bluuebunny/crossref_metadata_2025 huggingface dataset:
Proplatelet formation is regulated by the Rho/ROCK pathway
Authors: Chang Yunhua , Auradé Frédéric , Larbret Frédéric , Zhang Yanyan , Le Couedic Jean-Pierre , Momeux Laurence , Larghero Jerôme , Bertoglio Jacques , Louache Fawzia , Cramer Elisabeth , Vainchenker William , Debili Najet
Published: January 2007

Platelets are released by megakaryocytes (MKs) via cytoplasmic extensions called proplatelets, which require profound changes in the microtubule and actin organization. Here, we provide evidence that the Rho/ROCK pathway, a well-known regulator of actin cytoskeleton, acts as a negative regulator of proplatelet formation (PPF). Rho is expressed at a high level during the entire MK differentiation including human CD34+ cells. Thrombopoietin stimulates its activity but at a higher extent in immature than in mature MKs. Overexpression of a dominant-negative or a spontaneously active RhoA leads to an increase or a decrease in PPF indicating that Rho activation inhibits PPF. This inhibitory effect is mediated through the main Rho effector, Rho kinase (ROCK), the inhibition of which also increases PPF. Furthermore, inhibition of Rho or ROCK in MKs leads to a decrease in myosin light chain 2 (MLC2) phosphorylation, which is required for myosin contractility. Interestingly, inhibition of the MLC kinase also decreases MLC2 phosphorylation while increasing PPF. Taken together, our results suggest that MLC2 phosphorylation is regulated by both ROCK and MLC kinase and plays an important role in platelet biogenesis by controlling PPF and fragmentation.
A randomized, placebo-controlled trial of oral beclomethasone dipropionate as a prednisone-sparing therapy for gastrointestinal graft-versus-host disease
Authors: Hockenbery David M. , Cruickshank Scott , Rodell Timothy C. , Gooley Ted , Schuening Friedrich , Rowley Scott , David Donald , Brunvand Mark , Berryman Brian , Abhyankar Sunil , Bouvier Michelle , McDonald George B. ,   for the orBec GVHD Study Group
Published: January 2007

We tested the hypothesis that oral beclomethasone dipropionate (BDP) would control gastrointestinal graft-versus-host disease (GVHD) in patients with anorexia, vomiting, and diarrhea. Patients were randomized to prednisone for 10 days and either oral BDP 8 mg/d (n = 62) or placebo (n = 67) tablets for 50 days. At study day 10, prednisone was rapidly tapered while continuing study drug. On an intent-to-treat basis, the risk of GVHD-treatment failure was reduced for the BDP group at study day 50 (hazard ratio [HR] 0.63, 95% confidence interval [CI] 0.35-1.13) and at 30 days follow-up (HR 0.55, 95% CI 0.32-0.93). Among patients eligible for prednisone taper at study day 10, the risk of GVHD-treatment failure was significantly reduced at both study days 50 and 80 (HR 0.39 and 0.38, respectively). By day 200 after transplantation, 5 patients randomized to BDP had died compared with 16 deaths on placebo, a 67% reduction in the hazard of mortality (HR 0.33, P = .03). In 47 recipients of unrelated and HLA-mismatched stem cells, mortality at transplantation day 200 was reduced by 91% in the BDP group compared with placebo (HR 0.09, P = .02). The survival benefit was durable to 1 year after randomization. Oral BDP prevents relapses of gastrointestinal GVHD following tapering of prednisone; survival is statistically significantly better among patients receiving BDP.
Prolonged shear stress and KLF2 suppress constitutive proinflammatory transcription through inhibition of ATF2
Authors: Fledderus Joost O. , van Thienen Johannes V. , Boon Reinier A. , Dekker Rob J. , Rohlena Jakub , Volger Oscar L. , Bijnens Ann-Pascale J. J. , Daemen Mat J. A. P. , Kuiper Johan , van Berkel Theo J. C. , Pannekoek Hans , Horrevoets Anton J. G.
Published: January 2007

Absence of shear stress due to disturbed blood flow at arterial bifurcations and curvatures leads to endothelial dysfunction and proinflammatory gene expression, ultimately resulting in atherogenesis. KLF2 has recently been implicated as a transcription factor involved in mediating the anti-inflammatory effects of flow. We investigated the effect of shear on basal and TNF-α–induced genomewide expression profiles of human umbilical vein endothelial cells (HUVECs). Cluster analysis confirmed that shear stress induces expression of protective genes including KLF2, eNOS, and thrombomodulin, whereas basal expression of TNF-α–responsive genes was moderately decreased. Promoter analysis of these genes showed enrichment of binding sites for ATF transcription factors, whereas TNF-α–induced gene expression was mostly NF-κB dependent. Furthermore, human endothelial cells overlying atherosclerotic plaques had increased amounts of phosphorylated nuclear ATF2 compared with endothelium at unaffected sites. In HUVECs, a dramatic reduction of nuclear binding activity of ATF2 was observed under shear and appeared to be KLF2 dependent. Reduction of ATF2 with siRNA potently suppressed basal proinflammatory gene expression under no-flow conditions. In conclusion, we demonstrate that shear stress and KLF2 inhibit nuclear activity of ATF2, providing a potential mechanism by which endothelial cells exposed to laminar flow are protected from basal proinflammatory, atherogenic gene expression.
Antiendothelial cell antibodies mediate enhanced leukocyte adhesion to cytokine-activated endothelial cells through a novel mechanism requiring cooperation between FcγRIIa and CXCR1/2
Authors: Florey Oliver J. , Johns Michael , Esho Olubukola O. , Mason Justin C. , Haskard Dorian O.
Published: January 2007

Antiendothelial cell antibodies (AECAs) are commonly detectable in diseases associated with vascular injury, including systemic lupus erythematosus (SLE), systemic sclerosis, Takayasu arteritis, Wegener granulomatosis, Behçet syndrome, and transplant arteriosclerosis. Here, we explore the hypothesis that these antibodies might augment polymorphonuclear leukocyte (PMN) adhesion to endothelium in inflammation. Initially, we established that a mouse IgG mAb bound to endothelial cells (ECs) significantly increased PMN adhesion to cytokine-stimulated endothelium in an FcγRIIa-dependent manner. Neutralizing antibodies, and adenoviral transduction of resting ECs, demonstrated that the combination of E-selectin, CXCR1/2, and β2 integrins is both necessary and sufficient for this process. We observed an identical mechanism using AECA IgG isolated directly from patients with SLE. Assembled immune complexes also enhanced PMN adhesion to endothelium, but, in contrast to adhesion because of AECAs, this process did not require CXCR1/2, was not inhibited by pertussis toxin, and was FcγRIIIb rather than FcγRIIa dependent. These data are the first to demonstrate separate nonredundant FcγRIIa and FcγRIIIb-mediated mechanisms by which EC-bound monomeric IgG and assembled immune complexes amplify leukocyte adhesion under dynamic conditions. Furthermore, the observation that FcγRIIa and CXCR1/2 cooperate to enhance PMN recruitment in the presence of AECAs suggests a mechanism whereby AECAs may augment tissue injury during inflammatory responses.
Expansion of spleen myeloid suppressor cells represses NK cell cytotoxicity in tumor-bearing host
Authors: Liu Cunren , Yu Shaohua , Kappes John , Wang Jianhua , Grizzle William E. , Zinn Kurt R. , Zhang Huang-Ge
Published: January 2007

Tumor growth promotes the expansion of myeloid suppressor cells. An inverse correlation between natural killer (NK) cell activation and myeloid suppressor cell (MSC) expansion in tumor-bearing patients and mice prompted us to investigate the role of MSCs in controlling NK antitumor cytotocixity. After adoptive transfer to naive recipients, CD11b+Gr-1+ MSCs freshly isolated from spleens of tumor-bearing mice but not naive mice were able to inhibit NK cell cytotoxicity. An in vivo imaging analysis indicates that the removal of tumors resulted in a significant increased ability (P < .05) in NK cell cytotoxicity to eliminate injected YAC-1 cells from the lungs. Fluorescence-activated cell sorter (FACS) analysis of the composition of lung leukocytes further indicates that the removal of tumors also leads to the reduction of MSCs accumulated in the lung. These data suggest that MSCs suppress NK cell cytotoxicity. The inhibition of NK cell cytotoxicity is cell-cell contact dependent. Inhibition of perforin but not granzyme B production was responsible for MSC-mediated inhibition of NK cytotoxicity. Western blot analyses further suggests that MSCs suppress IL-2–mediated NK cell cytotoxicity by affecting the activity of Stat5.
Activation of MAP kinase p38 is critical for the cell-cycle–controlled suppressor function of regulatory T cells
Authors: Adler Henric S. , Kubsch Sebastian , Graulich Edith , Ludwig Stephan , Knop Juergen , Steinbrink Kerstin
Published: January 2007

Regulatory T cells play an essential role in the control of self-tolerance and processes of adaptive immunity. Tolerogenic IL-10–modulated human dendritic cells (IL-10DCs) induce anergic T cells with strong suppressive properties (iTregs) that inhibit the activation of effector T cells. In this study, we evaluated the interaction between cell-cycle regulation and intracellular signaling in these iTregs. Analysis of signal transduction events revealed a down-regulation of the mitogen-activated protein kinases (MAPKs) Jun N-terminal kinase (JNK) and a nonactivation of extracellular-signal–regulated kinase (ERK) in contrast to a marked activation of p38 MAPK and the p38 effector MAPK-activated protein kinases 2/3 (MAPKAP2/3). The elevated activation of p38 is critical for the induction and maintenance of anergy controlled by an increased expression of the cell-cycle inhibitor p27Kip1. Moreover, blocking experiments with the specific inhibitor SB203580 demonstrated that the regulatory function of iTregs is associated with an enhanced p38 MAPK activity. In contrast to other Treg populations, the suppressor function of iTregs is independent of IL-10. In conclusion, our data indicate that a cross-talk of cell-cycle regulation and p38-dependent signal transduction is required for the suppressor function of iTregs.
CD137 stimulation delivers an antigen-independent growth signal for T lymphocytes with memory phenotype
Authors: Zhu Yuwen , Zhu Gefeng , Luo Liqun , Flies Andrew S. , Chen Lieping
Published: January 2007

CD137 has long been recognized as a costimulatory receptor for growth and functional maturation of recently activated T cells in the presence of T-cell receptor signal. In this report, we present the fact that, in the absence of MHC and antigen, triggering of CD137 by an agonist monoclonal antibody induces vigorous growth of both CD8+ and CD4+ T cells with memory phenotype, whereas it does not affect naive T cells. Moreover, T cells with memory phenotype accumulate progressively in transgenic mice overexpressing CD137 ligand. CD137-mediated proliferation of memory T cells is directly through CD137 on T cells and does not require IL-15 and IFN-γ. Our results define a new role of CD137 signal in the growth of memory T cells.
B-cell development fails in the absence of the Pbx1 proto-oncogene
Authors: Sanyal Mrinmoy , Tung James W. , Karsunky Holger , Zeng Hong , Selleri Licia , Weissman Irving L. , Herzenberg Leonore A. , Cleary Michael L.
Published: January 2007

Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B–cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19+) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B–cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.
NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils
Authors: von Vietinghoff Sibylle , Tunnemann Gisela , Eulenberg Claudia , Wellner Maren , Cristina Cardoso M. , Luft Friedrich C. , Kettritz Ralph
Published: January 2007

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)–linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor.
Rapid infusion rituximab in combination with corticosteroid-containing chemotherapy or as maintenance therapy is well tolerated and can safely be delivered in the community setting
Authors: Sehn Laurie H. , Donaldson Jane , Filewich Allison , Fitzgerald Catherine , Gill Karamjit K. , Runzer Nancy , Searle Barb , Souliere Sheila , Spinelli John J. , Sutherland Judy , Connors Joseph M.
Published: January 2007

The increasing usage of rituximab in the management of non-Hodgkin lymphoma (NHL) has created huge logistical challenges with respect to the delivery of this time- and labor-intensive drug. To address these challenges, we developed and tested the feasibility of a 90-minute infusion schedule for rituximab (20% of the dose administered in the first 30 minutes, remaining 80% administered over 60 minutes). A safety analysis performed in 150 patients receiving rituximab with corticosteroid-containing chemotherapy and 56 patients receiving rituximab as maintenance therapy demonstrated that this schedule was well tolerated, with no grade 3 or 4 infusion reactions observed. In addition, no increase in minor reactions was noted. More than 1200 patients have been treated with this rapid rituximab infusion schedule in the province of British Columbia (BC), demonstrating its safety in the community setting. The adoption of this 90-minute schedule as standard practice has had a positive impact on resource utilization.
Insulin Resistance Increases the Risk of Spontaneous Abortion after Assisted Reproduction Technology Treatment
Authors: Tian Li , Shen Huan , Lu Qun , Norman Robert J. , Wang Jim
Published: January 2007

Objective: This study aims to examine the impact of insulin resistance (IR) on the risk of spontaneous abortion in patients who received infertility treatment.

Patients and Methods: This is a cohort study of 107 patients who achieved their first pregnancy after infertility treatment in a tertiary medical center. A homeostasis model assessment of IR (HOMA-IR) was carried out. Patient demographic characteristics and pregnancy outcome were also recorded. Statistical comparison was made between patients with and without IR. Logistical regression analysis was used to assess the effect of IR and several other factors simultaneously on the risk of spontaneous abortion.

Results: The incidence of spontaneous abortion was 17.8%. The association of IR with the risk of spontaneous abortion was significant after adjusting for other risk factors. The effect of overweight/obesity and polycystic ovarian syndrome was not statistically significant in the multivariate model.

Discussion and Conclusion: This study suggested that IR was an independent risk factor for spontaneous abortion. Because of the high prevalence of IR in obese or polycystic ovarian syndrome patients, the risk of spontaneous abortion in these patients can be raised. Patients with IR should be advised to improve their insulin sensitivity through lifestyle change or medical intervention before infertility treatment to reduce their risk of spontaneous abortion.
Thyroglobulin Gene Mutations Producing Defective Intracellular Transport of Thyroglobulin Are Associated with Increased Thyroidal Type 2 Iodothyronine Deiodinase Activity
Authors: Kanou Yasuhiko , Hishinuma Akira , Tsunekawa Katsuhiko , Seki Koji , Mizuno Yutaka , Fujisawa Haruki , Imai Tsuneo , Miura Yoshitaka , Nagasaka Tetsuro , Yamada Chizumi , Ieiri Tamio , Murakami Masami , Murata Yoshiharu
Published: January 2007

Context: Most patients with defective synthesis and/or secretion of thyroglobulin (Tg) present relatively high serum free T3 (FT3) concentrations with disproportionately low free T4 (FT4) resulting in a high FT3/FT4 ratio. The mechanism of this change in FT3/FT4 ratio remains unknown.

Objective: We hypothesize that increased type 2 iodothyronine deiodinase (D2) activity in the thyroid gland may explain the higher FT3/FT4 ratio that is frequently observed in patients with abnormal Tg synthesis.

Design: We recently identified a compound heterozygous patient (patient A) with a Tg G2356R mutation and one previously described (C1245R) that is known to cause a defect in intracellular transport of Tg. In the current study, after determining the abnormality caused by G2356R, we measured D2 activity as well as its mRNA level in the thyroid gland. We also measured the thyroidal D2 activity in three patients with Tg transport defect and in normal thyroid tissue.

Results: Morphological and biochemical analysis of the thyroid gland from patient A, complemented by a pulse-chase experiment, revealed that G2356R produces a defect in intracellular Tg transport. D2 activity but not type 1 deiodinase in thyroid glands of patients with abnormal Tg transport was significantly higher than in normal thyroid glands, whereas D2 mRNA level in patient A was comparable with that in normal thyroid glands. Furthermore, there was a positive correlation between D2 activity and FT3/FT4 ratios.

Conclusion: Increased thyroidal D2 activity in the thyroid gland is responsible for the higher FT3/FT4 ratios in patients with defective intracellular Tg transport.
Relationship between Vascular Reactivity and Lipids in Mexican-Americans with Type 2 Diabetes Treated with Pioglitazone
Authors: Wajcberg Estela , Sriwijitkamol Apiradee , Musi Nicolas , DeFronzo Ralph A. , Cersosimo Eugenio
Published: January 2007

Context: Vascular dysfunction and insulin resistance precede atherosclerosis in type 2 diabetes (T2DM). Better knowledge of the interaction between these is of considerable clinical interest.

Objective: The objective of this study was to examine the association between inflammation, glucose, and lipid metabolism and vascular dysfunction.

Design and Setting: We conducted a randomized, double-blind, controlled trial of pioglitazone vs. placebo and other therapies aimed at equal glycemic control for 24 wk at an academic tertiary referral clinic.

Patients and Interventions: Mexican-American subjects with T2DM and no complications were randomly assigned to pioglitazone 45 mg daily (PIO, n = 16) or placebo (CON, n = 15) and matched for age, gender, body mass index, diabetes duration, and glycemic control. All subjects completed the study.

Main Outcome Measure: We looked for improved vascular reactivity independent of glycemic control but closely related to plasma adiponectin, lipids, and insulin sensitivity.

Results: After 24 wk, there was an equal decrease in fasting plasma glucose (∼135 mg/dl), glycosylated hemoglobin (∼7.0%), and glucose production (∼15%). The decrease in free fatty acids (30 vs. 10%) and increase in glucose disposal (40 vs. 25%) were greater in PIO vs. CON (P < 0.05). In PIO, plasma high-density lipoprotein rose by 15% (P < 0.05), and low-density lipoprotein and high-density lipoprotein particle size rose significantly (P < 0.01). Plasma adiponectin doubled in PIO (from 6.1 ± 0.8 to 12.7 ± 2.1 μg/ml). Forearm blood flow rose equally (∼130%) during reactive hyperemia in both groups, although after therapy, the increase was greater (P < 0.001) in PIO (153%) than in CON (137%); vasodilation was greater (P = 0.01) in PIO (92, 160, and 204%) than in CON with acetylcholine (74, 130, and 144%) and with sodium nitroprusside (PIO = 164 and 253% vs. 116 and 230%; P = 0.04). The elevation in diameter was also greater in PIO (13 vs. 10%; P < 0.05). Vascular responses correlated with plasma free fatty acids, adiponectin, and low-density lipoprotein particle size but not with glycemic control.

Conclusion: These data indicate that pioglitazone improves vascular reactivity irrespective of glycemic control and suggest a close association with changes in fat cell metabolism.
Increased Adiposity Enhances Intrafollicular Estradiol Levels in Normoandrogenic Ovulatory Women Receiving Gonadotropin-Releasing Hormone Analog/Recombinant Human Follicle-Stimulating Hormone Therapy for in Vitro Fertilization
Authors: Dumesic Daniel A. , Lesnick Timothy G. , Abbott David H.
Published: January 2007

Context: Body mass index (BMI) reflects the amount of insulin in the human follicle and may enhance insulin action as a cogonadotropin.

Objective: This study examined whether increased adiposity enhances intrafollicular steroidogenesis in normoandrogenic ovulatory women receiving GnRH analog/recombinant human FSH therapy for in vitro fertilization.

Design, Setting, and Participants: Study participants were from an institutional practice and comprised 30 normoandrogenic ovulatory women who were lean (n = 17; BMI < 25 kg/m2) or overweight (n = 13; BMI ≥ 25 kg/m2). Women received GnRH analog after basal serum hormone determinations and oral glucose tolerance testing, followed by recombinant human FSH therapy and human chorionic gonadotropin administration when two or more follicles 18 mm or larger in diameter were present.

Intervention: Follicle fluid was aspirated at oocyte retrieval from the first follicle of each ovary.

Main Outcome Measures: Follicle fluid was assayed for estradiol (E2), progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone, insulin, glucose, and lactate.

Results: Overweight women had hyperinsulinemia (P = 0.03) with decreased serum SHBG (P = 0.001) and increased serum free testosterone levels (P = 0.02). Elevated intrafollicular insulin levels in overweight women (P = 0.004) were accompanied by normal glucose and lactate levels. Intrafollicular E2 levels were greater in overweight vs. lean women (P = 0.03), whereas the remaining intrafollicular steroid levels were similar in both female groups.

Conclusion: In normoandrogenic ovulatory women undergoing in vitro fertilization, increased adiposity elevates insulin and E2 levels in terminally differentiated follicles without altering intrafollicular androgen levels or luteinization. Additional studies are required to determine whether these abnormalities impair oocyte development.
RbAp48 is a Target of Nuclear Factor-κB Activity in Thyroid Cancer
Authors: Pacifico Francesco , Paolillo Michele , Chiappetta Gennaro , Crescenzi Elvira , Arena Simona , Scaloni Andrea , Monaco Mario , Vascotto Carlo , Tell Gianluca , Formisano Silvestro , Leonardi Antonio
Published: January 2007

Context: We have recently shown that nuclear factor (NF)-κB activity is constitutively elevated in anaplastic human thyroid carcinomas. The inhibition of NF-κB in the anaplastic thyroid carcinoma cell line (FRO) leads to increased susceptibility to apoptosis induced by chemotherapeutic drugs and to the block of oncogenic activity.

Objectives: To understand better the molecular mechanisms played by NF-κB in thyroid oncogenesis, we performed a differential proteomic analysis between FRO transfected with a superrepressor form of inhibitor of κBα (IκBαM) and the parental counterpart (FRO Neo cells).

Results: Differential proteomic analysis revealed that the retinoblastoma-associated protein 48 (RbAp48) is down-regulated in the absence of functional NF-κB. Immunohistochemical analysis of normal and pathological human thyroid specimens confirmed that RbAp48 is strongly overexpressed in primary human carcinomas. Reduction of RbAp48 expression using small interfering RNA determined the suppression of tumorigenicity, very likely due to the decrease of their growth rate rather than to an increased susceptibility to apoptosis. In addition, we showed that NF-κB, at least in part, transcriptionally controls RbAp 48. A functional NF-κB consensus sequence was located within the promoter region of RbAp48 human gene, and embryonic fibroblasts isolated from the p65 knockout mouse (murine embryonic fibroblasts p65−/−) showed decreased expression of RbAp48.

Conclusion: Our results show that RbAp48 is a NF-κB-regulated gene playing an important role in thyroid cancer cell autonomous proliferation.
Transcriptional Profiling of Androgen Receptor (AR) Mutants Suggests Instructive and Permissive Roles of AR Signaling in Germ Cell Development
Authors: Eacker Stephen M. , Shima James E. , Connolly Charles M. , Sharma Manju , Holdcraft Robert W. , Griswold Michael D. , Braun Robert E.
Published: January 2007

The androgen receptor (AR) is a transcription factor that plays a critical role in male sexual development, spermatogenesis, and maintenance of hormonal homeostasis. Despite the extensive knowledge of the phenotypic consequences of mutations in Ar, very little is known about the transcriptional targets of AR within the testis. To identify potential targets of androgen signaling in the testis, we have analyzed the transcriptional profile of adult testes from Ar hypomorphs alone or in combination with Sertoli cell-specific Ar ablation. Using Affymetrix MOE430A mouse genome arrays we interrogated more than 22,000 transcripts. We found the expression level of 62 transcripts in the Ar mutants differed by greater than 2-fold compared with wild type. We also found that more transcripts were up-regulated than down-regulated, highlighting AR’s role as a transcriptional repressor in the testis. Twelve transcripts were uniquely affected, and 16 transcripts were more severely affected in Sertoli cell-specific Ar ablation compared with hypomorphic Ar mutants. Using a comparative genomic approach, we analyzed the 6 kb around the transcriptional start sites of affected transcripts for conserved AREs (androgen response elements). We identified at least one conserved ARE in 65% of the genes misregulated in our microarray analysis where clear mouse-human orthologs were available. We used a reporter assay in cell culture to functionally verify the AREs for the kallikrein 27 gene. This suggests that the majority of the misregulated transcripts have a high probability of being direct AR targets. The transcripts affected by these Ar mutations encode a diverse array of proteins whose molecular functions support the contention that AR supports spermatogenesis in both a permissive and instructive fashion.
Essential Role of GATA2 in the Negative Regulation of Thyrotropin β Gene by Thyroid Hormone and Its Receptors
Authors: Matsushita Akio , Sasaki Shigekazu , Kashiwabara Yumiko , Nagayama Koji , Ohba Kenji , Iwaki Hiroyuki , Misawa Hiroko , Ishizuka Keiko , Nakamura Hirotoshi
Published: January 2007

Previously we reported that the negative regulation of the TSHβ gene by T3 and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHβ inhibition. The negative regulatory element (NRE), which had been reported to mediate T3-bound TR (T3-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHβ-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T3-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T3-independent manner. The suppression by T3-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TαT1, revealed that T3 treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15–30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHβ gene by T3-TR we report that 1) GATA2 is the major transcriptional activator of the TSHβ gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.
Maximal Activity of the Luteinizing Hormoneβ-Subunit Gene Requires β-Catenin
Authors: Salisbury Travis B. , Binder April K. , Grammer Jean C. , Nilson John H.
Published: January 2007

GnRH regulates expression of LHB via transcriptional regulation of early growth response 1 (EGR1), an immediate early gene that encodes a zinc-finger DNA-binding protein. EGR1 interacts functionally with the orphan nuclear receptor steroidogenic factor 1 (SF1) and pituitary homeobox 1, a member of the paired-like homeodomain family. The functional synergism of this tripartite interaction defines the maximal level of LHB transcription that can occur in response to GnRH. Results presented herein provide new evidence that the interaction between SF1 and EGR1 also requires β-catenin, a transcriptional coactivator and member of the canonical Wnt signaling pathway. For instance, targeted reduction of β-catenin attenuates activity of a GnRH-primed LHB promoter. Additional gene reporter assays indicate that overexpression of β-catenin, or its targeted reduction by small interfering RNA, modulates activity of both SF1 and EGR1 as well as their functional interaction. β-Catenin coimmunoprecipitates with SF1. Moreover, an SF1 mutant that lacks a β-catenin binding domain has compromised transcriptional activity and fails to interact synergistically with EGR1. Finally, GnRH promotes β-catenin colocalization with SF1 and EGR1 on the endogenous mouse Lhb promoter-regulatory region. Taken together, these data suggest that β-catenin binds to SF1 and that this interaction is required for subsequent functional interaction with EGR1. Thus, these data identify β-catenin as a new and required member of the basal transcriptional complex that allows the LHB promoter to achieve maximal activity in response to GnRH.
Anti-NR1 N-terminal-domain vaccination unmasks the crucial action of tPA on NMDA-receptor-mediated toxicity and spatial memory
Authors: Benchenane Karim , Castel Hervé , Boulouard Michel , Bluthé Rosemarie , Fernandez-Monreal Monica , Roussel Benoit D. , Lopez-Atalaya José P. , Butt-Gueulle Sabrina , Agin Véronique , Maubert Eric , Dantzer Robert , Touzani Omar , Dauphin François , Vivien Denis , Ali Carine
Published: January 2007

Fine-tuning of NMDA glutamatergic receptor signalling strategically controls crucial brain functions. This process depends on several ligands and modulators, one of which unexpectedly includes the serine protease tissue-type plasminogen activator (tPA). In vitro, tPA increases NMDA-receptor-mediated calcium influx by interacting with, and then cleaving, the NR1 subunit within its N-terminal domain. Owing to lack of in vivo evidence of the relevance and contribution of this mechanism in physiological and pathological brain processes, active immunisation was developed here in mice, to allow transient and specific prevention of the interaction of tPA with the NR1 subunit. Immunisation significantly reduced the severity of ischemic and excitotoxic insults in the mouse brain. Cognitive function was altered in some, but not all behavioural tasks affected in tPA-deficient mice. Our data demonstrate that in vivo, tPA controls neurotoxicity and the encoding of novel spatial experiences by binding to and cleaving the NMDA receptor NR1 subunit. Interesting therapeutic possibilities for several brain pathologies that involve excitotoxicity may now be envisaged.
IQGAP3, a novel effector of Rac1 and Cdc42, regulates neurite outgrowth
Authors: Wang Shujie , Watanabe Takashi , Noritake Jun , Fukata Masaki , Yoshimura Takeshi , Itoh Norimichi , Harada Takumi , Nakagawa Masato , Matsuura Yoshiharu , Arimura Nariko , Kaibuchi Kozo
Published: January 2007

Rac1 and Cdc42, members of the Rho family GTPases, control diverse cellular processes such as cell migration and morphogenesis through their effectors. Among the effectors, IQGAP1 plays pivotal roles in the establishment of cytoskeletal architecture and intercellular adhesions in various cells. However, its roles remain to be clarified, especially in neuronal cells. We have identified IQGAP3 as a novel member of the IQGAP family, which is highly expressed in brain. We found that IQGAP3, an effector of Rac1 and Cdc42, associates directly with actin filaments and accumulates asymmetrically at the distal region of axons in hippocampal neurons. The depletion of IQGAP3 impairs neurite or axon outgrowth in neuronal cells with the disorganized cytoskeleton, but depletion of IQGAP1 does not. Furthermore, IQGAP3 is indispensable for Rac1/Cdc42-promoted neurite outgrowth in PC12 cells. Taken together, these results indicate that IQGAP3 can link the activation of Rac1 and Cdc42 with the cytoskeletal architectures during neuronal morphogenesis.
Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells
Authors: Hajdo-Milašinović Amra , Ellenbroek Saskia I. J. , van Es Saskia , van der Vaart Babet , Collard John G.
Published: January 2007

Rac1 and Rac3 are highly homologous members of the Rho small GTPase family. Rac1 is ubiquitously expressed and regulates cell adhesion, migration and differentiation in various cell types. Rac3 is primarily expressed in brain and may therefore have a specific function in neuronal cells. We found that depletion of Rac1 by short interference RNA leads to decreased cell-matrix adhesions and cell rounding in neuronal N1E-115 cells. By contrast, depletion of Rac3 induces stronger cell adhesions and dramatically increases the outgrowth of neurite-like protrusions, suggesting opposite functions for Rac1 and Rac3 in neuronal cells. Consistent with this, overexpression of Rac1 induces cell spreading, whereas overexpression of Rac3 results in a contractile round morphology. Rac1 is mainly found at the plasma membrane, whereas Rac3 is predominantly localized in the perinuclear region. Residues 185-187, present in the variable polybasic rich region at the carboxyl terminus are responsible for the difference in phenotype induced by Rac1 and Rac3 as well as for their different intracellular localization. The Rac1-opposing function of Rac3 is not mediated by or dependent on components of the RhoA signaling pathway. It rather seems that Rac3 exerts its function through negatively affecting integrin-mediated cell-matrix adhesions. Together, our data reveal that Rac3 opposes Rac1 in the regulation of cell adhesion and differentiation of neuronal cells.
Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism
Authors: Bryja Vítězslav , Schulte Gunnar , Rawal Nina , Grahn Alexandra , Arenas Ernest
Published: January 2007

Previously, we have shown that Wnt-5a strongly regulates dopaminergic neuron differentiation by inducing phosphorylation of Dishevelled (Dvl). Here, we identify additional components of the Wnt-5a-Dvl pathway in dopaminergic cells. Using in vitro gain-of-function and loss-of-function approaches, we reveal that casein kinase 1 (CK1) δ and CK1ϵ are crucial for Dvl phosphorylation by non-canonical Wnts. We show that in response to Wnt-5a, CK1ϵ binds Dvl and is subsequently phosphorylated. Moreover, in response to Wnt-5a or CK1ϵ, the distribution of Dvl changed from punctate to an even appearance within the cytoplasm. The opposite effect was induced by a CK1ϵ kinase-dead mutant or by CK1 inhibitors. As expected, Wnt-5a blocked the Wnt-3a-induced activation of β-catenin. However, both Wnt-3a and Wnt-5a activated Dvl2 by a CK1-dependent mechanism in a cooperative manner. Finally, we show that CK1 kinase activity is necessary for Wnt-5a-induced differentiation of primary dopaminergic precursors. Thus, our data identify CK1 as a component of Wnt-5a-induced signalling machinery that regulates dopaminergic differentiation, and suggest that CK1δ/ϵ-mediated phosphorylation of Dvl is a common step in both canonical and non-canonical Wnt signalling.
Common patterns of sense making: A discursive reading of quantitative and interpretative data on sexual boredom
Authors: Tunariu Aneta D. , Reavey Paula
Published: January 2007

This paper explores the notion of sexual boredom through combining the use of qualitative and quantitative methods. Drawing on ideas from discursive psychology, we provide an interpretative reading of both numerical and textual data obtained via a postal questionnaire. Within the mixed‐methods strategy adopted here, the questionnaire is treated as a medium that can deliver interesting material about prevalent linguistic resources, their content and pattern of use, available to romantic partners in making sense of sexual boredom. A total of 144 women and 66 men from the general population completed a set of structured questions, including a Sexual Boredom Scale (SBS;Watt & Ewing, 1996), followed by an open‐ended question prompting more elaborated views on the topic. Statistical analysis found gender to explain some of the variation across SBS scores. An interpretative analysis of respondent ratings of disagreement/agreement and the actual meaning content of the scale's statements also reveals ranked and gendered regularities. Written responses to the open‐ended question were subjected to a thematic analysis, revealing how specific changes to quality of sex, intensity of sexual interest and degree of romantic relatedness with a current partner are used by participants to delineate key dimensions of sexual boredom. Overall, the unfolding narratives of sexual boredom are greatly indebted to a static view of relationship satisfaction founded on wishful expectations for consistent, idealized displays of sexual excitement and interest from oneself and one's partner. The interplay between these understandings and a missing discourse of sexuo‐erotic calmness is also considered.
Male and female managers' ability to read emotions: Relationships with supervisor's performance ratings and subordinates' satisfaction ratings
Authors: Byron Kristin
Published: January 2007

The present study examines the direct and indirect effect of managers' accuracy in ‘reading’ non‐verbal emotional expressions and their supervisors' performance ratings and subordinates' satisfaction with the manager. Data from working managers and their supervisors and subordinates were used to test the study's hypotheses. Although managers' accuracy was not directly related to others' ratings, a significant interactive effect was found: Female but not male managers who more accurately perceived non‐verbal emotional expressions received higher performance ratings from their supervisor and higher satisfaction ratings from their subordinates, perhaps due to gender stereotypes prescribing emotional sensitivity to female managers. Furthermore, how managers used emotional information to enhance subordinates' satisfaction varied for female and male managers. Results suggested that male managers who were more accurate at emotion perception received higher satisfaction ratings if they used the information to be more persuasive, whereas more emotionally perceptive female managers received higher satisfaction ratings when they demonstrated more supportiveness.
Experimental study of excitation functions of some proton induced reactions on natTi for beam monitoring purposes
Authors: Zarie K. , Al-Hammad N. , Azzam A.
Published: January 2007

Excitation functions of proton induced reactions on natTi, leading to the formation of 43,44m,44g,47Sc and 48V, were measured using the stacked-foil technique over the proton energies starting from the respective reaction threshold up to 27.5 MeV. The new experimental values were compared with earlier reported data. In general good agreement was found. Also in the case of 48V the results were compared with the cross-section values recommended by the International Atomic Energy Agency. A good agreement was observed.
AMS analysis of 36Cl induced in concrete of accelerator facilities
Authors: Bessho Kotaro , Matsumura Hiroshi , Miura Taichi , Wang Qingbin , Masumoto Kazuyoshi , Matsuhiro Takeshi , Nagashima Yasuo , Seki Riki , Takahashi Tsutomu , Sasa Kimikazu , Sueki Keisuke
Published: January 2007

Accelerator mass spectrometry (AMS) was applied to the analysis of 36Cl induced in concrete samples obtained from accelerator facilities. In order to use a small amount of concrete sample and to separate chlorine as pure as possible, an improvement of separation process was developped. Chlorine was extracted from 1–5 g of concrete into 0.01 M nitric acid in a pressurized decomposition vessel. After determining chlorine using ion chromatography, a certain amount of NaCl solution was added to obtain sufficient amounts of AgCl precipitate and to dilute to a suitable isotope ratio of 36Cl to 35Cl (36Cl/35Cl) for the AMS (10-12–10-10). A careful purification procedure was applied to reduce 36S interference in AMS. Good reproducibility and small error throughout the chemical process for sample preparation was attained. Depth profiles of 36Cl/35Cl in concrete of a medium-energy cyclotron were measured by the developed method and compared with the results of γ-emitters induced by thermal neutrons. Since it was confirmed that 36Cl was produced by thermal neutron capture of 35Cl, the thermal neutron fluence irradiated during accelerator operation could be obtained using 36Cl/35Cl. In order to estimate the neutron fluences, the 36Cl/35Cl measurement by AMS is more useful than radioactivity measurements of other isotopes such as γ-emitters because AMS directly provides the isotope ratio and the half-life of 36Cl is very long.
Ion exchange studies for the removal of plutonium and americium from organic liquid scintillator waste solution
Authors: Chetty K. V. , Swarup R. , Venugopal V. , Vasudeva Rao P. R.
Published: January 2007

Removal of radioactive nuclides from effluents is important for the safe disposal of the radioactive waste. In the present work ion exchange studies were carried out using simulated alpha active organic liquid scintillator and ethyl alcohol waste solutions containing Pu and Am using different resins. The time of equilibration and batch data were obtained under different conditions for individual resins as well as their mixtures. The indigenously developed Macroporous bifunctional phosphinic acid (MPBPA) resin was found to be suitable for the removal of alpha activity mainly due to Pu and Amberlite IR-120 and Biorad 50W × 8 for removal of Am. The results indicated that the presence of alcohol in the scintillator solution helps in increasing the distribution ratio. Experiments were carried out with column containing either MPBPA resin alone or with additional resin bed of Amberlite IR-120 using active organic liquid scintillator waste (with and without dilution with alcohol). Studies were also carried out on alcoholic waste generated in the laboratory during the washing of the used liquid scintillator vials. The alcohol after removal of activity either could be reused or could be disposed off directly as a ‘potentially’ active waste.
The interaction ofDesulfovibrio äspöensisDSM 10631Twith plutonium
Authors: Moll Henry , Merroun Mohamed , Hennig Christoph , Rossberg Andre , Selenska-Pobell Sonja , Bernhard Gert
Published: January 2007

Microbes are widely distributed in nature and they can strongly influence the migration of actinides in the environment. This investigation describes the interaction of plutonium in mixed oxidation states (Pu(VI) and Pu(IV)-polymers) with cells of the sulfate-reducing bacterial (SRB) strainDesulfovibrio äspöensisDSM 10631T, which frequently occurs in the deep granitic rock aquifers at the Äspö Hard Rock Laboratory (Äspö HRL), Sweden. In this study, accumulation experiments were performed in order to obtain information about the amount of Pu bound by the bacteria in dependence on the contact time and the initial plutonium concentration. We used solvent extractions, UV-Vis absorption spectroscopy and X-ray absorption near edge structure (XANES) spectroscopy to determine the speciation of Pu oxidation states. Extended X-ray absorption fine structure (EXAFS) spectroscopy was used to study the coordination of the Pu bound by the bacteria. In the first step, the Pu(VI) and Pu(IV)-polymers are bound to the biomass. Solvent extractions showed that 97% of the initially present Pu(VI) is reduced to Pu(V) due to the activity of the cells within the first 24 h of contact time. Most of the formed Pu(V) dissolves from the cell envelope back to the aqueous solution due to the weak complexing properties of this plutonium oxidation state. Indications were found for a penetration of Pu species inside the bacterial cells.
Uranium(VI) sorption onto kaolinite in the presence and absence of humic acid
Authors: Křepelová A. , Sachs S. , Bernhard Gert
Published: January 2007

We studied the U(VI) sorption onto kaolinite in batch experiments in the absence and presence of humic acid (HA) under different experimental conditions: [U]0= 1 × 10-6M or 1 × 10-5M, [HA]0=10 or 50 mg/L,I=0.1 M or 0.01 M NaClO4, pH=3–10, CO2or N2atmosphere. The study showed that the U(VI) sorption onto kaolinite is influenced by pH, CO2and HA presence. In the absence of CO2, the U(VI) uptake increases with increasing pH value up to pH 6. Above pH 6 it remains unchanged. Because of the formation of negatively charged uranyl carbonate complexes, the decrease in the U(VI) sorption onto the negative surface of kaolinite was observed above pH 8 in the presence of CO2. In presence of HA, the adsorption of U(VI) closely follows the adsorption of HA. In the acidic pH range the U(VI) uptake is enhanced compared to the system without HA due to the formation of additional binding sites for U(VI) coming from HA adsorbed onto kaolinite. The formation of aqueous uranyl-humate complexes reduces the U(VI) sorption in the near neutral pH range. The enhancement of the U(VI) concentration from 1 × 10-6M to 1 × 10-5M results in the shift of the sorption pH edge by one pH unit to higher pH values. The ionic strength has only a slight influence on the U(VI) sorption onto kaolinite, whereas the HA sorption shows a dependence on the ionic strength.