from rats at midgestation. We found that NADPH-diaphorase activity, which reflects the distribution and activity of NO synthases (NOS), was increased in both placenta and fetuses from diabetic rats when compared with controls. In addition, while a NO donor enhanced MMP2 and MMP9 activities, a NOS inhibitor reduced these activities in the maternal side of the placenta from control rats. This regulatory effect of NO was only observed on MMP9 in the diabetic group. On the other hand, the NO donor did not modify MMP2 and MMP9 activities, while the NOS inhibitor reduced MMP9 activity in the fetal side of both control and diabetic placentas. In the fetuses, MMP2 was enhanced by the NO donor and reduced by the NO inhibitor in both fetuses from control and diabetic rats. Overall, this study demonstrates that NO is able to modulate the activation of MMPs in the feto-placental unit, and provides supportive evidence that increased NOS activity leads to NO overproduction in the feto-placental unit from diabetic rats, an alteration closely related to the observed MMPs dysregulation that may have profound implications in the formation and function of the placenta and the fetal organs.
Evaluation in mammalian oocytes of gene transcripts linked to epigenetic reprogramming
Authors: Oliveri Roberto S , Kalisz Mark , Schjerling Charlotte Karlskov , Andersen Claus Yding , Borup Rehannah , Byskov Anne Grete
Published: September 2007

The mature mammalian metaphase II (MII) oocyte has a unique ability to reprogram sperm chromatin and support early embryonic development. This feature even extends to the epigenetic reprogramming of a terminally differentiated cell nucleus as observed in connection with somatic cell nuclear transfer. Epigenetic nuclear reprogramming is highly linked to chromatin structure and includes covalent modifications of DNA and core histone proteins as well as reorganization of higher-order chromatin structure. A group of conserved enzymes mediating DNA methylation, methyl-CpG-binding protein (MeCP), histone acetylation and methylation, and chromatin remodeling are extensively involved in epigenetic reprogramming in mammalian cells. Using the oligonucleotide microarray technique, the present study compared the expression levels of 86 genes associated with epigenetic reprogramming in murine in vivo matured MII oocytes with that of germinal vesicle oocytes. Correlation between biological replicates was high. A total of 57 genes with potential reprogramming effect were detected. In MII oocytes, four genes were significant up-regulated, whereas 18 were down-regulated and 35 unchanged. The significantly regulated genes were validated by real-time quantitative RT-PCR. For example, MII oocytes showed a significant down-regulation of oocyte-specific maintenance DNA methyltransferase, Dnmt1o, and up-regulation of MeCP transcript, methyl-CpG binding domain protein 2. Furthermore, histone acetyltransferases were proportionally overrepresented when compared with histone deacetylases. These data elucidate for the first time some of the mechanisms that the oocyte may employ to reprogram a foreign genome either in form of a spermatozoa or a somatic nucleus and may thus be of importance for advancing the fields of stem cell research and regenerative medicine.
Nutritional manipulation between early to mid-gestation: effects on uncoupling protein-2, glucocorticoid sensitivity, IGF-I receptor and cell proliferation but not apoptosis in the ovine placenta
Authors: Gnanalingham M G , Williams P , Wilson V , Bispham J , Hyatt M A , Pellicano A , Budge H , Stephenson T , Symonds M E
Published: September 2007

In sheep, modest maternal nutrient restriction (NR) over the period of rapid placental growth restricts placentome growth and results in offspring in which glucocorticoid action is enhanced. Therefore, this study investigated the placental effects of early to mid-gestational NR on glucocorticoid receptor (GR), 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), uncoupling protein-2 (UCP2), and IGF type-I receptor (IGF-IR) mRNA abundance together with cell proliferation and apoptosis as determined histologically, and the mitochondrial proteins voltage-dependent anion channel and cytochrome c that are involved in apoptosis. Placenta was sampled at 80 and 140 days gestation (dGA; term ~147 dGA). NR was imposed between 28 and 80 days gestation when control and nutrient-restricted groups consumed 150 or 60% respectively of their total metabolizable energy requirements. All mothers were then fed to requirements up to term. Total fetal placentome weights were decreased by NR at 80 dGA but were heavier at 140 dGA following 60 days of nutritional rehabilitation. GR and UCP2 mRNA abundance increased whilst 11βHSD2 mRNA decreased with gestational age. NR persistently up-regulated GR and UCP2 mRNA abundance. 11βHSD2 mRNA was reduced by NR at 80 dGA but increased near to term. IGF-IRmRNA abundance was only decreased at 80 dGA. Placental apoptosis and mitochondrial protein abundance were unaffected by NR, whereas cell proliferation was markedly reduced. In conclusion, placental UCP2 and local glucocorticoid action are affected by the gestational nutritional status and may result in the offspring showing enhanced glucocorticoid sensitivity, thereby predisposing them to disease in later life.
Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1
Authors: Susor Andrej , Ellederova Zdenka , Jelinkova Lucie , Halada Petr , Kavan Daniel , Kubelka Michal , Kovarova Hana
Published: September 2007

In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I–anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin–dependent kinase(CDK1)–cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I–anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.
Testicular and ovarian gonocytes from 20-day incubated chicken embryos contribute to germline lineage after transfer into bloodstream of recipient embryos
Authors: Naito Mitsuru , Minematsu Takeo , Harumi Takashi , Kuwana Takashi
Published: September 2007

The present study was conducted to elucidate whether testicular and ovarian gonocytes obtained from 20-day incubated chicken embryos (stage 45) have the ability to migrate to the germinal ridges and contribute to germline lineage after transfer into the bloodstream of recipient embryos. Testicular and ovarian gonocytes were first identified as relatively large cells in a population of gonadal cells. The proportions of testicular and ovarian gonocytes in the total gonadal cells were 0.94 and 0.75% respectively, recognised as chicken vasa homologue-positive cells. Then, the dissociated gonadal cells obtained from 20-day incubated embryos containing testicular or ovarian gonocytes, with or without transfection, were transferred into recipient embryos. Expression of the introduced GFP gene was observed in the gonads of 6.5-day cultured recipient embryos (stage 30) in males and females, suggesting that the transferred testicular and ovarian gonocytes have the ability to migrate to the germinal ridges and enter the gonads. Furthermore, the presence of the donor-derived DNA was detected in the gonads of 20-day cultured recipient embryos in males and females, and also in the sperm samples obtained from the hatched male putative chimaeric chickens, suggesting that the transferred testicular and ovarian gonocytes were incorporated into the germline of chimaeric embryos and chickens. It is concluded that testicular and ovarian gonocytes obtained from 20-day incubated embryos have the ability to migrate to the germinal ridges after transfer into the bloodstream of recipient embryos, enter the gonads and contribute to the germline lineage of chimaeric embryos and chickens.
The role of Zn-α2 glycoprotein in sperm motility is mediated by changes in cyclic AMP
Authors: Qu Fei , Ying Xiaoqian , Guo Wei , Guo Qiangsu , Chen Guowu , Liu Yue , Ding Zhide
Published: September 2007

Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-α2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the β3-adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90- or 120-min incubation (P<0.05 and P<0.01 respectively), together with the decreasing of intracellular cAMP and PKA levels. In conclusion, these data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway.
Bedraggled, a Putative Transporter, Influences the Tissue Polarity Complex During the R3/R4 Fate Decision in the Drosophila Eye
Authors: Rawls Amy S , Schultz Sarah A , Mitra Robi D , Wolff Tanya
Published: September 2007

The tissue polarity pathway is required for the establishment of epithelial polarity in a variety of vertebrate and invertebrate organs. Core tissue polarity proteins act in a dynamically regulated complex to direct the polarization of the Drosophila eye. We report the identification and characterization of bedraggled (bdg), a novel gene that regulates one output of the tissue polarity pathway—the establishment of the R3/R4 photoreceptor fates. bdg encodes a novel, putative transporter protein and interacts genetically with all of the core polarity genes to influence the specification of the R3 and R4 cell fates. Finally, bdg is required for both viability and the initial stages of imaginal disc development.
The Dynamics of therooTransposable Element In Mutation-Accumulation Lines and Segregating Populations ofDrosophila melanogaster
Authors: Papaceit Montserrat , Ávila Victoria , Aguadé Montserrat , García-Dorado Aurora
Published: September 2007

We estimated the number of copies for the long terminal repeat (LTR) retrotransposable element roo in a set of long-standing Drosophila melanogaster mutation-accumulation full-sib lines and in two large laboratory populations maintained with effective population size ∼500, all of them derived from the same isogenic origin. Estimates were based on real-time quantitative PCR and in situ hybridization. Considering previous estimates of roo copy numbers obtained at earlier stages of the experiment, the results imply a strong acceleration of the insertion rate in the accumulation lines. The detected acceleration is consistent with a model where only one (maybe a few) of the ∼70 roo copies in the ancestral isogenic genome was active and each active copy caused new insertions with a relatively high rate (∼10−2), with new inserts being active copies themselves. In the two laboratory populations, however, a stabilized copy number or no accelerated insertion was found. Our estimate of the average deleterious viability effects per accumulated insert [E(s) < 0.003] is too small to account for the latter finding, and we discuss the mechanisms that could contain copy number.