bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Chk1
Authors: Tapia-Alveal Claudia , Calonge Teresa M , O'Connell Matthew J
Published: April 2009

Chk1 is a serine/threonine protein kinase that is the effector of the G2 DNA damage checkpoint. Chk1 homologs have a highly conserved N-terminal kinase domain, and a less conserved C-terminal regulatory domain of ~200 residues. In response to a variety of genomic lesions, a number of proteins collaborate to activate Chk1, which in turn ensures that the mitotic cyclin-dependent kinase Cdc2 remains in an inactive state until DNA repair is completed. Chk1 activation requires the phosphorylation of residues in the C-terminal domain, and this is catalyzed by the ATR protein kinase. How phosphorylation of the C-terminal regulatory domain activates the N-terminal kinase domain has not been elucidated, though some studies have suggested that this phosphorylation relieves an inhibitory intramolecular interaction between the N- and C-termini. However, recent studies in the fission yeast Schizosaccharomyces pombe have revealed that there is more to Chk1 regulation than this auto-inhibition model, and we review these findings and their implication to the biology of this genome integrity determinant.
Application of serum proteomics to the Women's Health Initiative conjugated equine estrogens trial reveals a multitude of effects relevant to clinical findings
Authors: Katayama Hiroyuki , Paczesny Sophie , Prentice Ross , Aragaki Aaron , Faca Vitor M , Pitteri Sharon J , Zhang Qing , Wang Hong , Silva Melissa , Kennedy Jacob , Rossouw Jacques , Jackson Rebecca , Hsia Judith , Chlebowski Rowan , Manson JoAnn , Hanash Samir
Published: April 2009

Background

The availability of serum collections from the Women's Health Initiative (WHI) conjugated equine estrogens (CEE) randomized controlled trial provides an opportunity to test the potential of in-depth quantitative proteomics to uncover changes in the serum proteome related to CEE and to assess their relevance to trial findings, including elevations in the risk of stroke and venous thromboembolism and a reduction in fractures.

Methods

Five independent large scale quantitative proteomics analyses were performed, each comparing a set of pooled serum samples collected from 10 subjects, 1 year following initiation of CEE at 0.625 mg/d, relative to their baseline pool. A subset of proteins that exhibited increased levels with CEE by quantitative proteomics was selected for validation studies.

Results

Of 611 proteins quantified based on differential stable isotope labeling, the levels of 116 (19%) were changed after 1 year of CEE (nominal P < 0.05), while 64 of these had estimated false discovery rates <0.05. Most of the changed proteins were not previously known to be affected by CEE and had relevance to processes that included coagulation, metabolism, osteogenesis, inflammation, and blood pressure maintenance. To validate quantitative proteomic data, 14 proteins were selected for ELISA. Findings for ten - IGF1, IGFBP4, IGFBP1, IGFBP2, F10, AHSG, GC, CP, MMP2, and PROZ - were confirmed in the initial set of 50 subjects and further validated in an independent set of 50 additional subjects who received CEE.

Conclusions

CEE affected a substantial fraction of the serum proteome, including proteins with relevance to findings from the WHI CEE trial related to cardiovascular disease and fracture.

Clinical Trials Registration

ClinicalTrials.gov identifier: NCT00000611
The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA– OMVs, without further stimulating their proinflammatory activity on circulating monocytes
Authors: Tavano Regina , Franzoso Susanna , Cecchini Paola , Cartocci Elena , Oriente Francesca , Aricò Beatrice , Papini Emanuele
Published: April 2009

Anti-Meningococcus B vaccine candidate stimulates macrophages.

Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadAΔ351–405) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA+ OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA– OMVs. To do this we investigated the activity of purified free NadAΔ351–405 and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadAΔ351–405 stimulated monocytes and macrophages to secrete cytokines (IL-1β, TNF-α, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1α, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadAΔ351–405 induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA+ OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA– OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadAΔ351–405 ones, were much less IFN-γ-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations ∼106 times lower than those required to stimulate cells with free NadAΔ351–405.
A role for glycogen synthase kinase-3 in antagonizing mycobacterial immune evasion by negatively regulating IL-10 induction
Authors: Chan Mabel M P , Cheung Benny K W , Li James C B , Chan Lally L Y , Lau Allan S Y
Published: April 2009

GSK3 regulation of inflammation is targeted by mycobacterial for evasion of immunity resulting in IL-10 induction in the host.

Mtb dysregulates monocyte/macrophage functions to produce a large amount of the immunosuppressive cytokine IL-10. An important function of IL-10 in promoting Mtb survival is the suppression of antigen presentation of monocytes/macrophages to T cells. This dampens the host immune responses and provides an opportunity for immune evasion. GSK3 has been shown to control the balance between pro- and anti-inflammatory cytokine productions. Here, we investigated whether GSK3 regulates IL-10 expression and mediates a protective role upon live mycobacterial challenge using BCG as a model. Our results showed that BCG increased Akt phosphorylation and inhibited GSK3 activity, resulting in increased IL-10 production. We confirmed further that suppression of GSK3 activities by a specific chemical inhibitor strongly enhanced BCG-induced IL-10 production. We also showed that IL-10 secreted by BCG-infected human PBMo was a major suppressor of subsequent IFN-γ production by PBMC and HLA-DR expression on PBMo in response to BCG. Neutralization of PBMo-secreted IL-10 by anti-IL-10 antibodies restored the IFN-γ production and HLA-DR surface expression. Taken together, GSK3 negatively regulates mycobacteria-induced IL-10 production in human PBMo. The kinase may play a role in restoring IFN-γ secretions and subsequent antigen presentation in response to mycobacterial infection. In conclusion, our results suggest a significant role for GSK3 in guarding against mycobacterial evasion of immunity via IL-10 induction in the host.
Src family kinases are necessary for cell migration induced by extracellular HMGB1
Authors: Palumbo Roberta , De Marchis Francesco , Pusterla Tobias , Conti Antonio , Alessio Massimo , Bianchi Marco E
Published: April 2009

The signaling pathways activated by extracellular HMGB1 to mediate cell migration include Src and related kinases, which target cystoskeletal proteins directly.

HMGB1 is a nuclear protein that signals tissue damage, as it is released by cells dying traumatically or secreted by activated innate immunity cells. Extracellular HMGB1 elicits the migration to the site of tissue damage of several cell types, including inflammatory cells and stem cells. The identity of the signaling pathways activated by extracellular HMGB1 is not known completely: We reported previously that ERK and NF-κB pathways are involved, and we report here that Src is also activated. The ablation of Src or inhibition with the kinase inhibitor PP2 blocks migration toward HMGB1. Src associates to and mediates the phosphorylation of FAK and the formation of focal adhesions.
AGE–modified albumin containing infusion solutions boosts septicaemia and inflammation in experimental peritonitis
Authors: Humpert Per M , Lukic Ivan K , Thorpe Suzanne R , Hofer Stefan , Awad Ezzat M , Andrassy Martin , Deemer Elizabeth K , Kasper Michael , Schleicher Erwin , Schwaninger Markus , Weigand Markus A , Nawroth Peter P , Bierhaus Angelika
Published: April 2009

Advanced glycation endproducts (AGEs) act as potential contaminants of infusion solutions boosting inflammation in experimental sepsis.

HSA preparations for i.v. use are administered in critically ill patients. Although increasing intravascular osmotic pressure seems to be a pathophysiologically orientated treatment, clinical trials do not indicate a benefit for mortality in HSA–treated patients. Instead, there is evidence for inflammatory reactions upon infusion of different HSA batches. A neglected issue concerning the safety and quality of these therapeutics is processing–related post–transcriptional protein modifications, such as AGEs. We therefore tested the hypothesis that commercially available infusion solutions contain AGEs and studied whether these protein modifications influence outcome and inflammation in a murine model of sepsis induced by CLP. Screening of different HSA and Ig preparations in this study revealed an up to approximate tenfold difference in the amount of AGE modifications. Application of clinically relevant concentrations of CML–modified HSA in CLP led to increased inflammation and enhanced mortality in wild–type mice but not in mice lacking the RAGE. Lethality was paralleled by increased activation of the proinflammatory transcription factor NF–κB, NF–κB–dependent gene expression, and infiltration of inflammatory cells in the peritoneal cavity. This study implies that infusion solutions containing a high load of the AGE–modified protein have the potential to activate RAGE/NF–κB–mediated inflammatory reactions, causing increased mortality in experimental peritonitis.
T cell suppression by naturally occurring HLA-G-expressing regulatory CD4+ T cells is IL-10-dependent and reversible
Authors: Huang Yu-Hwa , Zozulya Alla L , Weidenfeller Christian , Schwab Nicholas , Wiendl Heinz
Published: April 2009

HLA-Gpos Treg-mediated suppression is critically dependent on the secretion of IL-10 but not TGF-β.

CD4+ T cells constitutively expressing the immune-tolerogenic HLA-G have been described recently as a new type of nTreg (HLA-Gpos Treg) in humans. HLA-Gpos Treg accumulate at sites of inflammation and are potent suppressors of T cell proliferation in vitro, suggesting their role in immune regulation. We here characterize the mechanism of how CD4+ HLA-Gpos Treg influence autologous HLA-Gneg Tresp function. Using a suppression system free of APC, we demonstrate a T–T cell interaction, resulting in suppression of HLA-Gneg Tresp, which is facilitated by TCR engagement on HLA-Gpos Treg. Suppression is independent of cell–cell contact and is reversible, as the removal of HLA-Gpos Treg from the established coculture restored the proliferative capability of responder cells. Further, HLA-Gpos Treg-mediated suppression critically depends on the secretion of IL-10 but not TGF-β.
Ca2+-dependent mast cell death induced by Ag (I) via cardiolipin oxidation and ATP depletion
Authors: Inoue Toshio , Suzuki Yoshihiro , Yoshimaru Tetsuro , Ra Chisei
Published: April 2009

Ag (I) induction leads to a unique necrotic death pathway in which Ca2+-dependent cardiolipin oxidation and ATP depletion play critical roles.

In genetically susceptible humans and/or experimental animals, ions of heavy metals, Hg (II), Au (III), and Ag (I) have been shown to strongly induce autoimmunity, in which mast cells have been implicated to play a role. Here, we demonstrate that Ag (I) application results in mast cell death through a unique Ca2+- and mitochondria-dependent pathway. As cellular susceptibilities to Ag (I) cytotoxicity varied considerably, we analyzed the cell death pathway in the low and high responding cells. In the low responding cells, long application (e.g., 20 h) of Ag (I) at concentrations (≥30 μM) induced cell death, which was accompanied by mitochondrial membrane depolarization, cyt c release, and caspase-3/7 activation but was not prevented by selective inhibitors of caspase-3/7 and the mitochondrial permeability transition. The cell death was preceded by elevations in the cytoplasmic and mitochondrial Ca2+ levels, and Ca2+ responses and cell death were prevented by thiol reagents, including DTT, N-acetylcysteine, and reduced glutathione monoethyl ester. In the high responding cells, Ag (I) evoked considerable cell death by necrosis within 1 h, without inducing caspase activation, and this cell death was reduced significantly by depleting extracellular but not intracellular Ca2+. Moreover, Ag (I) strongly induced Ca2+-dependent CL oxidation and intracellular ATP depletion, both of which were blocked by thiol reagents. These results suggest that Ag (I) activates thiol-dependent Ca2+ channels, thereby promoting Ca2+-dependent CL oxidation, cyt c release, and ATP depletion. This necrotic cell death may play roles in Ag-induced inflammation and autoimmune disorders.
Lethal pulmonary infection with Francisella novicida is associated with severe sepsis
Authors: Sharma Jyotika , Li Qun , Mishra Bibhuti B , Pena Christopher , Teale Judy M
Published: April 2009

Excessive host inflammatory responses negatively impact the disease outcome in pneumonic tularemia.

The bacterial or host determinants of lethality associated with respiratory Francisella infections are currently unknown. No exo– or endotoxins that contribute to the severity of this disease have been identified. However, a deregulated host immune response upon infection is characterized by an initial 36– to 48–h delay followed by a rapid and excessive inflammatory response prior to death at 72–120 h. Here, we extend these findings by comparing host immune responses between sublethal and lethal respiratory infections of mice with an attenuated transposon mutant (Mut) of F. novicida (F.n.) strain U112 (sublethal) versus the wild–type (WT) strain (lethal). Infection with WT bacteria, but not the Mut, was characterized by sustained bacteremia and systemic dissemination of the pathogen with temporal increases in bacterial burdens in liver and spleen. Severe pathology with large foci of infiltrates associated with extensive tissue damage was evident in WT–infected lungs, and Mut–infected mice displayed much reduced pathology with intact lung architecture. Similar to other experimental models of severe sepsis, WT– but not the Mut–infected mice exhibited a robust increase in numbers of Gr1+ and CD11b+ cells, while displaying a significant depletion of αβ T cells. Further, a dramatic up–regulation of multiple cytokines and chemokines was observed only in lethal WT infection. In addition, an earlier and larger increased expression of S100A9, a known mediator of sepsis, was observed in WT–infected mice. Taken together, these results show that a hyperinflammatory host immune response, culminating in severe sepsis, is responsible for the lethal outcome of respiratory tularemia.
Morphometric Vertebral Fractures in Postmenopausal Women with Primary Hyperparathyroidism
Authors: Vignali Edda , Viccica Giuseppe , Diacinti Daniele , Cetani Filomena , Cianferotti Luisella , Ambrogini Elena , Banti Chiara , Del Fiacco Romano , Bilezikian John P. , Pinchera Aldo , Marcocci Claudio
Published: April 2009

Context: An increased risk of fracture in patients with primary hyperparathyroidism (PHPT) compared to the general population has been reported, but available data are controversial.

Objective: The aim of the study was to evaluate the rate of vertebral fractures (VFs) by dual-energy x-ray absorptiometry in postmenopausal women with sporadic PHPT and compare the results with a control group.

Design and Setting: A case-control study was performed at a referral center.

Participants: A total of 150 consecutive patients and 300 healthy women matched for age and menopausal age participated in the study.

Results: VFs were detected in 37 of 150 (24.6%) patients and 12 of 300 (4.0%) controls (P < 0.0001). The majority of VFs were mild. Stepwise multiple logistic regression analysis showed that in PHPT patients lumbar spine bone mineral density was the only variable independently associated with the prevalence of VFs (P = 0.003). The rate of fracture was higher in symptomatic (34.1%) than asymptomatic (21.1%) patients, but this difference was not statistically significant (P = 0.15). Among asymptomatic patients, fracture rate was significantly higher in those who met the criteria for parathyroidectomy (28.1%) than in those who did not (11.1%) (P = 0.03). Compared to controls, the fracture rate was significantly higher in patients with symptomatic and asymptomatic PHPT who met the criteria for surgery (P < 0.0001), but not in those who did not meet the criteria (P = 0.06).

Conclusions: VF rate is increased in postmenopausal women with PHPT compared to controls, independently of whether they are classified as symptomatic or asymptomatic. The question of whether the finding of mild morphometric VFs in the latter represents an indication for parathyroid surgery remains to be established.
Identification and Characterization of Two Novel Truncated but Functional Isoforms of the Somatostatin Receptor Subtype 5 Differentially Present in Pituitary Tumors
Authors: Durán-Prado Mario , Gahete Manuel D. , Martínez-Fuentes Antonio J. , Luque Raúl M. , Quintero Ana , Webb Susan M. , Benito-López Pedro , Leal Alfonso , Schulz Stefan , Gracia-Navarro F. , Malagón María M. , Castaño Justo P.
Published: April 2009

Context: Somatostatin and its related peptide cortistatin exert multiple actions on normal and tumoral tissue targets through a family of receptors termed somatostatin receptor (sst)1-5. Despite the considerable advances in the knowledge on these receptors and their (patho)physiological roles, there is still evidence that additional receptors for these peptides should exist to fully explain their actions.

Objective: The growing number of spliced variants found in similar receptor families, often present in tumors, and results from our group obtained on sst5 from other species (pig) led us to explore the existence of new human sst5 isoforms.

Design and Results: A rapid amplification of cDNA ends PCR approach on samples from a human pituitary tumor and a cell line enabled identification of two novel alternatively spliced sst5 receptor variants. The sequences obtained encode putative proteins that correspond to truncated isoforms of five and four transmembrane domains (TMDs), accordingly named sst5TMD5 and sst5TMD4, respectively. Both novel receptors show a differential expression pattern in normal tissues and are also present in pituitary tumors of diverse etiology including nonfunctioning adenomas, corticotropinomas, somatotropinomas, and a prolactinoma. In contrast to the predominant plasma membrane localization of full-length sst5, both sst5TMD5 and sst5TMD4 show a preferentially intracellular localization. Despite their truncated nature, both receptors are functional, as shown by their ability to mediate selective, ligand-induced rises in free cytosolic calcium concentration. Specifically, whereas sst5TMD5 is selectivity activated by somatostatin compared with cortistatin, cells transfected with sst5TMD4 almost exclusively respond to cortistatin and not to somatostatin.

Conclusions: Our results demonstrate the existence of two previously unidentified sst5 spliced variants with distinct distribution in normal tissues and pituitary tumors, unique ligand-selective signaling properties, and subcellular distribution, which could contribute to somatostatin and cortistatin signaling in normal and tumoral cells.
Hypothyroidism Secondary to Hypothalamic-Pituitary Dysfunction May Be Part of the Phenotype in Klinefelter Syndrome: A Case-Control Study
Authors: Bjørn Anne-Mette Bay , Bojesen Anders , Gravholt Claus H. , Laurberg Peter
Published: April 2009

Context: Klinefelter syndrome (KS) may involve a number of abnormalities besides the characteristic testicular insufficiency. Some studies have suggested that thyroid abnormalities may be common, but this has not been clarified.

Design: A case-control study of men with KS (n = 75) compared with age-matched men from the general population (n = 75) was organized, and thyroid function, thyroid volume by ultrasonography, and thyroid antibodies were examined.

Results: Men with KS were on average taller and heavier and tended to have a higher body mass index than the men in the control group. Serum free T4 (fT4) was lower in men with KS than controls [mean (sd): 16.3 (2.35) vs. 17.6 (1.75) pmol/liter; P < 0.001], with clustering in or just below the lower part of the reference range for the assay. The ratio fT4 to free T3 was low in KS (P < 0.001), whereas no differences between groups were observed in TSH, free T3, TSH to fT4 ratio, thyroid volume, or the prevalence of thyroid antibodies. No difference in any of the variables were observed between testosterone-treated and untreated KS men. Adjustment for differences in height, weight, and concomitant disease in multivariate models did not alter the results.

Conclusions: Men with KS had a general shift toward lower values in distribution of serum fT4 with no compensatory increase in serum TSH. The most likely mechanism is a decrease or change in set point of thyrotroph control of thyroid function.
Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains
Authors: Iinuma Takayuki , Aoki Takehiro , Arasaki Kohei , Hirose Hidenori , Yamamoto Akitsugu , Samata Rie , Hauri Hans-Peter , Arimitsu Nagisa , Tagaya Mitsuo , Tani Katsuko
Published: April 2009

The presence of subdomains in the endoplasmic reticulum (ER) enables this organelle to perform a variety of functions, yet the mechanisms underlying their organization are poorly understood. In the present study, we show that syntaxin 18, a SNAP (soluble NSF attachment protein) receptor localized in the ER, is important for the organization of two ER subdomains, smooth/rough ER membranes and ER exit sites. Knockdown of syntaxin 18 caused a global change in ER membrane architecture, leading to the segregation of the smooth and rough ER. Furthermore, the organization of ER exit sites was markedly changed concomitantly with dispersion of the ER-Golgi intermediate compartment and the Golgi complex. These morphological changes in the ER were substantially recovered by treatment of syntaxin-18-depleted cells with brefeldin A, a reagent that stimulates retrograde membrane flow to the ER. These results suggest that syntaxin 18 has an important role in ER subdomain organization by mediating the fusion of retrograde membrane carriers with the ER membrane.
A novel mode of cell detachment from fibrillar fibronectin matrix under shear
Authors: Engler Adam J. , Chan May , Boettiger David , Schwarzbauer Jean E.
Published: April 2009

Cells within tissues are surrounded by fibrillar extracellular matrix (ECM) that supports cell adhesion via integrin receptors. The strength of cell interactions with fibrillar matrix and the effects of force on these interactions have not been quantified. To this end, we used a spinning disc device to apply radially increasing shear to human HT1080 fibrosarcoma cells attached to a cell-derived fibrillar fibronectin (FN) matrix. The shear required to detach 50% of HT1080 cells was eight times greater on a FN-coated, rigid glass substrate than on fibrillar FN matrix. Covalent crosslinking of the FN matrix increased its stiffness tenfold and produced a modest increase in shear detachment force for these cells. On FN-coated surfaces, cells detach by releasing interactions between α5β1 integrin and FN. By contrast, cell detachment from fibrillar matrix occurred through a novel mechanism of fibril breakage, which left holes in the matrix visible by fluorescence microscopy. These results show that cells require less force to detach from fibrillar matrix than from FN adsorbed on glass and that detachment occurs through breaking fibrils instead of by release of integrin-matrix bonds. Thus, ECM fibril breakage is another molecular feature to consider when understanding cell and tissue homeostasis.
Interaction between PAR-3 and the aPKC–PAR-6 complex is indispensable for apical domain development of epithelial cells
Authors: Horikoshi Yosuke , Suzuki Atsushi , Yamanaka Tomoyuki , Sasaki Kazunori , Mizuno Keiko , Sawada Hajime , Yonemura Shigenobu , Ohno Shigeo
Published: April 2009

The evolutionarily conserved polarity proteins PAR-3, atypical protein kinase C (aPKC) and PAR-6 critically regulate the apical membrane development required for epithelial organ development. However, the molecular mechanisms underlying their roles remain to be clarified. We demonstrate that PAR-3 knockdown in MDCK cells retards apical protein delivery to the plasma membrane, and eventually leads to mislocalized apical domain formation at intercellular regions in both two-dimensional and three-dimensional culture systems. The defects in PAR-3 knockdown cells are efficiently rescued by wild-type PAR-3, but not by a point mutant (S827/829A) that lacks the ability to interact with aPKC, indicating that formation of the PAR-3–aPKC–PAR-6 complex is essential for apical membrane development. This is in sharp contrast with tight junction maturation, which does not necessarily depend on the aPKC–PAR-3 interaction, and indicates that the two fundamental processes essential for epithelial polarity are differentially regulated by these polarity proteins. Importantly, highly depolarized cells accumulate aPKC and PAR-6, but not PAR-3, on apical protein-containing vacuoles, which become targeted to PAR-3-positive primordial cell-cell contact sites during the initial stage of the repolarization process. Therefore, formation of the PAR-3–aPKC–PAR-6 complex might be required for targeting of not only the aPKC–PAR-6 complex but also of apical protein carrier vesicles to primordial junction structures.
AncientLeishmaniacoronin (CRN12) is involved in microtubule remodeling during cytokinesis
Authors: Sahasrabuddhe Amogh A. , Nayak Ramesh C. , Gupta Chhitar M.
Published: April 2009

In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture.
The collagen receptor DDR1 regulates cell spreading and motility by associating with myosin IIA
Authors: Huang Yun , Arora Pamela , McCulloch Christopher A. , Vogel Wolfgang F.
Published: April 2009

The spreading and migration of cells on adhesive substrates is regulated by the counterbalance of contractile and protrusive forces. Non-muscle myosin IIA, an ubiquitously expressed contractile protein and enzyme, is implicated in the regulation of cell spreading and directional migration in response to various stimuli. Here we show that discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor activated by type I collagen, associates with the non-muscle myosin IIA heavy chain (NMHC-IIA) upon ligand stimulation. An association was also indicated by coimmunoprecipitation of NMHC-IIA with full-length DDR1, but not with the truncated DDR1d-isoform lacking the kinase domain. DDR1 was important for assembly of NMHC-IIA into filaments on cells plated on collagen. DDR1 expression inhibited cell spreading over collagen but promoted cell migration. By contrast, blockade of non-muscle myosin II activity by blebbistatin enhanced cell spreading but inhibited migration over collagen. We propose that myosin and DDR1 impact cell spreading and migration by regulating adhesive contacts with collagen.
A splice variant of cyclin D2 regulates cardiomyocyte cell cycle through a novel protein aggregation pathway
Authors: Sun Qian , Zhang Feixiong , Wafa Karim , Baptist Timothy , Pasumarthi Kishore B. S.
Published: April 2009

The mammalian heart lacks intrinsic ability to replace diseased myocardium with newly divided myocytes. There is scant information on mechanisms regulating cell cycle exit in cardiomyocytes. We cloned a splice variant of cyclin D2 (D2SV) from the mouse heart and found a novel role for this protein in cardiomyocyte cell cycle exit. We report that D2SV is highly expressed in embryonic myocardium compared with the adult heart. Localization studies indicate that D2SV is retained in the endoplasmic reticulum (ER), Golgi and lysosomal compartments and subjected to ER-stress-associated protein aggregation. D2SV aggregation relies on the motor activities of dynein and is blocked by ER stress modulators. The ability of D2SV to sequester other cell cycle proteins provides a mechanistic explanation for its effects on cardiomyocyte cell cycle. We show that D2SV-induced cell cycle exit can be rescued by overexpression of D-type and B-type cyclins. We suggest that protein aggregation may be a major block for cardiomyocyte cell cycle reactivation.
An integrin-α4–14-3-3ζ–paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration
Authors: Deakin Nicholas O. , Bass Mark D. , Warwood Stacey , Schoelermann Julia , Mostafavi-Pour Zohreh , Knight David , Ballestrem Christoph , Humphries Martin J.
Published: April 2009

α4 integrins are used by leukocytes and neural crest derivatives for adhesion and migration during embryogenesis, immune responses and tumour invasion. The pro-migratory activity of α4 integrin is mediated in part through the direct binding of the cytoplasmic domain to paxillin. Here, using intermolecular FRET and biochemical analyses, we report a novel interaction of the α4 integrin cytoplasmic domain with 14-3-3ζ. This interaction depends on serine phosphorylation of α4 integrin at a site (S978) distinct from that which regulates paxillin binding (S988). Using a combination of metabolic labelling and targeted mass spectrometry by multiple reaction monitoring we demonstrate the low stoichiometry phosphorylation of S978. The interaction between α4 integrin and 14-3-3ζ is enhanced by the direct association between 14-3-3ζ and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between α4 integrin and paxillin is sufficient for normal Rac1 regulation, the integrity of the ternary complex is essential for focused Cdc42 activity at the lamellipodial leading edge and directed cell movement. Taken together, these data identify a key signalling nexus mediating α4 integrin-dependent migration.
Can public managers reduce red tape? The role of internal management in overcoming external constraints
Authors: Walker Richard M. , Brewer Gene A.
Published: April 2009

This study assesses the relative impact of four waves of variables on the level of red tape in English local government services. These variables range from factors that are largely uncontrollable, such as the inherent nature of the service being provided and turbulence in the external environment, to factors that are at least partly within public managers’ control, such as organisational integration and management strategy. Data for this study come from a 2004 survey of English local government (785 services). The findings show that many factors affect the level of red tape in public organisations, and while public managers cannot eliminate all burdensome rules and regulations, they can significantly reduce the level of red tape by being vigilant in their efforts and employing appropriate management techniques and strategies. The implications for research on red tape and public management theory and practice are discussed.
Public policy making in Turkey: faltering attempts to generate a national drugs policy
Authors: Robins Philip
Published: April 2009

The caricature of public policy making in Turkey persists: a monolithic state and top-down political authority. There is some secondary literature, albeit limited, that challenges this image, and suggests greater state fragmentation. This cross-cutting case study of drugs policy in Turkey confirms this more differentiated experience. It illuminates the difficulties of process in the development of policy. It points to the disparities in capacity between different government agencies in generating change. For drugs policy itself, the article concludes that in the absence of domestic political will, other rewards, notably the benefits of international prestige and the resources that flow from it, are crucial in bringing about a nominal compliance with global norms.
Organising immigration policy: the unstable balance between political control and agency autonomy
Authors: Christensen Tom , Lægreid Per
Published: April 2009

In this article we ask to what extent and how political executives balance political control and agency autonomy in immigration policy and how this is reflected in the different ways of organising the central immigration administration. The analysis is based on instrumental, negotiation and cultural perspectives. The case under study is the organisation of the central immigration administration in Norway, which experienced three major reforms: in 1988, 2001 and 2005. These involved an increase in the autonomy of the immigration agency, the granting of further autonomy and eventually an attempt to increase political control. We ask what the political motives behind organising the immigration administration are and analyse the implications of different modes of organisation.
English mental health policy under New Labour
Authors: Pilgrim David , Ramon Shulamit
Published: April 2009

This article reviews the changes in mental health policy in England under the jurisdiction of New Labour governments between 1997 and 2007. These are considered in relation to recurring policy questions about: the structures and processes involved in responding to mental health problems; professional jurisdiction for mental health problems; the conditions under which mental disorder should be lawfully controlled; links with wider health and welfare policy changes; and the enhancement of mental health in the population. Distinctions are made in the analysis between continuities about this policy agenda and the particular features linked to the period under consideration, initiated or responded to by government.
Competition and contracts in the voluntary sector: exploring the implications for homelessness service providers in Southampton
Authors: Buckingham Heather
Published: April 2009

This study explores the impacts of contractual government funding and competitive tendering on voluntary organisations (VOs) providing homelessness services in Southampton, UK. Although service quality has arguably improved, the interview data suggest that implementing competitive tendering within the voluntary sector is not unproblematic. Three key issues are discussed: changing demands for expertise, increasing job insecurity, and tensions between competition and cooperation among VOs. The article argues that if VOs are to retain the distinctive qualities for which politicians have lauded them, procurement and monitoring procedures must be carefully attuned to the social relations and practices of the voluntary sector.
Src Family Kinases Are Required for Limb Trajectory Selection by Spinal Motor Axons
Authors: Kao Tzu-Jen , Palmesino Elena , Kania Artur
Published: April 2009

Signal relay by guidance receptors at the axonal growth cone is a process essential for the assembly of a functional nervous system. We investigated thein vivofunction of Src family kinases (SFKs) as growth cone guidance signaling intermediates in the context of spinal lateral motor column (LMC) motor axon projection toward the ventral or dorsal limb mesenchyme. Usingin situmRNA detection we determined thatSrcandFynare expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. Inhibition of SFK activity by C-terminal Src kinase (Csk) overexpression in chick LMC axons usingin ovoelectroporation resulted in LMC axons selecting the inappropriate dorsoventral trajectory within the limb mesenchyme, with medial LMC axon projecting into the dorsal and ventral limb nerve with apparently random incidence. We also detected LMC axon trajectory choice errors inSrcmutant mice demonstrating a nonredundant role for Src in motor axon guidance in agreement with gain and loss of Src function in chick LMC neurons which led to the redirection of LMC axons. Finally, Csk-mediated SFK inhibition attenuated the retargeting of LMC axons caused by EphA or EphB over-expression, implying the participation of SFKs in Eph-mediated LMC motor axon guidance. In summary, our findings demonstrate that SFKs are essential for motor axon guidance and suggest that they play an important role in relaying ephrin:Eph signals that mediate the selection of motor axon trajectory in the limb.
Excitotoxic Death of Retinal NeuronsIn VivoOccurs via a Non-Cell-Autonomous Mechanism
Authors: Lebrun-Julien Frédéric , Duplan Laure , Pernet Vincent , Osswald Ingrid , Sapieha Przemyslaw , Bourgeois Philippe , Dickson Kathleen , Bowie Derek , Barker Philip A. , Di Polo Adriana
Published: April 2009

The central hypothesis of excitotoxicity is that excessive stimulation of neuronal NMDA-sensitive glutamate receptors is harmful to neurons and contributes to a variety of neurological disorders. Glial cells have been proposed to participate in excitotoxic neuronal loss, but their precise role is defined poorly. In thisin vivostudy, we show that NMDA induces profound nuclear factor κB (NF-κB) activation in Müller glia but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons is effectively blocked by inhibitors of NF-κB activity. We demonstrate that tumor necrosis factor α (TNFα) protein produced in Müller glial cells via an NMDA-induced NF-κB-dependent pathway plays a crucial role in excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNFα-dependent increase in Ca2+-permeable AMPA receptors on susceptible neurons. Thus, our data reveal a novel non-cell-autonomous mechanism by which glial cells can profoundly exacerbate neuronal death following excitotoxic injury.
δ-Catenin Regulates Spine and Synapse Morphogenesis and Function in Hippocampal Neurons during Development
Authors: Arikkath Jyothi , Peng I-Feng , Gie Ng Yu , Israely Inbal , Liu Xin , Ullian Erik M. , Reichardt Louis F.
Published: April 2009

The maintenance of spine and synapse number during development is critical for neuronal circuit formation and function. Here we show that δ-catenin, a component of the cadherin-catenin cell adhesion complex, regulates spine and synapse morphogenesis during development. Genetic ablation or acute knockdown of δ-catenin leads to increases in spine and synapse density, accompanied by a decrease in tetrodotoxin induced spine plasticity. Our results indicate that δ-catenin may mediate conversion of activity-dependent signals to morphological spine plasticity. The functional role of δ-catenin in regulating spine density does not require binding to cadherins, but does require interactions with PDZ domain-containing proteins. We propose that the perturbations in spine and synaptic structure and function observed after depletion of δ-catenin during development may contribute to functional alterations in neural circuitry, the cognitive deficits observed in mutant mice, and the mental retardation pathology of Cri-du-chat syndrome.
Parallel Use of Two Behavioral Mechanisms for Chemotaxis inCaenorhabditis elegans
Authors: Iino Yuichi , Yoshida Kazushi
Published: April 2009

Caenorhabditis elegansshows chemotaxis to various odorants and water-soluble chemoattractants such as NaCl. Previous studies described the pirouette mechanism for chemotaxis, in whichC. elegansquickly changes the direction of locomotion by using a set of stereotyped behaviors, a pirouette, in response to a decrease in the concentration of the chemical. Here, we report the discovery of a second mechanism for chemotaxis, called the weathervane mechanism. In this strategy animals respond to a spatial gradient of chemoattractant and gradually curve toward higher concentration of the chemical. By computer simulation, we find that both of these mechanisms contribute to chemotaxis and both mechanisms need to act in parallel for efficient chemotaxis. Using laser ablation of individual neurons to examine the underlying neural circuit, we find the ASE sensory neurons and AIZ interneurons are essential for both the pirouette and weathervane mechanisms in chemotaxis to NaCl. Salt-conditioned animals show reversed responses in both of these behaviors, leading to avoidance of NaCl. These results provide a platform for detailed molecular and cellular analyses of chemotaxis and its plasticity in this model organism.
Stimulus-Specific Adaptation in the Inferior Colliculus of the Anesthetized Rat
Authors: Malmierca Manuel S. , Cristaudo Salvatore , Pérez-González David , Covey Ellen
Published: April 2009

To identify sounds as novel, there must be some neural representation of commonly occurring sounds. Stimulus-specific adaptation (SSA) is a reduction in neural response to a repeated sound. Previous studies using an oddball stimulus paradigm have shown that SSA occurs at the cortex, but this study demonstrates that neurons in the inferior colliculus (IC) also show strong SSA using this paradigm. The majority (66%) of IC neurons showed some degree of SSA. Approximately 18% of neurons showed near-complete SSA. Neurons with SSA were found throughout the IC. Responses of IC neurons were reduced mainly during the onset component of the response, and latency was shorter in response to the oddball stimulus than to the standard. Neurons with near-complete SSA were broadly tuned to frequency, suggesting a high degree of convergence. Thus, some of the mechanisms that may underlie novelty detection and behavioral habituation to common sounds are already well developed at the midbrain.
How Multiple Conductances Determine Electrophysiological Properties in a Multicompartment Model
Authors: Taylor Adam L. , Goaillard Jean-Marc , Marder Eve
Published: April 2009

Most neurons have large numbers of voltage- and time-dependent currents that contribute to their electrical firing patterns. Because these currents are nonlinear, it can be difficult to determine the role each current plays in determining how a neuron fires. The lateral pyloric (LP) neuron of the stomatogastric ganglion of decapod crustaceans has been studied extensively biophysically. We constructed ∼600,000 versions of a four-compartment model of the LP neuron and distributed 11 different currents into the compartments. From these, we selected ∼1300 models that match well the electrophysiological properties of the biological neuron. Interestingly, correlations that were seen in the expression of channel mRNA in biological studies were not found across the ∼1300 admissible LP neuron models, suggesting that the electrical phenotype does not require these correlations. We used cubic fits of the function from maximal conductances to a series of electrophysiological properties to ask which conductances predominantly influence input conductance, resting membrane potential, resting spike rate, phasing of activity in response to rhythmic inhibition, and several other properties. In all cases, multiple conductances contribute to the measured property, and the combinations of currents that strongly influence each property differ. These methods can be used to understand how multiple currents in any candidate neuron interact to determine the cell's electrophysiological behavior.
Activity-Dependent Repression of Cbln1 Expression: Mechanism for Developmental and Homeostatic Regulation of Synapses in the Cerebellum
Authors: Iijima Takatoshi , Emi Kyoichi , Yuzaki Michisuke
Published: April 2009

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is released from cerebellar granule cells and plays a crucial role in forming and maintaining excitatory synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells not only during development but also in the adult cerebellum. Although neuronal activity is known to cause morphological changes at synapses, how Cbln1 signaling is affected by neuronal activity remains unclear. Here, we show that chronic stimulation of neuronal activity by elevating extracellular K+levels or by adding kainate decreased the expression ofcbln1mRNA within several hours in mature granule cells in a manner dependent onl-type voltage-dependent Ca2+channels and calcineurin. Chronic activity also reduced Cbln1 protein levels within a few days, during which time the number of excitatory synapses on Purkinje cell dendrites was reduced; this activity-induced reduction of synapses was prevented by the addition of exogenous Cbln1 to the culture medium. Therefore, the activity-dependent downregulation ofcbln1may serve as a new presynaptic mechanism by which PF–Purkinje cell synapses adapt to chronically elevated activity, thereby maintaining homeostasis. In addition, the expression ofcbln1mRNA was prevented when immature granule cells were maintained in high-K+medium. Since immature granule cells are chronically depolarized before migrating to the internal granule layer, this depolarization-dependent regulation ofcbln1mRNA expression may also serve as a developmental switch to facilitate PF synapse formation in mature granule cells in the internal granule layer.
Early Deficits in Spatial Memory and Theta Rhythm in Experimental Temporal Lobe Epilepsy
Authors: Chauvière Laetitia , Rafrafi Nadia , Thinus-Blanc Catherine , Bartolomei Fabrice , Esclapez Monique , Bernard Christophe
Published: April 2009

Patients with temporal lobe epilepsy (TLE), the most common form of epilepsy in adults, often display cognitive deficits. The time course and underlying mechanisms of cognitive decline remain unknown during epileptogenesis (the process leading to epilepsy). Using the rat pilocarpine model of TLE, we performed a longitudinal study to assess spatial and nonspatial cognitive performance during epileptogenesis. In parallel, we monitored interictal-like activity (ILA) in the hippocampal CA1 region, as well as theta oscillations, a brain rhythm central to numerous cognitive processes. Here, we report that spatial memory was altered soon after pilocarpine-induced status epilepticus, i.e., already during the seizure-free, latent period. Spatial deficits correlated with a decrease in the power of theta oscillations but not with the frequency of ILA. Spatial deficits persisted when animals had spontaneous seizures (chronic stage) without further modification. In contrast, nonspatial memory performances remained unaffected throughout. We conclude that the reorganization of hippocampal circuitry that immediately follows the initial insult can affect theta oscillation mechanisms, in turn, resulting in deficits in hippocampus-dependent memory tasks. These deficits may be dissociated from the process that leads to epilepsy itself but could instead constitute, as ILA, early markers in at-risk patients and/or provide beneficial therapeutic targets.