bluuebunny/crossref_metadata_2025 huggingface dataset:
Three-Arm Randomized Study of Two Cisplatin-Based Regimens and Paclitaxel Plus Gemcitabine in Advanced Non–Small-Cell Lung Cancer: A Phase III Trial of the European Organization for Research and Treatment of Cancer Lung Cancer Group—EORTC 08975
Authors: Smit Egbert F. , van Meerbeeck Jan P.A.M. , Lianes Pilar , Debruyne Channa , Legrand Catherine , Schramel Franz , Smit Hans , Gaafar Rabab , Biesma Bonne , Manegold Chris , Neymark Niels , Giaccone Giuseppe
Published: October 2003

Purpose: To compare the therapeutic efficacy of paclitaxel plus cisplatin (arm A) versus gemcitabine plus cisplatin (arm B) and arm A versus paclitaxel plus gemcitabine (arm C) in chemotherapy-naive patients with advanced non–small-cell lung cancer (NSCLC).

Materials and Methods: Patients were randomly assigned to receive either paclitaxel 175 mg/m2 (3-hour infusion, day 1) or gemcitabine 1,250 mg/m2 (days 1 and 8) both combined with cisplatin 80 mg/m2 (day 1) or paclitaxel 175 mg/m2 (3-hour infusion, day 1) combined with gemcitabine 1,250 mg/m2 (days 1 and 8). Primary end point was comparison of overall survival for B versus A and C versus A. Secondary end points included response rate and duration, progression-free survival, toxicities, quality of life [QoL], and cost of treatment.

Results: Four hundred eighty patients (arm A, 159; arm B, 160; arm C, 161 patients) were enrolled; all baseline characteristics were balanced. Median survival times were as follows: arm A, 8.1 months; arm B, 8.9 months; arm C, 6.7 months. Response rates were 31.8% for arm A, 36.6% for arm B, and 27.7% for arm C. Other than myelosuppression (B v A, P < .005), no statistically or clinically significant differences were observed for secondary end points. The average treatment costs were 25% higher in arm C as compared with arms A and B.

Conclusion: Gemcitabine plus cisplatin and paclitaxel plus gemcitabine do not increase overall survival in patients with advanced NSCLC as compared with paclitaxel plus cisplatin. Treatment was well tolerated, and most QoL parameters were similar, but costs associated with the nonplatinum arm were highest.
Efficacy and Safety of Rasburicase (recombinant urate oxidase) for the Prevention and Treatment of Hyperuricemia During Induction Chemotherapy of Aggressive Non-Hodgkin’s Lymphoma: Results of the GRAAL1 (Groupe d’Etude des Lymphomes de l’Adulte Trial on Rasburicase Activity in Adult Lymphoma) Study
Authors: Coiffier Bertrand , Mounier Nicolas , Bologna Serge , Fermé Christophe , Tilly Hervé , Sonet Anne , Christian Bernard , Casasnovas Olivier , Jourdan Eric , Belhadj Karim , Herbrecht Raoul
Published: October 2003

Purpose: Hyperuricemia and tumor lysis syndrome are well-known complications during induction treatment of aggressive non-Hodgkin’s lymphomas (NHLs). Usual prophylaxis and treatment of hyperuricemia consist of hydration, alkalinization, and administration of allopurinol. This study was designed to evaluate the efficacy and the safety of rasburicase (recombinant urate oxidase) in adult patients with aggressive NHL during their first cycle of chemotherapy.

Patients and Methods: A total of 100 patients from Groupe d’Etude des Lymphomes de l’Adulte centers, with diffuse large B-cell lymphoma (n = 79); anaplastic large-cell lymphoma (n = 6); peripheral T-cell lymphoma (n = 8); transformation of indolent lymphoma (n = 5); Burkitt’s lymphoma (n = 1); and lymphoblastic lymphoma (n = 1) were enrolled from May 2001 to June 2002. Before chemotherapy, 66% of patients had elevated lactate dehydrogenase (LDH), including 28% with LDH above 1,000 U/mL. Eleven percent of patients were hyperuricemic (uric acid [UA] > 450 mmol/L or > 7.56 mg/dL). Rasburicase 0.20 mg/kg/d intravenously for 3 to 7 days was started the day before or at day 1 of chemotherapy. UA levels were measured 4 hours after rasburicase injection, then daily during treatment.

Results: All patients responded to rasburicase, as defined by normalization of UA levels maintained during chemotherapy. The control of UA was obtained within 4 hours after the first injection of the drug. Creatinine levels and other metabolites were also controlled with the administration of rasburicase. No patient exhibited increased creatinine levels or required dialysis during chemotherapy.

Conclusion: Rasburicase is the treatment of choice to control UA and prevent tumor lysis syndrome in adult patients with aggressive NHL.
Combined Analysis of Two Multicenter, Randomized, Placebo-Controlled Studies of Pamidronate Disodium for the Palliation of Bone Pain in Men With Metastatic Prostate Cancer
Authors: Small Eric J. , Smith Matthew R. , Seaman John J. , Petrone Stephanie , Kowalski Mildred Ortu
Published: October 2003

Purpose: Bone metastases occur in approximately 80% of patients with advanced prostate cancer. Pain is common in these patients. The purpose of this study was to evaluate the effect of an intravenous bisphosphonate, pamidronate disodium, on pain control in metastatic prostate cancer patients.

Patients and Methods: Two multicenter, double-blind, randomized, placebo-controlled trials were conducted in patients with bone pain due to metastatic prostate cancer, with disease progression after first-line hormonal therapy. Intravenous pamidronate disodium (90 mg) or placebo was administered every 3 weeks for 27 weeks. Efficacy was measured via self-reported pain score (Brief Pain Inventory), analgesic use, the proportion of patients with a skeletal-related event (SRE; defined as pathologic fracture, radiation or surgery to bone, spinal cord compression, or hypercalcemia), and a pilot quantitative measurement of mobility. Laboratory evaluations included serum prostate-specific antigen, interleukin-6, bone alkaline phosphatase, and urinary bone resorption markers.

Results: Results of the two trials were pooled. There were no sustained significant differences between the pamidronate and placebo groups in self-reported pain measurements, analgesic use, proportion of patients with an SRE, or mobility at week 9 or 27. Urinary bone resorption markers were suppressed in the pamidronate group compared with placebo.

Conclusion: Pamidronate disodium failed to demonstrate a significant overall treatment benefit compared with placebo in palliation of bone pain or reduction of SREs. Evaluation of more potent bisphosphonates in patients with prostate cancer is warranted.
Adjuvant Chemotherapy for Breast Cancer: How Presentation of Recurrence Risk Influences Decision-Making
Authors: Chao Celia , Studts Jamie L. , Abell Troy , Hadley Terence , Roetzer Lynne , Dineen Sean , Lorenz Doug , YoussefAgha Ahmed , McMasters Kelly M.
Published: October 2003

Purpose: The purpose of this study was to examine the impact of four methods of communicating survival benefits on chemotherapy decisions. We hypothesized that the four methods of communicating mathematically equivalent risk information would lead to different chemotherapy decisions.

Methods: Each participant received two hypothetical scenarios regarding their mother (a postmenopausal woman with an invasive, lymph node-negative, hormone receptor-positive breast cancer) and was asked to decide whether they would encourage their mother to take chemotherapy in addition to surgery and tamoxifen. In the part 1, participants received one of four methods of describing the chemotherapy survival benefit: (1) relative risk reduction, (2) absolute risk reduction, (3) absolute survival benefit, or (4) number needed to treat. In part 2, each participant received all four methods. Following each decision, participants were asked to rate their confidence and confusion regarding their decision.

Results: Participants included 203 preclinical medical students. In part 1, participants who received relative risk reduction information were significantly more likely to endorse chemotherapy. In part 2, there were no treatment decision differences when participants received all four methods of communicating survival benefits of chemotherapy. However, receiving all four methods led to significantly higher ratings of confusion. In deciding on endorsing chemotherapy, participants understood the information best when presented with data in the absolute survival benefit format.

Conclusion: These results support the hypothesis that the method used to present information about chemotherapy influences treatment decisions. Absolute survival benefit is the most easily understood method of conveying the information regarding benefit of treatment.
High-Dose Therapy and Autologous Peripheral-Blood Stem-Cell Transplantation As Salvage Treatment for HIV-Associated Lymphoma in Patients Receiving Highly Active Antiretroviral Therapy
Authors: Re Alessandro , Cattaneo Chiara , Michieli Mariagrazia , Casari Salvatore , Spina Michele , Rupolo Maurizio , Allione Bernardino , Nosari Annamaria , Schiantarelli Clara , Viganò Mariagrazia , Izzi Immacolata , Ferremi Piero , Lanfranchi Arnalda , Mazzuccato Maurizio , Carosi Gianpiero , Tirelli Umberto , Rossi Giuseppe
Published: October 2003

Purpose: High-dose therapy (HDT) and peripheral-blood stem-cell transplantation (PBSCT) in HIV-associated lymphoma (HIV-Ly) has been recently reported in selected patients. We describe the results of a multi-institutional program of HDT and PBSCT as salvage therapy in HIV-Ly responsive to highly active antiretroviral therapy (HAART) in unselected patients.

Patients and Methods: Patients with resistant or relapsed HIV-Ly after first-line chemotherapy (CT) underwent PBSC collection after a course of second-line CT or cyclophosphamide and granulocyte colony-stimulating factor. Patients with chemotherapy-sensitive disease received carmustine, etoposide, cytarabine, and melphalan (BEAM regimen) and PBSC reinfusion. Effective HAART was maintained during the entire program.

Results: Sixteen consecutive patients entered the program. Adequate collection of PBSC was obtained in 80% of patients (median CD34+ cells 6.8 × 106/kg). Three patients had early progression. Ten patients (62%) received PBSCT with prompt engraftment in all patients (neutrophils and platelet engraftment after a median of 10 days [range, 8 to 10 days] and 13 days [range, 8 to 18 days], respectively). No patients died as a result of opportunistic or other infections or treatment-related complications. Eight of nine assessable patients achieved complete remission (one patient after radiotherapy for residual disease) and one patient achieved partial remission. Two patients experienced relapse and died at +10 and +14 months. Six patients are alive and disease free at a median of 8 months after transplantation.

Conclusion: Our data confirm that HDT plus PBSCT is feasible and active as salvage therapy in HIV-Ly on a multi-institutional basis and in unselected HAART-responding patients. HIV infection should no longer preclude the opportunity of HDT in patients with lymphoma.
Breast Cancer in Older Women: Quality of Life and Psychosocial Adjustment in the 15 Months After Diagnosis
Authors: Ganz Patricia A. , Guadagnoli Edward , Landrum Mary Beth , Lash Timothy L. , Rakowski William , Silliman Rebecca A.
Published: October 2003

Purpose: We examined the health-related quality of life (QOL) of a cohort of older women with breast cancer after their diagnosis.

Patients and Methods: Six hundred ninety-one women aged 65 years and older were interviewed approximately 3 months after breast cancer surgery and two additional times in the following year using standardized QOL measures. Demographic factors, breast cancer treatments, and comorbid conditions were used to model ratings of health-related QOL over time. Self-perceived health and psychosocial adjustment at 15 months after surgery were modeled.

Results: Physical and mental health scores declined significantly in the follow-up year, independent of age. However, a cancer-specific psychosocial instrument showed significant improvement in scores. Better 3-month physical and mental health scores, as well as better emotional social support, predicted more favorable self-perceived health 15 months after surgery. Psychosocial adjustment at 15 months was significantly predicted by better mental health, emotional social support, and better self-rated interaction with health care providers assessed at 3 months.

Conclusion: Contrary to reports from younger women with breast cancer, we observed significant declines in the physical and mental health of older women in the 15 months after breast cancer surgery, whereas scores on a cancer-specific psychosocial QOL measure improved over time, consistent with patterns in younger women. Predictive models indicate that older women with impaired physical functioning, mental health, and emotional social support after surgery have poorer self-perceived health and psychosocial adjustment 1 year later. Interventions to address the physical and emotional needs of older women with breast cancer should be developed and evaluated to determine their impact on subsequent health-related QOL.
Clinical and Immunologic Results of a Randomized Phase II Trial of Vaccination Using Four Melanoma Peptides Either Administered in Granulocyte-Macrophage Colony-Stimulating Factor in Adjuvant or Pulsed on Dendritic Cells
Authors: Slingluff Craig L. , Petroni Gina R. , Yamshchikov Galina V. , Barnd Donna L. , Eastham Shannon , Galavotti Holly , Patterson James W. , Deacon Donna H. , Hibbitts Sarah , Teates David , Neese Patrice Y. , Grosh William W. , Chianese-Bullock Kimberly A. , Woodson Elizabeth M.H. , Wiernasz Catherine J. , Merrill Priscilla , Gibson Jennifer , Ross Maureen , Engelhard Victor H.
Published: October 2003

Purpose: To determine clinical and immunologic responses to a multipeptide melanoma vaccine regimen, a randomized phase II trial was performed.

Patients and Methods: Twenty-six patients with advanced melanoma were randomly assigned to vaccination with a mixture of four gp100 and tyrosinase peptides restricted by HLA-A1, HLA-A2, and HLA-A3, plus a tetanus helper peptide, either in an emulsion with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Montanide ISA-51 adjuvant (Seppic Inc, Fairfield, NJ), or pulsed on monocyte-derived dendritic cells (DCs). Systemic low-dose interleukin-2 (Chiron, Emeryville, CA) was given to both groups. T-lymphocyte responses were assessed, by interferon gamma ELIspot assay (Chiron, Emeryville, CA), in peripheral-blood lymphocytes (PBLs) and in a lymph node draining a vaccine site (sentinel immunized node [SIN]).

Results: In patients vaccinated with GM-CSF in adjuvant, T-cell responses to melanoma peptides were observed in 42% of PBLs and 80% of SINs, but in patients vaccinated with DCs, they were observed in only 11% and 13%, respectively. The overall immune response was greater in the GM-CSF arm (P < .02). Vitiligo developed in two of 13 patients in the GM-CSF arm but in no patients in the DC arm. Helper T-cell responses to the tetanus peptide were detected in PBLs after vaccination and correlated with T-cell reactivity to the melanoma peptides. Objective clinical responses were observed in two patients in the GM-CSF arm and one patient in the DC arm. Stable disease was observed in two patients in the GM-CSF arm and one patient in the DC arm.

Conclusion: The high frequency of cytotoxic T-lymphocyte responses and the occurrence of clinical tumor regressions support continued investigation of multipeptide vaccines administered with GM-CSF in adjuvant.
Phase III Trial of Long-Term Adjuvant Androgen Deprivation After Neoadjuvant Hormonal Cytoreduction and Radiotherapy in Locally Advanced Carcinoma of the Prostate: The Radiation Therapy Oncology Group Protocol 92–02
Authors: Hanks Gerald E. , Pajak Thomas F. , Porter Arthur , Grignon David , Brereton Harmart , Venkatesan Varagur , Horwitz Eric M. , Lawton Colleen , Rosenthal Seth A. , Sandler Howard M. , Shipley William U.
Published: October 2003

Purpose: Radiation Therapy Oncology Group (RTOG) Protocol 92–02 was a randomized trial testing long-term (LT) adjuvant androgen deprivation (AD) after initial AD with external-beam radiotherapy (RT) in patients with locally advanced prostate cancer (PC; T2c-4) and with prostate-specific antigen level less than 150 ng/mL.

Patients and Methods: Patients received a total of 4 months of goserelin and flutamide, 2 months before and 2 months during RT. A radiation dose of 65 to 70 Gy was given to the prostate and a dose of 44 to 50 Gy to the pelvic lymph nodes. Patients were randomly assigned to receive no additional therapy (short-term [ST]AD-RT) or 24 months of goserelin (LTAD-RT); 1,554 patients were entered onto the study.

Results: The LTAD-RT arm showed significant improvement in all efficacy end points except overall survival (OS; 80.0% v 78.5% at 5 years, P = .73), compared with the STAD-RT arm. In a subset of patients not part of the original study design, with tumors assigned Gleason scores of 8 to 10 by the contributing institutions, the LTAD-RT arm had significantly better OS (81.0% v 70.7%, P = .044). There was a small but significant increase in the frequency of late radiation grades 3, 4, and 5 gastrointestinal toxicity ascribed to the LTAD-RT arm (2.6% v 1.2% at 5 years, P = .037), the cause of which is not clear.

Conclusion: The RTOG 92–02 trial supports the addition of LT adjuvant AD to STAD with RT for T2c-4 PC. In the exploratory subset analysis of patients with Gleason scores 8 to 10, LT adjuvant AD resulted in a survival advantage.
Attenuation by Reactive Oxygen Species of Glucocorticoid Suppression on Proopiomelanocortin Gene Expression in Pituitary Corticotroph Cells
Authors: Asaba Koichi , Iwasaki Yasumasa , Yoshida Masanori , Asai Masato , Oiso Yutaka , Murohara Toyoaki , Hashimoto Kozo
Published: October 2003

Up-regulation of hypothalamo-pituitary-adrenal axis is maintained during acute inflammation and/or infection, in the face of sustained elevation of plasma glucocorticoid hormone. Inflammatory stress is usually associated with high plasma cytokine levels and increased generation of reactive oxygen species (ROS) as well. In this study, we examined the effect of ROS on the negative feedback regulation of glucocorticoid in hypothalamo-pituitary-adrenal axis using AtT20 corticotroph cells in vitro. When the cells were treated with H2O2, glucocorticoid suppression on the proopiomelanocortin gene promoter activity was attenuated in a dose-dependent manner. H2O2 also inhibited the ligand-stimulated nuclear translocation of glucocorticoid receptor. The released glucocorticoid suppression by H2O2 was not observed when the cells were cotreated with antioxidants. Together, these results suggest that increased ROS generation in the oxidative redox state attenuates the glucocorticoid negative feedback system, at least in part, by interfering with the nuclear translocation of glucocorticoid receptor and eliminating the repression on proopiomelanocortin gene expression.
Corticotropin-Releasing Hormone and Its Structurally Related Urocortin Are Synthesized and Secreted by Human Mast Cells
Authors: Kempuraj Duraisamy , Papadopoulou Nikoletta G. , Lytinas Michael , Huang Man , Kandere-Grzybowska Kristiana , Madhappan Bhuvaneshwari , Boucher William , Christodoulou Spyridon , Athanassiou Achilles , Theoharides Theoharis C.
Published: October 2003

Stress activates the hypothalamic-pituitary-adrenal axis through CRH, leading to production of glucocorticoids that down-regulate immune responses. However, acute stress also has proinflammatory effects. We previously showed that restraint stress, as well as CRH and its structurally related urocortin (Ucn), could activate mast cells and trigger mast cell-dependent vascular permeability. Here we show for the first time that human cord blood-derived cultured mast cells (hCBMC) at 10 wk, but not at 2 wk, are immunocytochemically positive for CRH and Ucn; human leukemic mast cells are weakly positive for both peptides. The ability of these mast cells to synthesize CRH and Ucn was confirmed by showing mRNA expression with RT-PCR. hCBMC (8–14 wk) synthesize and store 1–10 ng/106 cells (10–20 μg/g) of both CRH and Ucn detected by ELISA of cell homogenates. Stimulation of IgE-sensitized hCBMC with anti-IgE results in secretion of most CRH and Ucn. These findings indicate that mast cells are not only the target, but also a potential source of CRH and Ucn that could have both autocrine and paracrine functions, especially in allergic inflammatory disorders exacerbated by stress.
Cell-Specific Pituitary Gene Expression Profiles after Treatment with Leukemia Inhibitory Factor Reveal Novel Modulators for Proopiomelanocortin Expression
Authors: Abbud Rula A. , Kelleher Robert , Melmed Shlomo
Published: October 2003

Leukemia inhibitory factor (LIF) mediates the hypothalamo-pituitary-adrenal stress response. Transgenic mice overexpressing LIF in the developing pituitary have altered pituitary differentiation with expansion of corticotropes, maintenance of Rathke’s cleft cysts, and suppression of all other pituitary cell types. Affymetrix GeneChips were used to identify modulators of LIF effects in corticotrope (AtT-20) and somatolactotrope (GH3) cells. In addition to genes known to respond to LIF in corticotrope cells [e.g. suppressor of cytokine signaling-3 (SOCS-3), signal transducer and activator of transcription-3, SH2 domain-containing tyrosine phosphatase-1, and proopiomelanocortin (POMC)], corticotrope-specific changes were also observed for genes involved in glycolysis and gluconeogenesis, transcription factors, signaling molecules, and expressed sequence tags. Two transcription factors identified, CCAAT/enhancer-binding protein β (C/EBPβ) and glial cell-derived neurotrophic factor (GDNF)-inducible factor (GIF), dose-dependently induced expression of the rat POMC promoter when overexpressed in AtT-20 cells. LIF further induced POMC transcription with C/EBPβ, but not with GIF. C/EBPβ also induced expression of the SOCS-3 promoter that was further enhanced by cotreatment with LIF. However, GIF did not affect SOCS-3 expression. These results indicate that C/EBPβ and GIF are downstream effectors of LIF corticotrope action. LIF also stimulates the expression of inhibitors of its actions, such as SOCS-3 and SH2 domain-containing tyrosine phosphatase-1. α2-HS-glycoprotein (AHSG)/fetuin, a secreted protein that antagonizes bone TGFβ/bone morphogenic protein signaling, was induced by LIF in a signal transducer and activator of transcription-3-dependent fashion. Pretreatment with AHSG/fetuin blocked LIF-induced expression of the POMC promoter independently of SOCS-3. Thus, using GeneChips, C/EBPβ and GIF have been identified as novel mediators and AHSG/fetuin as an inhibitor of LIF action in corticotropes.
Valproate Potentiates Androgen Biosynthesis in Human Ovarian Theca Cells
Authors: Nelson-DeGrave Velen L. , Wickenheisser Jessica K. , Cockrell Jennifer E. , Wood Jennifer R. , Legro Richard S. , Strauss Jerome F. , McAllister Jan M.
Published: October 2003

In patients with epilepsy, treatment with valproate (VPA) has been reported to be associated with polycystic ovary syndrome-like symptoms including weight gain, hyperandrogenemia, and hyperinsulinemia. We examined the effect of VPA on androgen biosynthesis in ovarian theca cells isolated from follicles of normal cycling women to determine whether the hyperandrogenemia reported with VPA treatment could be a result of direct effects of VPA on the ovary. In long-term cultures of theca cells treated for 72 h with sodium valproate (30–3000 μm), we observed an increase in basal and forskolin-stimulated dehydroepiandrosterone (DHEA), androstenedione, and 17α-hydroxyprogesterone production compared with control values. In contrast, low doses of VPA treatment (i.e. 30–300 μm) had no effect on basal and forskolin-stimulated progesterone production, whereas higher doses of VPA (1000–3000 μm) inhibited progesterone production. The most pronounced effect of VPA on androgen biosynthesis was observed in the dose range of 300-3000 μm, which represent therapeutic levels in the treatment of epilepsy and bipolar disorder. Western analyses demonstrated that VPA treatment increased both basal and forskolin-stimulated P450c17 and P450scc protein levels, whereas the amount of steroidogenic acute regulatory protein was unaffected. In transient transfection studies, VPA was found to increase P450 17α-hydroxylase and P450 cholesterol side-chain cleavage promoter activity, whereas steroidogenic acute regulatory protein promoter activity was unaffected. Consistent with the ability of VPA to act as a histone deacetylase (HDAC) inhibitor in other cell systems, VPA (500 μm) treatment was observed to increase histone H3 acetylation and P450 17α-hydroxylase mRNA accumulation. The HDAC inhibitor butyric acid (500 μm) similarly increased histone H3 acetylation and DHEA biosynthesis, whereas the VPA derivative valpromide (500 μm), which lacks HDAC inhibitory activity, had no effect on histone acetylation or DHEA biosynthesis. These data suggest that VPA-induced ovarian androgen biosynthesis results from changes in chromatin modifications (histone acetylation) that augment transcription of steroidogenic genes. These studies provide the first biochemical evidence to support a role for VPA in the genesis of polycystic ovary syndrome-like symptoms, and establish a direct link between VPA treatment and increased ovarian androgen biosynthesis.
Desensitization and Endocytosis Mechanisms of Ghrelin-Activated Growth Hormone Secretagogue Receptor 1a
Authors: Camiña Jesus P. , Carreira Marcos C. , El Messari Said , Llorens-Cortes Catherine , Smith Roy G. , Casanueva Felipe F.
Published: October 2003

In this study, a sequential analysis of pathways involved in the regulation of GH secretagogue receptor subtype 1a (GHSR-1a) signaling has been undertaken to characterize the process of rapid desensitization that is observed after ghrelin binding. This process was evaluated by studying the binding of [125I]ghrelin, measurement of intracellular calcium mobilization, and confocal microscopy. The results indicate that GHSR-1a is mainly localized at the plasma membrane under unstimulated conditions and rapidly desensitizes after stimulation. The agonist-dependent desensitization is not mediated by protein kinase C because phorbol ester, phorbol-12-myristate-13-acetate, failed to block the ghrelin-induced calcium response. The ghrelin/GHSR-1a complex progressively disappears from the plasma membrane after 20 min exposure to ghrelin and accumulates in the perinuclear region after 60 min. Colocalization of the internalized GHSR-1a with the early endosome marker (EEA1) after 20 min exposure to ghrelin suggests that endocytosis occurs via clathrin-coated pits, which is consistent with the lack of internalization of this receptor observed after potassium depletion. Different from other G protein-coupled receptors, GHSR-1a showed slow recycling. Surface binding slowly recovered after agonist treatment and returned to control levels within 360 min. Furthermore, inhibition of vacuolar H+-ATPases prevented recycling of the receptor, suggesting that the nondissociation of the ligand/receptor complex is responsible for this effect. The GHSR-1a internalization may explain the characteristic physiological responses mediated by this receptor.
Nuclear Factor-κB Activates Transcription of the Androgen Receptor Gene in Sertoli Cells Isolated from Testes of Adult Rats
Authors: Zhang Liying , Charron Martin , Wright William W. , Chatterjee Bandana , Song Chung S. , Roy Arun K. , Brown Terry R.
Published: October 2003

The androgen receptor (AR) in Sertoli cells mediates the actions of testosterone on spermatogenesis. However, the transcription factors responsible for AR gene regulation in Sertoli cells remain unknown. In this study, we determined that nuclear factor-κB (NF-κB) regulates transcription of AR in primary cultures of Sertoli cells isolated from testes of adult rats. Electrophoretic mobility shift and antibody supershift assays with nuclear extracts prepared from Sertoli cells identified two binding sites, termed κB1 at −491/−482 bp and κB2 at −574/−565 bp, upstream of the transcription start site of the AR gene that bind the NF-κB subunits, p50 and p65. DNAse I footprint analyses showed that binding of the p50 NF-κB subunit protected the same regions on the rat AR promoter. Analyses of AR promoter-luciferase reporter gene activity after transfection of primary cultures of Sertoli cells demonstrated that mutation of the κB2 site or combined mutation of the κB1 and κB2 sites reduced activity by 40%. Preferential binding of the transcriptionally active p65/p50 heterodimer to the κB2 site rather than to the κB1 site supported these observations. Overexpression of the NF-κB p65 and p50 subunits in Sertoli cells increased activity from the wild-type AR promoter and the promoter with mutation of the κB1 site, but not the κB2 site. Activity was further stimulated by CBP (CREB binding protein), a coactivator of p65 transcriptional activity. Taken together, our data show that NF-κB is an activator of AR gene transcription in Sertoli cells and may be an important determinant of androgen activity during spermatogenesis.
Low-Dose Immunization with Adenovirus Expressing the Thyroid-Stimulating Hormone Receptor A-Subunit Deviates the Antibody Response toward That of Autoantibodies in Human Graves’ Disease
Authors: Chen Chun-Rong , Pichurin Pavel , Chazenbalk Gregorio D. , Aliesky Holly , Nagayama Yuji , McLachlan Sandra M. , Rapoport Basil
Published: October 2003

Immunization with adenovirus expressing the TSH receptor (TSHR) induces hyperthyroidism in 25–50% of mice. Even more effective is immunization with a TSHR A-subunit adenovirus (65–84% hyperthyroidism). Nevertheless, TSHR antibody characteristics in these mice do not mimic accurately those of autoantibodies in typical Graves’ patients, with a marked TSH-blocking antibody response. We hypothesized that this suboptimal antibody response was consequent to the standard dose of TSHR-adenovirus providing too great an immune stimulus. To test this hypothesis, we compared BALB/c mice immunized with the usual number (1011) and with far fewer viral particles (109 and 107). Regardless of viral dose, hyperthyroidism developed in a similar proportion (68–80%) of mice. We then examined the qualitative nature of TSHR antibodies in each group. Sera from all mice had TSH binding-inhibitory (TBI) activity after the second immunization, with TBI values in proportion to the viral dose. After the third injection, all groups had near-maximal TBI values. Remarkably, in confirmation of our hypothesis, immunization with progressively lower viral doses generated TSHR antibodies approaching the characteristics of autoantibodies in human Graves’ disease as follows: 1) lower TSHR antibody titers on ELISA and 2) lower TSH-blocking antibody activity without decrease in thyroid-stimulating antibody activity. In summary, low-dose immunization with adenovirus expressing the free TSHR A-subunit provides an induced animal model with a high prevalence of hyperthyroidism as well as TSHR antibodies more closely resembling autoantibodies in Graves’ disease.
Tbx5specifies the left/right ventricles and ventricular septum position during cardiogenesis
Authors: Takeuchi Jun K. , Ohgi Makoto , Koshiba-Takeuchi Kazuko , Shiratori Hidetaka , Sakaki Ichiro , Ogura Keiko , Saijoh Yukio , Ogura Toshihiko
Published: October 2003

Extensive misexpression studies were carried out to explore the roles played by Tbx5, the expression of which is excluded from the right ventricle (RV) during cardiogenesis. When Tbx5 was misexpressed ubiquitously,ventricular septum was not formed, resulting in a single ventricle. In such heart, left ventricle (LV)-specific ANF gene was induced. In search of the putative RV factor(s), we have found that chick Tbx20 is expressed in the RV, showing a complementary fashion to Tbx5. In the Tbx5-misexpressed heart, this gene was repressed. When misexpression was spatially partial, leaving small Tbx5-negative area in the right ventricle,ventricular septum was shifted rightwards, resulting in a small RV with an enlarged LV. Focal expression induced an ectopic boundary of Tbx5-positive and-negative regions in the right ventricle, at which an additional septum was formed. Similar results were obtained from the transient transgenic mice. In such hearts, expression patterns of dHAND and eHAND were changed with definitive cardiac abnormalities. Furthermore, we report that human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and GATA4. This activation was abrogated by Tbx20, implicating the pivotal roles of interactions among these heart-specific factors. Taken together, our data indicate that Tbx5 specifies the identity of LV through tight interactions among several heart-specific factors, and highlight the essential roles of Tbx5 in cardiac development.
Bar homeodomain proteins are anti-proneural in theDrosophilaeye: transcriptional repression ofatonalby Bar prevents ectopic retinal neurogenesis
Authors: Lim Janghoo , Choi Kwang-Wook
Published: October 2003

Atonal (Ato)/Math (Mammalian atonal homolog) family proneural proteins are key regulators of neurogenesis in both vertebrates and invertebrates. In the Drosophila eye, Ato is essential for the generation of photoreceptor neurons. Ato expression is initiated at the anterior ridge of the morphogenetic furrow but is repressed in the retinal precursor cells behind the furrow to prevent ectopic neurogenesis. We show that Ato repression is mediated by the conserved homeobox proteins BarH1 and BarH2. Loss of Bar causes cell-autonomous ectopic Ato expression, resulting in excess photoreceptor clusters. The initial ommatidial spacing at the furrow occurs normally in the absence of Bar, suggesting that the ectopic neurogenesis within Bar mutant clones is not due to the lack of Notch(N)-dependent lateral inhibition. Targeted misexpression of Bar is sufficient to repress ato expression. Furthermore, we provide evidence that Bar represses ato expression at the level of transcription without affecting the expression of an ato activator, Cubitus interruptus(Ci). Thus, we propose that Bar is essential for transcriptional repression of ato and the prevention of ectopic neurogenesis behind the furrow.
Embryonic atrial function is essential for mouse embryogenesis, cardiac morphogenesis and angiogenesis
Authors: Huang Chengqun , Sheikh Farah , Hollander Melinda , Cai Chengleng , Becker David , Chu Po-Hsien , Evans Sylvia , Chen Ju
Published: October 2003

The requirement for atrial function in developing heart is unknown. To address this question, we have generated mice deficient in atrial myosin light chain 2 (MLC2a), a major structural component of the atrial myofibrillar apparatus. Inactivation of the Mlc2a gene resulted in severely diminished atrial contraction and consequent embryonic lethality at ED10.5-11.5, demonstrating that atrial function is essential for embryogenesis. Our data also address two longstanding questions in cardiovascular development: the connection between function and form during cardiac morphogenesis, and the requirement for cardiac function during vascular development. Diminished atrial function in MLC2a-null embryos resulted in a number of consistent secondary abnormalities in both cardiac morphogenesis and angiogenesis. Our results unequivocally demonstrate that normal cardiac function is directly linked to normal morphogenic development of heart and vasculature. These data have important implications for the etiology of congenital heart disease.
Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis
Authors: Legg James , Jensen Uffe B. , Broad Simon , Leigh Irene , Watt Fiona M.
Published: October 2003

Human interfollicular epidermis is renewed by stem cells that are clustered in the basal layer in a patterned, non-random distribution. Stem cells can be distinguished from other keratinocytes by high expression of β1 integrins and lack of expression of terminal differentiation markers; they divide infrequently in vivo but form actively growing colonies in culture. In a search for additional stem cell markers, we observed heterogeneous epidermal expression of melanoma chondroitin sulphate proteoglycan (MCSP). MCSP was expressed by those keratinocytes with the highest β1 integrin levels. In interfollicular epidermis, expression was confined to non-cycling cells and,in culture, to self-renewing clones. However, fluorescence-activated cell sorting on the basis of MCSP and β1 integrin expression gave no more enrichment for clonogenic keratinocytes than sorting for β1 integrins alone. To interfere with endogenous MCSP, we retrovirally infected keratinocytes with a chimera of the CD8 extracellular domain and the MCSP cytoplasmic domain. CD8/MCSP did not affect keratinocyte proliferation or differentiation but the cohesiveness of keratinocytes in isolated clones or reconstituted epidermal sheets was greatly reduced. CD8/MCSP caused stem cell progeny to scatter without differentiating. CD8/MCSP did not alter keratinocyte motility but disturbed cadherin-mediated cell-cell adhesion and the cortical actin cytoskeleton, effects that could be mimicked by inhibiting Rho. We conclude that MCSP is a novel marker for epidermal stem cells that contributes to their patterned distribution by promoting stem cell clustering.
Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons
Authors: Lalli Giovanna , Gschmeissner Stephen , Schiavo Giampietro
Published: October 2003

Using a novel assay based on the sorting and transport of a fluorescent fragment of tetanus toxin, we have investigated the cytoskeletal and motor requirements of axonal retrograde transport in living mammalian motor neurons. This essential process ensures the movement of neurotrophins and organelles from the periphery to the cell body and is crucial for neuronal survival. Unlike what is observed in sympathetic neurons, fast retrograde transport in motor neurons requires not only intact microtubules, but also actin microfilaments. Here, we show that the movement of tetanus toxin-containing carriers relies on the nonredundant activities of dynein as well as kinesin family members. Quantitative kinetic analysis indicates a role for dynein as the main motor of these carriers. Moreover, this approach suggests the involvement of myosin(s) in retrograde movement. Immunofluorescence screening with isoform-specific myosin antibodies reveals colocalization of tetanus toxin-containing retrograde carriers with myosin Va. Motor neurons from homozygous myosin Va null mice showed slower retrograde transport compared with wild-type cells, establishing a unique role for myosin Va in this process. On the basis of our findings, we propose that coordination of myosin Va and microtubule-dependent motors is required for fast axonal retrograde transport in motor neurons.
Migrating fibroblasts perform polarized, microtubule-dependent exocytosis towards the leading edge
Authors: Schmoranzer Jan , Kreitzer Geri , Simon Sanford M.
Published: October 2003

Cell migration might involve biased membrane traffic toward the leading edge to facilitate the building of extracellular matrix, membrane protrusions and adhesion plaques. We tested the hypothesis that secretory vesicles are preferentially delivered toward the leading lamella in wound-edge fibroblasts. Single fusion events of vesicles containing LDLR-GFP were mapped by total internal reflection fluorescence microscopy (TIR-FM). In migrating fibroblasts, exocytic events were polarized towards the leading edge. After disrupting microtubules with nocodazole, exocytosis continued, but fusion sites were clustered around central Golgi elements; there was no peripheral exocytosis. We conclude that microtubules are necessary for the domain-specific fusion of post-Golgi vesicles with the plasma membrane during migration.
Mutation of an unusual mitochondrial targeting sequence of SODB2 produces multiple targeting fates inToxoplasma gondii
Authors: Brydges Susannah D. , Carruthers Vern B.
Published: October 2003

Proteins destined for the mitochondria travel an intricate pathway through two membranes, each with its own receptors and channels. These proteins interact with receptors via N-terminal presequences that form amphipathic helices. Generally, these helices contain abundant positive charges on one face and hydrophobic residues on the other, but share little primary sequence homology. While extensive research on mitochondrial import has been done in yeast and mammalian cells, little is known about import or contents of the single mitochondrion of Toxoplasma gondii, a parasite in the phylum Apicomplexa. We describe here the characterization of TgSODB2, a novel, mitochondrial superoxide dismutase in T. gondii with an unusual targeting sequence consisting of a hydrophobic segment resembling a signal peptide, followed by a presequence. We show that although the hydrophobic segment is competent to target a reporter protein to the secretory system, it is prevented from directing ER translocation when coupled with the presequence. When we mutated the only charged residue in the hydrophobic sequence, ER translocation is restored and the reporter targeted to the apicoplast, a chloroplast-like organelle found in most apicomplexans. The presequence that follows is predicted to form an amphipathic helix, but targeted the cytoplasm when the hydrophobic peptide is removed. In addition to having an unusual targeting sequence, TgSODB2 is only the second mitochondrially imported, iron-containing SOD to be described.
Potential role of MCP-1 in endothelial cell tight junction `opening': signaling via Rho and Rho kinase
Authors: Stamatovic Svetlana M. , Keep Richard F. , Kunkel Steven L. , Andjelkovic Anuska V.
Published: October 2003

The expression of the monocyte chemoattractant protein-1 (MCP-1) receptor CCR2 by brain endothelial cells suggests that MCP-1 may have other functions than purely driving leukocyte migration into brain parenchyma during inflammation. This study examines one of these potential novel roles of MCP-1 regulation of endothelial permeability using primary cultures of mouse brain endothelial cells. MCP-1 induces reorganization of actin cytoskeleton (stress fiber formation) and redistribution of tight junction proteins, ZO-1, ZO-2 occludin and claudin-5, from the Triton X-100-soluble to the Triton X-100-insoluble fractions. These morphological changes are associated with a decrease in transendothelial electrical membrane resistance and an increase in [14C]inulin permeability. MCP-1 did not induce these events in brain endothelial cells prepared from mice genotype CCR2–/–. The Rho kinase inhibitor Y27632 and inhibition of Rho (C3 exoenzyme, and dominant negative mutant of Rho, RhoT19N) prevented MCP-1-induced stress fiber assembly, reorganization of tight junction proteins and alterations in endothelial permeability. In all, this suggests that a small GTPase Rho and Rho kinase have a pivotal role in MCP-1-induced junction disarrangement. These data are the first to strongly suggest that MCP-1, via CCR2 present on brain endothelial cells, contributes to increased brain endothelial permeability.
Direct translocation of histone molecules across cell membranes
Authors: Hariton-Gazal Elana , Rosenbluh Joseph , Graessmann Adolf , Gilon Chaim , Loyter Abraham
Published: October 2003

The present work shows that histones are able to directly cross cell plasma membranes and mediate penetration of macromolecules covalently attached to them. Adding a mixture containing the five nucleosomal histones, H1, H2A, H2B, H3 and H4, as well as each of the last four individual histones to intact HeLa and Colo-205 cultured cells resulted in cell penetration and nuclear import of these externally added histones. This was observed by fluorescent and confocal microscopy using fixed and unfixed cells, showing that penetration was not due to the fixation process. Accumulation was also estimated by a quantitative assay that did not require cell fixation and allowed neutralization of surface-bound histones. Translocation into the HeLa and Colo-205 cells occurred at 4°C, in ATP-depleted cells and in cells incubated with sucrose (0.5 M) – conditions that block the endocytic pathway. Furthermore, various endocytosis inhibitors such as colchicine, nocodazole, cytochalasin D, brefeldin A, chloroquine and nystatin did not have any effect on the penetration process. Thus, cellular uptake was mostly due to direct translocation of the histones through the cell plasma membrane and not to endocytosis. The histones were also able to mediate penetration of covalently attached bovine serum albumin (BSA) molecules, indicating their potential as carriers for the delivery of macromolecules into living mammalian cells.
Control of embryonicXenopusmorphogenesis by a Ral-GDS/Xral branch of the Ras signalling pathway
Authors: Lebreton Stéphanie , Boissel Laurent , Moreau Jacques
Published: October 2003

Ras proteins mediate biological responses through various effectors and play a key role in relaying the Fibroblast Growth Factor (FGF) mesoderm induction signal during embryogenesis of the frog, Xenopus laevis. One Ras effector pathway involves the activation of the small G protein Ral. In the present study, we have investigated the role of key components in the Ral branch of FGF and Ras signalling during early Xenopus development. Treatment of animal caps with bFGF, which converts prospective ectoderm to mesoderm, activates Xral. The Ras mutant 12V37G, which can bind to Ral-GDS but not Raf, also activates Xral as well as causing developmental defects and cortical F-actin disassembly. A similar phenotype is induced by Ral-GDS itself. FGF-induced expression of several signature mesodermal genes, by contrast, is independent of Xral signalling. This and other data suggest that the RalB branch of Ras and FGF signalling regulates the actin cytoskeleton and morphogenesis in a transcriptionally independent manner. We also find Xral to be specifically activated in the marginal zone of Xenopus embryos, and find that disruption of the Ral pathway in this region prevents closure of the blastopore during gastrulation. We conclude that Ral signalling is autonomously required by mesodermal cells to effect essential morphogenetic changes during Xenopus gastrulation.
Bpag1 localization to actin filaments and to the nucleus is regulated by its N-terminus
Authors: Young Kevin G. , Pool Madeline , Kothary Rashmi
Published: October 2003

Plakins are a family of giant cytoskeleton binding proteins. One member of this group is bullous pemphigoid antigen 1 (Bpag1)/dystonin, which has neuronal and muscle isoforms that consist of actin-binding and microtubule-binding domains at either end separated by a plakin domain and several spectrin repeats. The better-characterized epithelial isoform has only the plakin domain in common with the neuronal and muscle isoforms. Here, we have analyzed the localization of muscle/neuronal (Bpag1a/b) isoforms and the epithelial (Bpag1e) isoform within C2C12 myoblast cells. Although an antibody specific to Bpag1a/b isoform 2 detected protein co-aligning actin stress fibers, this same antibody and two Bpag1e antibodies predominantly detected protein in the nuclei. A Bpag1a/b isoform 2 N-terminal fusion protein containing the plakin domain also localized to actin stress fibers and to nuclei. Within the plakin domain, we characterized a functional nuclear localization signal, which was responsible for localization of the fusion protein to the nucleus. Bpag1a/b isoform 1 N-terminal fusion proteins differed in their interaction with the actin cytoskeleton and with their ability to localize to the nucleus, suggesting that Bpag1 isoforms with different N-termini have differing roles. These results show the importance of N-terminal domains in dictating the localization and function of Bpag1 isoforms. We provide the first indication that Bpag1 is not strictly a cytoplasmic/membrane protein but that it can also localize to the nucleus.