bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythropoietin improves skeletal muscle microcirculation and tissue bioenergetics in a mouse sepsis model
Authors: Kao Raymond , Xenocostas Anargyros , Rui Tao , Yu Pei , Huang Weixiong , Rose James , Martin Claudio M
Published: May 2007

Introduction

The relationship between oxygen delivery and consumption in sepsis is impaired, suggesting a microcirculatory perfusion defect. Recombinant human erythropoietin (rHuEPO) regulates erythropoiesis and also exerts complex actions promoting the maintenance of homeostasis of the organism under stress. The objective of this study was to test the hypothesis that rHuEPO could improve skeletal muscle capillary perfusion and tissue oxygenation in sepsis.

Methods

Septic mice in three experiments received rHu-EPO 400 U/kg subcutaneously 18 hours after cecal ligation and perforation (CLP). The first experiment measured the acute effects of rHuEPO on hemodynamics, blood counts, and arterial lactate level. The next two sets of experiments used intravital microscopy to observe capillary perfusion and nicotinamide adenine dinucleotide (NADH) fluorescence post-CLP after treatment with rHuEPO every 10 minutes for 40 minutes and at 6 hours. Perfused capillary density during a three-minute observation period and NADH fluorescence were measured.

Results

rHuEPO did not have any effects on blood pressure, lactate level, or blood cell numbers. CLP mice demonstrated a 22% decrease in perfused capillary density compared to the sham group (28.5 versus 36.6 capillaries per millimeter; p < 0.001). Treatment of CLP mice with rHuEPO resulted in an immediate and significant increase in perfused capillaries in the CLP group at all time points compared to baseline from 28.5 to 33.6 capillaries per millimeter at 40 minutes; p < 0.001. A significant increase in baseline NADH, suggesting tissue hypoxia, was noted in the CLP mice compared to the sham group (48.3 versus 43.9 fluorescence units [FU]; p = 0.03) and improved with rHuEPO from 48.3 to 44.4 FU at 40 minutes (p = 0.02). Six hours after treatment with rHuEPO, CLP mice demonstrated a higher mean perfused capillary density (39.4 versus 31.7 capillaries per millimeter; p < 0.001) and a lower mean NADH fluorescence as compared to CLP+normal saline mice (49.4 versus 52.7 FU; p = 0.03).

Conclusion

rHuEPO produced an immediate increase in capillary perfusion and decrease in NADH fluorescence in skeletal muscle. Thus, it appears that rHuEPO improves tissue bioenergetics, which is sustained for at least six hours in this murine sepsis model.
Role of ADAM17 in the ectodomain shedding of TNF-α and its receptors by neutrophils and macrophages
Authors: Bell Jessica H , Herrera Amy H , Li Ying , Walcheck Bruce
Published: May 2007

TNF-α and its receptors TNFRI and TNFRII are cleaved from the surface of leukocytes by a proteolytic process referred to as ectodomain shedding. The role of a disintegrin and metalloproteinase 17 (ADAM17) in this process by the major professional phagocytes neutrophils and macrophages, the primary producers of TNF-α during inflammation induction, is based entirely on indirect evidence, and other sheddases have been implicated as well. As Adam17 gene-targeting in mice is lethal, we assessed the protease’s relative contribution to TNF-α, TNFRI, and TNFRII shedding using radiation chimeric mice with leukocytes lacking functional ADAM17. We report ablated, soluble TNF-α, TNFRI, and TNFRII production by neutrophils and macrophages stimulated with various microbial antigens and greatly reduced TNF-α levels in vivo following inflammation induction. This is the first simultaneous analysis of TNF-α, TNFRI, and TNFRII shedding by neutrophils and macrophages and the first direct evidence that ADAM17 is a primary and nonredundant sheddase.
Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin α4β1
Authors: Parmo-Cabañas Marisa , García-Bernal David , García-Verdugo Rosa , Kremer Leonor , Márquez Gabriel , Teixidó Joaquin
Published: May 2007

The α4β1 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that α4β1 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4+CD8+ and CD4+CD8− adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9+/α4β1+ human T cell line Molt-4. To study the role on CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP-interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM), we knocked down their expression and tested transfectant attachment to α4β1 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial α4β1-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by α4β1 could contribute to control their trafficking in the thymus during maturation, and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside-out signaling, leading to stimulated adhesion.
Sex Difference in Link between Interleukin-6 and Stress
Authors: Jankord Ryan , Turk James R. , Schadt James C. , Casati Jennifer , Ganjam Venkataseshu K. , Price Elmer M. , Keisler Duane H. , Laughlin M. Harold
Published: May 2007

Inflammation contributes to disease development, and the neuroimmunoendocrine interface is a potential site of action for inflammatory products like IL-6 to affect health. Although plasma IL-6 can stimulate the activity of the hypothalamo-pituitary-adrenocortical (HPA) axis, the precise role, if any, for IL-6 in the HPA response to nonimmunological stressors is unclear. The purpose of this study was to test the hypothesis that IL-6 in the stalk median eminence (SME) can be directly involved in stimulating ACTH secretion in response to acute stress in female swine. This study was undertaken as a result of finding IL-6 localized to the external zone of the SME next to the hypophyseal portal vessels. Results indicate that content of IL-6 in the SME decreases in response to acute stress along with an increase in nuclear phosphorylated signal transducer and activator of transcription-3 (pSTAT-3) in pituitary corticotrophs and a simultaneous increase in plasma concentrations of IL-6 and ACTH. Furthermore, we show that females concomitantly display greater SME content of IL-6 and greater HPA responsiveness to stress, thereby suggesting that IL-6 release from the SME is an integral factor contributing to enhanced stress responsiveness in females. Our results provide evidence for a direct link between IL-6 and ACTH release and reveal a sex difference in this relationship.
Steroid-Mediated Regulation of the Epithelial Sodium Channel Subunits in Mammary Epithelial Cells
Authors: Boyd Cary , Náray-Fejes-Tóth Anikó
Published: May 2007

The epithelial sodium channel (ENaC) is a key mediator of sodium transport in epithelia; however, little is known about ENaC expression in mammary epithelia. Using real-time PCR, we demonstrated the expression of the ENaC subunit mRNAs in mouse and human mammary cell lines and in vivo mouse mammary tissue. We determined the effects of glucocorticoids, progesterone, and prolactin on ENaC expression in four mammary cell lines. Dexamethasone induced all detectable ENaC subunits in noncancerous cell lines, HC11 and MCF10A. Interestingly, in cancerous cell lines (T-47D and MCF-7), both β- and γ- but not αENaC mRNAs were induced by dexamethasone. Progesterone induced ENaC mRNA only in T-47D cells, and prolactin had no effects. γENaC was rapidly induced by steroids, whereas induction of α- and βENaC was slower; moreover, the induction of the β-subunit required de novo protein synthesis. Dexamethasone treatment did not affect ENaC mRNA stability. Western blot analysis revealed immunoreactive bands corresponding to different forms of α-, β-, and γENaC; dexamethasone significantly increased the intensity of αENaC (85 kDa) and βENaC (90 kDa). We also showed an in vivo reduction in αENaC levels in the mammary tissue of lactating mice as compared with controls, whereas β- and γENaC mRNA levels were significantly increased. Furthermore, dexamethasone in vivo significantly increased α-, β-, and γENaC mRNA expression. Our data indicate that both mouse and human mammary cells express all ENaC subunits, and they are regulated by steroid hormones in a temporal and cell-specific manner both in culture and in vivo.
Gene Expression from the Imprinted Dio3 Locus Is Associated with Cell Proliferation of Cultured Brown Adipocytes
Authors: Hernandez Arturo , Garcia Bibian , Obregon Maria-Jesus
Published: May 2007

Active thyroid hormones are critical for the differentiation and function of brown adipose tissue. However, we have observed high basal and induced levels of type 3 deiodinase (D3), an enzyme that inactivates thyroid hormones and is coded by the imprinted gene Dio3, in differentiating brown preadipocytes in primary culture. We find that D3 activity and mRNA expression strongly correlate with the rate of proliferation of undifferentiated precursor cells under various conditions. Furthermore, differentiation of precursor cells to adipocytes is associated with decreased levels of D3 expression, and only very low levels of D3 mRNA are found in mature adipocytes. Dlk1, an inhibitor of adipocyte differentiation and a paternally expressed gene located in the same imprinted domain as Dio3, displayed changes in expression that parallel those of Dio3. In contrast, a 4-kb transcript for Dio3os, an antisense gene also located in the same imprinted domain, is markedly up-regulated in differentiated adipocytes. We conclude that D3 expression in differentiating preadipocytes is primarily linked to proliferating cells, whereas Dio3os expression is associated with mature adipocytes. Our results suggest that genomic imprinting and gene expression at the Dlk1/Dio3 imprinted domain may play a role in the regulation of adipocyte proliferation and differentiation.
Follicle-Stimulating Hormone Increases Tuberin Phosphorylation and Mammalian Target of Rapamycin Signaling through an Extracellular Signal-Regulated Kinase-Dependent Pathway in Rat Granulosa Cells
Authors: Kayampilly Pradeep P. , Menon K. M. J.
Published: May 2007

FSH-mediated regulation of mammalian target of rapamycin (mTOR) signaling in proliferating granulosa cells and the effect of dihydrotestosterone (DHT) on this pathway were examined. Inhibiting mTOR activation using rapamycin significantly reduced the FSH-mediated increase in cyclin D2 mRNA expression, suggesting that mTOR plays a role in the FSH-mediated increase in granulosa cell proliferation. FSH treatment of granulosa cells showed a 2-fold increase in phosphorylation of p70S6 kinase (p70S6K), the downstream target of mTOR. The increase in p70S6K phosphorylation by FSH treatment was abolished by prior exposure to DHT, suggesting that DHT inhibits FSH-mediated activation of mTOR signaling in cultured granulosa cells. The effect of FSH and DHT treatment on tuberin (TSC2), the upstream regulator of mTOR, was then examined. FSH treatment increased TSC2 phosphorylation, and pretreatment with DHT for 24 h reduced this stimulation. These results indicate that reduced p70S6K phosphorylation observed in DHT-treated cells might be the result of reduced TSC2 phosphorylation. Because Akt is the upstream activator of TSC2 phosphorylation, the effect of Akt inhibition was examined to test whether FSH-mediated TSC2 phosphorylation proceeds through an Akt-dependent pathway. Our results show that inhibiting Akt phosphorylation did not block FSH-stimulated TSC2 phosphorylation, whereas ERK inhibition reduced FSH-mediated stimulation. These results demonstrate the involvement of ERK rather than Akt in FSH-mediated TSC2 phosphorylation in granulosa cells. Based on these observations, we conclude that in granulosa cells, FSH uses a protein kinase A-/ERK-dependent pathway to stimulate TSC2 phosphorylation and mTOR signaling, and DHT treatment significantly reduces this response.
Corticosterone Inhibits the Lipid-Mobilizing Effects of Oleoyl-Estrone in Adrenalectomized Rats
Authors: del Mar Grasa Maria , Serrano Marta , Fernández-López José Antonio , Alemany Marià
Published: May 2007

Oleoyl-estrone (OE) is an adipose-derived signal that decreases energy intake and body lipid, maintaining energy expenditure and glycemic homeostasis. Glucocorticoids protect body lipid and the metabolic status quo. We studied the combined effects of OE and corticosterone in adrenalectomized female rats: daily OE gavages (0 or 10 nmol/g) and slow-release corticosterone pellets at four doses (0, 0.5, 1.7, and 4.8 mg/d). Intact and sham-operated controls were also included. After 8 d, body composition and plasma metabolites and hormones were measured. OE induced a massive lipid mobilization (in parallel with decreased food intake and maintained energy expenditure). Corticosterone increased fat deposition and inhibited the OE-elicited mobilization of body energy, even at the lowest dose. OE enhanced the corticosterone-induced rise in plasma triacylglycerols, and corticosterone blocked the OE-induced decrease in leptin. High corticosterone and OE increased insulin resistance beyond the effects of corticosterone alone. The presence of corticosterone dramatically affected OE effects, reversing its decrease of body energy (lipid) content, with little or no change on food intake or energy expenditure. The maintenance of glycemia and increasing insulin in parallel to the dose of corticosterone indicate a decrease in insulin sensitivity, which is enhanced by OE. The reversal of OE effects on lipid handling, insulin resistance, can be the consequence of a corticosterone-induced OE resistance. Nevertheless, OE effects on cholesterol were largely unaffected. In conclusion, corticosterone administration effectively blocked OE effects on body lipid and energy balance as well as insulin sensitivity and glycemia.
Distribution of Neuronal Apoptosis Inhibitory Protein in Human Tissues
Authors: Maier Johannes K. X. , Balabanian Sylvia , Coffill Cynthia R. , Stewart Alexandra , Pelletier Louise , Franks Douglas J. , Gendron Nathalie H. , MacKenzie Alex E.
Published: May 2007

The neuronal apoptosis inhibitory protein (NAIP) gene, also known as the baculovirus inhibitor of apoptosis repeat-containing protein 1 (BIRC1) gene, is a member of the inhibitors of apoptosis (IAP) family and was first characterized as a candidate gene for spinal muscular atrophy (SMA). The expression of NAIP has been thoroughly studied in the central nervous system and overlaps the pattern of neurodegeneration in SMA. Recent studies have pointed to a role for NAIP in non-neuronal cells. We report here the production of a specific anti-NAIP antibody and the profile of NAIP expression in human adult tissues by Western blot and immunohistochemical detection methods. NAIP was detected in a number of tissues by Western blot analysis, but immunohistochemistry revealed that NAIP's presence in certain tissues, such as liver, lung, and spleen, is most likely due to macrophage infiltration. In the small intestine, the expression of NAIP coincides with the expression of p21WAF1. This observation, coupled with findings from other groups, suggests a role for NAIP in increasing the survival of cells undergoing terminal differentiation as well as the possibility that the protein serves as an intestinal pathogen recognition protein. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.
Characterization of the Column and Autocellular Junctions That Define the Vasculature of Gill Lamellae
Authors: Kato Akira , Nakamura Korefumi , Kudo Hisayuki , Tran Yen Ha , Yamamoto Yoko , Doi Hiroyuki , Hirose Shigehisa
Published: May 2007

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the “column junction” and “autocellular junction” and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the “column junction.” In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.
A Comparative Analysis of Novel Fluorescent Proteins as Reporters for Gene Transfer Studies
Authors: Bell Peter , Vandenberghe Luk H. , Wu Di , Johnston Julie , Limberis Maria , Wilson James M.
Published: May 2007

We evaluated novel fluorescent proteins (FPs) as reporters for gene transfer in animals and cells with the aim to develop more-sensitive assays for vector development and the optimization of gene transfer strategies in gene therapy. Adeno-associated virus serotype 5 vectors carrying an expression cassette with a chicken β-actin promoter encoding the green FPs ZsGreen1, AcGFP1, hMGFP (with and without intron), and EGFP and the red FPs DsRed2 and TurboRFP were administered to mice at identical doses for each organ to target liver, lung, and muscle. Despite the fact that all FPs were expressed from an identical vector backbone, the observed number of fluorescent cells and fluorescence intensities varied between, but was consistent within, each combination of a specific protein and organ. The highest number of fluorescent cells was observed in liver with EGFP and in lung with ZsGreen1 and EGFP. In muscle, AcGFP1 and ZsGreen1 produced the most-intense fluorescence in fibers. In contrast, in culture cells, ZsGreen1 showed substantially stronger fluorescence than all other proteins. Our data demonstrate that each FP has tissue-specific expression profiles that need to be taken into consideration when comparing the performance of vectors in different organs.
Association of Cortactin and Fascin-1 Expression in Gastric Adenocarcinoma: Correlation With Clinicopathological Parameters
Authors: Tsai Wen-Chiuan , Jin Jong-Shiaw , Chang Wei-Kuo , Chan De-Chuan , Yeh Ming-Kung , Cherng Shiou-Chih , Lin Li-Fan , Sheu Lai-Fa , Chao You-Chen
Published: May 2007

Cortactin and fascin-1 are important factors in tumor progression. We tested the hypothesis that cortactin and fascin-1 expression correlates with clinicopathological parameters of gastric adenocarcinoma. Immunohistochemical analysis of cortactin and fascin-1 was done using tissue microarrays of 100 surgical specimens, including 20 well-differentiated, 20 moderately differentiated, and 60 poorly differentiated gastric adenocarcinomas. Among the 20 well-differentiated gastric adenocarcinomas, 15 cases (75%) showed negative or weak staining (1+); 5 cases (25%) had moderate (2+) or strong (3+) cortactin expression. Among the 60 poorly differentiated gastric adenocarcinomas, more than three-quarters of the cases (76.7%) had moderate or strong cortactin expression; 14 cases (23.3%) had weak staining. Of 20 well-differentiated gastric adenocarcinoma cases, 14 (70%) showed negative or weak staining of fascin-1, whereas nearly one-third (30%) had moderate or strong expression. Among the 60 poorly differentiated gastric adenocarcinomas, 32 (53.3%) exhibited moderate or strong fascin-1 expression; fewer than half of the cases showed negative or weak staining. Higher intensity of cortactin and fascin-1 staining correlated directly with more-advanced cancer stages (TNM) and inversely with survival rates. Our findings suggest the possibility that pharmacological inhibitors of cortactin and fascin-1 activity may slow down tumor progression and prolong survival time in patients with gastric adenocarcinomas.
Optimized Preservation of CNS Morphology for the Identification of Glycogen in the Pompe Mouse Model
Authors: Taksir Tatyana V. , Griffiths Denise , Johnson Jennifer , Ryan Susan , Shihabuddin Lamya S. , Thurberg Beth L.
Published: May 2007

Pompe disease (glycogenosis type II) is a rare lysosomal disorder caused by a mutational deficiency of acid α-glucosidase (GAA). This deficiency leads to glycogen accumulation in multiple tissues: heart, skeletal muscles, and the central nervous system. A knockout mouse model mimicking the human condition has been used for histological evaluation. Currently, the best method for preserving glycogen in Pompe samples uses epon–araldite resin. Although the preservation by this method is excellent, the size of the tissue is limited to 1 mm3. To accurately evaluate brain pathology in the Pompe mouse model, a modified glycol methacrylate (JB-4 Plus) method was developed. This approach allowed the production of larger tissue sections encompassing an entire mouse hemisphere (8 × 15 mm) while also providing a high level of morphological detail and preservation of glycogen. Application of the JB-4 Plus method is appropriate when a high level of cellular detail is desired. A modified paraffin method was also developed for use when rapid processing of multiple samples is a priority. Traditional paraffin processing results in glycogen loss. The modified paraffin method with periodic acid postfixation resulted in improved tissue morphology and glycogen preservation. Both techniques provide accurate anatomic evaluation of the glycogen distribution in Pompe mouse brain. (J Histochem Cytochem 55:991–998, 2007)
Estimation of Warfarin Maintenance Dose Based on VKORC1 (−1639 G>A) and CYP2C9 Genotypes
Authors: Zhu Yusheng , Shennan Michael , Reynolds Kristen K , Johnson Nancy A , Herrnberger Matthew R , Valdes Roland , Linder Mark W
Published: May 2007

Background: CYP2C9 polymorphisms are associated with decreased S-warfarin clearance and lower maintenance dosage. Decreased expression of VKORC1 resulting from the −1639G>A substitution has also been implicated in lower warfarin dose requirements. We investigated the additional contribution of this polymorphism to the variance in warfarin dose.

Methods: Sixty-five patients with stable anticoagulation were genotyped for CYP2C9 and VKORC1 with Tag-It™ allele-specific primer extension technology. Plasma S-warfarin concentrations and warfarin maintenance dose were compared among patients on the basis of the VKORC1 −1639G>A genotype.

Results: Eighty percent of CYP2C9*1/*1 patients stabilized on <4.0 mg/day warfarin had at least 1 VKORC1 −1639A allele. Mean warfarin doses (SD) were 6.7 (3.3), 4.3 (2.2), and 2.7 (1.2) mg/day for patients with the VKORC1 −1639GG, GA, and AA genotypes, respectively. Steady-state plasma concentrations of S-warfarin were lowest in patients with the VKORC1 −1639AA genotype and demonstrated a positive association with the VKORC1 −1639G allele copy number (trend P = 0.012). A model including VKORC1 and CYP2C9 genotypes, age, sex, and body weight accounted for 61% of the variance in warfarin daily maintenance dose.

Conclusions: The VKORC1 −1639A allele accounts for low dosage requirements of most patients without a CYP2C9 variant. Higher plasma S-warfarin concentrations corresponding to increased warfarin maintenance dosages support a hypothesis for increased expression of the VKORC1 −1639G allele. VKORC1 and CYP2C9 genotypes, age, sex, and body weight account for the majority of variance in warfarin dose among our study population.
Microtransponder-Based Multiplex Assay for Genotyping Cystic Fibrosis
Authors: Lin Xin , Flint James A , Azaro Marco , Coradetti Thomas , Kopacka Wesley M , Streck Deanna L , Wang Zhuying , Dermody James , Mandecki Wlodek
Published: May 2007

Background: We developed and evaluated a genotyping assay for detection of 50 cystic fibrosis (CF) mutations. The assay is based on small (500 μm) electronic chips, radio frequency (RF) microtransponders (MTPs). The chips are analyzed on a unique fluorescence and RF readout instrument.

Methods: We divided the CF assay into 4 panels: core, Hispanic, African-American, and Caucasian. We amplified 18 CF transmembrane regulator (CFTR) DNA fragments covering 50 mutations by use of multiplex PCR using 18 CFTR gene-specific primer pairs. PCR was followed by multiplex allele-specific primer extension (ASPE) reactions and hybridization to capture probes synthesized on MTPs. We used 100 ASPE primers and 100 capture probes. We performed fluorescence measurements of hybridized MTP kits and assay analysis using a custom automated bench-top flow instrument.

Results: We validated the system by performing the assay on 23 commercial DNA samples in an internal study and 32 DNA samples in an external study. For internal and external studies, correct calls were 98.8% and 95.7%, false-positive calls 1.1% and 3.9%, and false-negative calls 0.12% and 0.36%, respectively.

Conclusions: The MTP-based multiplex assay and analysis platform can be used for CF genotyping.
High Concentrations of Soluble P-Selectin Are Associated with Risk of Venous Thromboembolism and the P-Selectin Thr715 Variant
Authors: Ay Cihan , Jungbauer Lea V , Sailer Thomas , Tengler Theres , Koder Silvia , Kaider Alexandra , Panzer Simon , Quehenberger Peter , Pabinger Ingrid , Mannhalter Christine
Published: May 2007

Background: The cell adhesion molecule P-selectin has an important role in the pathophysiology of thrombosis. The effect on venous thromboembolism (VTE) of increased circulating concentrations of soluble P-selectin (sP-selectin) and their association with the P-selectin variant Thr715Pro is still uncertain.

Methods: This study was a case-control study of 116 patients with confirmed recurrent VTE and at least 1 event of unprovoked deep venous thrombosis or pulmonary embolism, and 129 age- and sex-matched healthy individuals. We measured sP-selectin by ELISA and P-selectin gene (SELP) variation by genotyping and sampled blood after a mean interval of 2.55 years after the most recent VTE event.

Results: The mean (SD) sP-selectin concentration was higher in patients than in controls: 47.3 (15.0) μg/L vs 36.8 (11.0) μg/L, P <0.001. The unadjusted odds ratio (OR) for sP-selectin >55.1 μg/L, representing the 95th percentile for controls, was 8.5 (95% CI, 3.7–23.3; P <0.001) and increased after adjustment for factor V Leiden, the prothrombin G20210A variant, increased factor VIII, and hyperhomocysteinemia (OR, 10.6; 95% CI, 4.1–31.2; P <0.001). Pro715 carriers were more prevalent among controls than patients (21.7% vs 14.7%). sP-selectin concentrations were lower in this subgroup than in noncarriers: 31.3 (7.9) μg/L vs 44.1 (14.1) μg/L; P <0.001).

Conclusions: Increased sP-selectin concentrations are associated with VTE and genotype status. sP-selectin concentrations are lower in individuals carrying the P-selectin Pro715 variant than in those without this variant.
Magnetic Control of an Electrochemical Microfluidic Device with an Arrayed Immunosensor for Simultaneous Multiple Immunoassays
Authors: Tang Dianping , Yuan Ruo , Chai Yaqin
Published: May 2007

Background: Methods based on magnetic bead probes have been developed for immunoassay, but most involve complicated labeling or stripping procedures and are unsuitable for routine use.

Methods: We synthesized magnet core/shell NiFe2O4/SiO2 nanoparticles and fabricated an electrochemical magnetic controlled microfluidic device for the detection of 4 tumor markers. The immunoassay system consisted of 5 working electrodes and an Ag/AgCl reference electrode integrated on a glass substrate. Each working electrode contained a different antibody immobilized on the NiFe2O4/SiO2 nanoparticle surface and was capable of measuring a specific tumor marker using noncompetitive electrochemical immunoassay.

Results: Under optimal conditions, the multiplex immunoassay enabled the simultaneous detection of 4 tumor markers. The sensor detection limit was <0.5 μg/L (or <0.5 kunits/L) for most analytes. Intra- and interassay imprecisions (CVs) were <4.5% and 8.7% for analyte concentrations >5 μg/L (or >5 kunits/L), respectively. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross-talk (CV) between neighboring sites was <10%.

Conclusion: This immunoassay system offers promise for label-free, rapid, simple, cost-effective analysis of biological samples. Importantly, the chip-based immunosensor could be suitable for use in the mass production of miniaturized lab-on-a-chip devices and open new opportunities for protein diagnostics and biosecurity.
High-Throughput Quantitative Profiling of Serum N-Glycome by MALDI-TOF Mass Spectrometry and N-Glycomic Fingerprint of Liver Fibrosis
Authors: Kam Richard KT , Poon Terence CW , Chan Henry LY , Wong Nathalie , Hui Alex Y , Sung Joseph JY
Published: May 2007

Background: The use of MALDI-TOF mass spectrometry (MS) in quantitative glycan profiling has not been reported. In this study, we attempted to establish a high-throughput quantitative assay for profiling serum N-glycome, and we applied the new assay to identifying serum N-glycans for diagnosis of liver fibrosis and cirrhosis.

Methods: N-glycans from whole serum proteins in 2 μL serum were released by enzymatic digestion, cleaned up by hydrophilic chromatography, and subsequently quantitatively profiled with a linear MALDI-TOF MS system, which was originally designed for quantitative proteomic profiling. Serum N-glycome profiles from 46 patients with chronic hepatitis B infection and with different degrees of liver fibrosis were examined.

Results: The intra- and interassay CVs of peak intensities of the standard N-glycans were <8% and <17%, respectively. When the assay was applied to the analysis of serum N-glycome profiles, 17 peaks were found to be potential biomarkers for detection of liver fibrosis/cirrhosis. Linear regression analysis revealed that 4 peaks of 1341.5, 1829.7, 1933.3, and 2130.3 m/z (all P <0.005) had complementary value in detecting liver fibrosis and included them, but not any serological markers, in the diagnostic model. Leave-one-out cross-validation showed the diagnostic model could identify significant fibrosis (Ishak score ≥3) and cirrhosis (Ishak score ≥5), both at 85% accuracy.

Conclusion: This is the first study to illustrate the quantitative aspect of MALDI-TOF MS in N-glycome profiling and the first study to reveal the potential value of the serum N-glycan profile for identifying liver fibrosis.
Multiplex Assay for Comprehensive Genotyping of Genes Involved in Drug Metabolism, Excretion, and Transport
Authors: Daly Thomas M , Dumaual Carmen M , Miao Xin , Farmen Mark W , Njau Reuben K , Fu Dong-Jing , Bauer Nancy L , Close Sandra , Watanabe Nancy , Bruckner Carsten , Hardenbol Paul , Hockett Richard D
Published: May 2007

Background: Drug metabolism is a multistep process by which the body disposes of xenobiotic agents such as therapeutic drugs. Genetic variation in the enzymes involved in this process can lead to variability in a patient’s response to medication.

Methods: We used molecular-inversion probe technology to develop a multiplex genotyping assay that can simultaneously test for 1227 genetic variants in 169 genes involved in drug metabolism, excretion, and transport. Within this larger set of variants, we performed analytical validation of a clinically defined core set of 165 variants in 27 genes to assess accuracy, imprecision, and dynamic range.

Results: In a test set of 91 samples, genotyping accuracy for the core set probes was 99.8% for called genotypes, with a 1.2% no-call (NC) rate. The majority of the core set probes (133 of 165) had ≤1 genotyping failure in the test set; a subset of 12 probes was responsible for the majority of failures (mainly NC). Genotyping results were reproducible upon repeat testing with overall within- and between-run variation of 1.1% and 1.4%, respectively—again, primarily NCs in a subset of probes. The assay showed stable genotyping results over a 6-fold range of input DNA.

Conclusions: This assay generates a comprehensive assessment of a patient’s metabolic genotype and is a tool that can provide a more thorough understanding of patient-to-patient variability in pharmacokinetic responses to drugs.
Rapid 2nd-Tier Test for Measurement of 3-OH-Propionic and Methylmalonic Acids on Dried Blood Spots: Reducing the False-Positive Rate for Propionylcarnitine during Expanded Newborn Screening by Liquid Chromatography–Tandem Mass Spectrometry
Authors: la Marca Giancarlo , Malvagia Sabrina , Pasquini Elisabetta , Innocenti Marzia , Donati Maria Alice , Zammarchi Enrico
Published: May 2007

Background: The expansion of newborn screening programs has increased the number of newborns diagnosed with inborn errors of metabolism in the presymptomatic phase, but it has also increased the number of costly, stress-producing false-positive results. Because propionylcarnitine (C3) is one of the analytes most frequently responsible for false-positive results, we aimed to develop a rapid liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to identify free methylmalonic (MMA) and 3-OH propionic (3OH-PA) acids in blood spots.

Methods: We studied newborn screening spots from 250 healthy controls; 124 from infants with abnormal C3, of whom only 5 (4%) were truly affected; 124 from infants with altered isolated methylmalonylcarnitine; and 4 from clinically diagnosed patients. Whole blood was eluted from a 3.2-mm dried blood spot by a CH3CN/H2O 7:3 and 5 mL/L formic. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray without derivatization. Total analysis time was 5 min per sample.

Results: The assays were linear up to 3300 nmol/L for both metabolites. Intra- and interassay imprecision data were 3.6%–8% and 3.1%–6%, respectively, for MMA and 5.2%–20% and 3.6%–17% for 3OH-PA. Limit of detection and limit of quantitation were 1.95 and 4.2 μmol/L, respectively, for MMA and 8 and 10 μmol/L for 3OH-PA. The recoveries were 92.9%–106.1%. No deterioration was noted on the columns after 500 chromatographic runs. If the new method had been used as a 2nd-tier test for the 124 samples, only the 5 true positives would have been recalled for additional samples, and the positive predictive value would have been 100%.

Conclusions: This method has the potential to markedly reduce false-positive results and the associated costs and anxiety. It may also be suitable for diagnosing and routinely monitoring blood spots for methylmalonic aciduria and propionic acidemia.
Semiotic Modalities in Translation Causality
Authors: Chesterman Andrew
Published: May 2007

A common feature of
much modern translation research is the notion of causality. This is true not
only of empirical descriptive research and applied studies, but also of
hermeneutic studies, since concepts influence action. Different approaches
focus on different kinds and levels of cause and effect. Some focus on the
broad socio-cultural context, some on the situational level (translation
event), some on the cognitive level (translation act) and some on the
linguistic level of the translation product itself (translation profile).
Aristotle's classification of kinds of cause has already been applied in
translation studies. This paper proposes an analysis of translation causality
based on Greimas' modalities of
faire
,
être
,
devoir
,

savoir
,
pouvoir
and
vouloir
. It is argued that the study
of causality does not imply a deterministic standpoint; that translation
causality must include the translator's subjectivity; and that the search for
regularities in cause-effect relations does not imply a neglect of what is
unique about every translation. A causal reading of the modalities of
être,
devoir, savoir, pouvoir
and
vouloir
as factors influencing the
translator's action (
faire
) allows us to relate different kinds of
causes at different levels, including the individual translator.
The Stakes of Translation in Literary Fields
Authors: Gouanvic Jean-Marc
Published: May 2007

This article proposes
to examine the stakes of translation in literary texts. Inspired by Pierre
Bourdieu's theory of culture, it uses the notions of habitus, field and
illusio

in the framework of the translation of American literature in post-WWII
France. After a short definition of what a field is for Bourdieu, we analyse
the power of literary translation during the period. As it is based on the
relationship between the habitus of the translator and a field, the translation
is endowed with a power which is not stated as such and which relies on the
exercise of legitimate violence from dominants, i.e., a violence that is not
known as violence, a violence that is tacitly recognised. Thus, American
literature imposes in France under the form of non-canonical genres such as
Science Fiction and the Série noire, but also highbrow literature, the
translation of which has begun before WWII. The article closes on the stakes of
Worldwide globalisation: the imposition of literary
illusio
, that is,
the imposition of Western hegemony, on an overall scale.
Interpreters as Professionals
Authors: Kalina Sylvia
Published: May 2007

In this article, I
shall examine how interpreting studies have so far accounted for different
modes and types of interpreting, and suggest that the traditional subdivision
into conference interpreting (CI) on the one hand and any other types of
interpreting on the other should be given up. Those other types are generally
referred to as “community interpreting”- a vague term which does not include
such types as court or business interpreting, so that I will, for the purposes
of this article, replace it by the term “non-conference interpreting”(NCI).
This might help obtain a more finely tuned approach to the many different
interpreting services that are being rendered today. I will then look more
closely at what exactly distinguishes various types of interpreting from one
another and what common elements they share. If interpreting as a whole is to
be professionalised, we need clear definitions of interpreting quality and
methods of quality assurance. On the basis of the commonalities found for the
different types of interpreting, it should be possible to develop modules of
training that are useful for all interpreting. Some of the common elements can
be taught jointly, others need specific training. Some elements can be taught
unilingually, others require the bilingual context. The training elements
suggested are aimed at obtaining an accepted degree of professionality for NC
interpreters whose contributions to interlingual communication are no less
important than those of conference interpreters.
Increased Dentate Neurogenesis After Grafting of Glial Restricted Progenitors or Neural Stem Cells in the Aging Hippocampus
Authors: Hattiangady Bharathi , Shuai Bing , Cai Jingli , Coksaygan Turhan , Rao Mahendra S. , Shetty Ashok K.
Published: May 2007

Neurogenesis in the dentate gyrus (DG) declines severely by middle age, potentially because of age-related changes in the DG microenvironment. We hypothesize that providing fresh glial restricted progenitors (GRPs) or neural stem cells (NSCs) to the aging hippocampus via grafting enriches the DG microenvironment and thereby stimulates the production of new granule cells from endogenous NSCs. The GRPs isolated from the spinal cords of embryonic day 13.5 transgenic F344 rats expressing human alkaline phosphatase gene and NSCs isolated from embryonic day 9 caudal neural tubes of Sox-2:EGFP transgenic mice were expanded in vitro and grafted into the hippocampi of middle-aged (12 months old) F344 rats. Both types of grafts survived well, and grafted NSCs in addition migrated to all layers of the hippocampus. Phenotypic characterization revealed that both GRPs and NSCs differentiated predominantly into astrocytes and oligodendrocytic progenitors. Neuronal differentiation of graft-derived cells was mostly absent except in the dentate subgranular zone (SGZ), where some of the migrated NSCs but not GRPs differentiated into neurons. Analyses of the numbers of newly born neurons in the DG using 5′-bromodeoxyuridine and/or doublecortin assays, however, demonstrated considerably increased dentate neurogenesis in animals receiving grafts of GRPs or NSCs in comparison with both naïve controls and animals receiving sham-grafting surgery. Thus, both GRPs and NSCs survive well, differentiate predominantly into glia, and stimulate the endogenous NSCs in the SGZ to produce more new dentate granule cells following grafting into the aging hippocampus. Grafting of GRPs or NSCs therefore provides an attractive approach for improving neurogenesis in the aging hippocampus.

Disclosure of potential conflicts of interest is found at the end of this article.
Effects of HOXB4 Overexpression on Ex Vivo Expansion and Immortalization of Hematopoietic Cells from Different Species
Authors: Zhang Xiao-Bing , Schwartz Jeffrey L. , Humphries R. Keith , Kiem Hans-Peter
Published: May 2007

Overexpression of the human HOXB4 has been shown to induce the expansion and self-renewal of murine hematopoietic stem cells. In preparation for clinical studies, we wished to investigate the effects of HOXB4 on cells from other species, in particular preclinical large animals such as dogs and nonhuman primates. Thus, we transduced CD34+ cells from nonhuman primates, dogs, and humans with a HOXB4-expressing gammaretroviral vector and a yellow fluorescent protein-expressing control vector. Compared with the control vector, HOXB4 overexpression resulted in a much larger increase in colony-forming cells in dog cells (28-fold) compared with human peripheral blood, human cord blood, and baboon cells (two-, four-, and fivefold, respectively). Furthermore, we found that HOXB4 overexpression resulted in immortalization with sustained growth (>12 months) of primitive hematopoietic cells from mice and dogs but not from monkeys and humans. This difference correlated with increased levels of retrovirally overexpressed HOXB4 in dog and mouse cells compared with human and nonhuman primate cells. The immortalized cells did not show any evidence of insertional mutagenesis or chromosomal abnormalities. Competitive congenic transplantation experiments showed that HOXB4-expanded mouse cells engrafted well after 1 or 3 months of expansion, and no leukemia was observed in mice. Our findings suggest that the growth promoting effects of HOXB4 are critically dependent on HOXB4 expression levels and that this can result in important species-specific differences in potency.

Disclosure of potential conflicts of interest is found at the end of this article.
Generation of Insulin-Producing Islet-Like Clusters from Human Embryonic Stem Cells
Authors: Jiang Jianjie , Au Melinda , Lu Kuanghui , Eshpeter Alana , Korbutt Gregory , Fisk Greg , Majumdar Anish S.
Published: May 2007

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to β cells. Generation of glucose-responsive, insulin-producing β cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%–8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/μg of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner.

Disclosure of potential conflicts of interest is found at the end of this article.
Directed Sibling Cord Blood Banking for Transplantation: The 10-Year Experience in the National Blood Service in England
Authors: Smythe Jon , Armitage Sue , McDonald Dorothy , Pamphilon Derwood , Guttridge Martin , Brown Juliette , Green Ann , Brown Colin , Warwick Ruth M. , Lankester Alan , Fehily Deirdre , Contreras Marcela , Navarrete Cristina , Watt Suzanne M.
Published: May 2007

Umbilical cord blood (UCB) is an important source of hematopoietic stem cells for transplantation. Although UCB is often collected from unrelated donors, directed umbilical cord blood (DCB) from sibling donors also provides an important source of UCB for transplantation. This report summarizes the experience in collection, testing, storage, and transplantation of DCB units by the National Blood Service for England and North Wales over 10 years. Eligibility for collection was based on an existing sibling suffering from a disease that may be treated by stem cell transplantation or a family history that could result in the birth of a sibling with a disease that could be treated by stem cell transplantation. Collections were made on the provision that the sibling's clinician was willing to financially support the collection and to take responsibility for medical review of the mother and potential recipient. Given the high investment in UCB banking and the introduction of new regulations and mandatory licensing under the European Union Tissues and Cells Directive and those proposed in the U.S., this report details the procedures that we have used for DCB donations, the outcome data where donations have been used for transplantation, and it provides some timely recommendations for best practices.

Disclosure of potential conflicts of interest is found at the end of this article.